Patent application title: SENECA VALLEY VIRUS BASED COMPOSITIONS AND METHODS FOR TREATING DISEASE
Inventors:
Paul L. Hallenbeck (Chester Springs, PA, US)
Seshidhar Reddy Police (Chester Springs, PA, US)
Laura M. Hales (Chester Springs, PA, US)
Carl M. Hay (Damascus, MD, US)
Shanthi Ganesh (San Francisco, CA, US)
Ling Xu (Boyds, MD, US)
Jingping Yang (Gaithersburg, MD, US)
Cheng Cheng (Rockville, MD, US)
Assignees:
NOVARTIS AG
IPC8 Class: AA61K3576FI
USPC Class:
424 936
Class name: Drug, bio-affecting and body treating compositions whole live micro-organism, cell, or virus containing virus or bacteriophage
Publication date: 2014-11-27
Patent application number: 20140348798
Abstract:
The present invention relates to a novel RNA picornavirus that is called
Seneca Valley virus ("SVV"). The invention provides isolated SVV nucleic
acids and proteins encoded by these nucleic acids. Further, the invention
provides antibodies that are raised against the SVV proteins. Because SVV
has the ability to selectively kill some types of tumors, the invention
provides methods of using SVV and SVV polypeptides to treat cancer.
Because SVV specifically targets certain tumors, the invention provides
methods of using SVV nucleic acids and proteins to detect cancer.
Additionally, due to the information provided by the tumor-specific
mechanisms of SVV, the invention provides methods of making new oncolytic
virus derivatives and of altering viruses to have tumor-specific
tropisms.Claims:
1. An isolated nucleic acid comprising a nucleic acid sequence having at
least 75% sequence identity to: (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, or 168, or (ii) a contiguous portion of SEQ ID NO: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, or 168, that is at least 20 nucleotides in
length.
2. The isolated nucleic acid of claim 1, wherein the nucleic acid is RNA or DNA.
3. An isolated polypeptide comprising an amino acid sequence having at least 75% sequence identity to: (i) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 169, or (ii) a contiguous portion of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 169 that is at least 10 amino acids in length.
4. An isolated Seneca Valley virus or derivative thereof, comprising identifying characteristics of ATCC Patent Deposit number PTA-5343.
5. An isolated Seneca Valley virus or derivative or relative thereof, having a genome comprising a sequence that is at least 95%, 90%, 85%, 80%, 75%, 70%, or 65% identical to SEQ ID NO:1 or SEQ ID NO:168.
6. The virus of claim 5 comprising the following characteristics: replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in normal cells.
7. The virus of claim 6, wherein said virus is replication competent in tumor cell types having neuroendocrine properties.
8. A pharmaceutical composition comprising an effective amount of the virus of claim 5 and a pharmaceutically acceptable carrier.
9. An isolated antibody that specifically binds to the epitope of the isolated virus of claim 5.
10. A method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genome that comprises a sequence that is at least 75% identical to a contiguous sequence of SEQ ID NO:1 or SEQ ID NO:168 that is at least 100 nucleotides in length.
11. The method of claim 10, wherein the virus is a picornavirus.
12. The method of claim 11, wherein the picornavirus is a cardiovirus.
13. The method of claim 12, wherein the cardiovirus is selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus.
14. The method of claim 11, wherein the picornavirus is a member of a genus to which Seneca Valley virus belongs.
15. The method of claim 11, wherein the picornavirus is Seneca Valley virus.
16. The method of claim 15, wherein the Seneca Valley virus has an ATCC deposit number PTA-5343.
17. The method of claim 11, wherein the picornavirus is a Seneca Valley virus-like picornavirus.
18. The method of claim 17, wherein the Seneca Valley virus-like picornavirus is selected from the group of isolates consisting of: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395, LA 1278, IL 66289, IL 94-9356, MN/GA 99-29256, MN 99197, and SC 363649.
19. A method of killing an abnormally proliferative cell comprising contacting the cell with the virus of claim 5.
20. A method for making an oncolytic virus, the method comprising: (a) comparing a Seneca Valley virus genomic sequence with a test virus genomic sequence; (b) identifying at least a first amino acid difference between a polypeptide encoded by the Seneca Valley virus genomic sequence and a polypeptide encoded by the test virus genomic sequence; (c) mutating the test virus genomic sequence such that the polypeptide encoded by the test virus genomic sequence has at least one less amino acid difference to the polypeptide encoded by the Seneca Valley virus genomic sequence; (d) transfecting the mutated test virus genomic sequence into a tumor cell; and (e) determining whether the tumor cell is lytically infected by the mutated test virus genomic sequence.
21. The method of claim 20, wherein the Seneca Valley virus genome comprises a sequence that is at least 95% identical to SEQ ID NO:1 or SEQ ID NO:168.
22. The method of claim 20, wherein the test virus is a picornavirus.
23. The method of claim 22, wherein the test virus is a Seneca Valley virus-like picornavirus.
24. The method of claim 20, wherein the amino acid differences are between a Seneca Valley virus capsid protein and a test virus capsid protein.
25. The method of claim 20, wherein mutating the test virus genomic sequence comprises mutating a cDNA having the test virus genomic sequence.
26. The method of claim 20, wherein transfecting the mutated test virus genomic sequence comprises transfecting RNA, wherein the RNA is generated from the cDNA having the mutated test virus genomic sequence.
Description:
[0001] This application is a continuation of U.S. Ser. No. 13/209,124,
which was filed on Aug. 12, 2011, which is a continuation of U.S. Ser.
No. 12/576,296, which was filed on Oct. 9, 2009, now U.S. Pat. No.
8,039,606 issued on Oct. 18, 2011, which is a continuation of U.S. Ser.
No. 11/335,891, which was filed on Jan. 19, 2006, now U.S. Pat. No.
7,638,318 issued on Dec. 29, 2009, which is a continuation-in-part of
International Application No. PCT/US2004/031504 (International
Publication No. WO 2005/030139), which was filed on Sep. 23, 2004, which
claims priority to U.S. Ser. No. 60/506,182, which was filed on Sep. 26,
2003. This application also claims priority to U.S. Ser. No. 60/664,442,
which was filed on Mar. 23, 2005 and U.S. Ser. No. 60/726,313, which was
filed on Oct. 13, 2005. These applications are hereby incorporated by
reference in their entirety.
[0002] This disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
[0003] All patent applications, published patent applications, issued and granted patents, texts, and literature references cited in this specification are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which the present invention pertains.
BACKGROUND OF THE INVENTION
[0004] Virotherapy holds great promise for treating cancer. Oncolytic viruses, which aim to specifically infect and kill cancer cells, whether native and/or engineered, may be more efficacious and less toxic than alternative treatments, such as chemotherapy and radiation. In addition, oncolytic virus therapy that uses replication competent viruses is the only therapy known that can amplify the therapeutic at the pharmacologically desired site.
[0005] A key aspect of cancer therapy is to achieve a high rate of killing of cancer cells versus normal cells. Accomplishing this goal has been difficult for many reasons, including the wide array of cell types involved, the systemic dissemination of cancer cells due to metastases, and the narrow biological differences between normal and cancer cells. While progress has been made, much still needs to be done to improve upon current cancer therapies.
[0006] In the past, surgeons have tried to remove tumors surgically without substantially harming the patient. Even complete removal of a primary tumor does not ensure survival since earlier metastases to unknown sites in the body are left undetected. There is also some research suggesting that surgical intervention may enhance the growth of distant metastases due to removal of tumor cells producing angiogenesis inhibitors. Finally, in many cases, the tumor grows back at the original site after surgical removal. Radiation aims to selectively destroy the most rapidly proliferating cells at the expense of the others. However, tumor cells can escape radiation therapy either by becoming resistant or by being in a non-dividing state during treatment. In addition, radiation is not always selective in that many normal cells are actively dividing and killed by the treatment (cells in bone marrow, gastrointestinal cells, hair follicles, etc.).
[0007] Like radiation, chemotherapy is not completely selective and thus destroys many normal cells, and does not kill all tumor cells due to drug resistance and/or division state of the cell. Thus, chemotherapy and radiation therapies exploit a small differential sensitivity that exists between normal and cancer cells, giving them a narrow therapeutic index. A small therapeutic index is clearly an undesirable property of any modality to treat cancer. Therefore, novel cancer therapeutic approaches overcoming these limitations are desired.
[0008] One such novel approach is oncolytic virus therapy. Initially, replication-defective viruses carrying cytotoxic transgenes were utilized in attempts to treat cancer. However, they were found to be inefficient in transduction of tumors, inadequate spread within the tumor mass and not adequately selective toward cancers. To overcome this limitation, viruses were either modified to replicate selectively in tumor cells or viruses were discovered to have natural tumor-selective properties. These oncolytic viruses thus had the properties to replicate, spread, and kill tumor cells selectively through a tumor mass by locally injecting the virus or by systemically delivering the virus (FIG. 1).
[0009] Despite the early promise of this newly defined class of anti-cancer therapeutics, several limitations remain that may limit their use as a cancer therapeutic. Therefore, there is an ongoing need for novel oncolytic viruses that can be utilized for cancer therapy.
SUMMARY OF THE INVENTION
[0010] A novel RNA picornavirus has been discovered (hereafter referred to as Seneca Valley virus ("SVV")) whose native properties include the ability to selectively kill some types of tumors. As demonstrated below in the examples, SVV selectively kills tumor lines with neurotropic properties, in most cases with a greater than 10,000 fold difference in the amount of virus necessary to kill 50% of tumor cells versus normal cells (i.e., the EC50 value). This result also translates in vivo, where tumor explants in mice are selectively eliminated. Further, in vivo results indicate that SVV is not toxic to normal cells, in that up to 1×1014 vp/kg (vector or virus particles per kilogram) systemically administered causes no mortality and no visible clinical symptoms in immune deficient or immune competent mice.
[0011] SVV elicits efficacy at doses as low as 1×108 vp/kg; therefore, a very high therapeutic index of >100,000 is achieved. Efficacy is very robust in that 100% of large pre-established tumors in mice can be completely eradicated (see Example 11). This efficacy may be mediated with a single systemic injection of SVV without any adjunct therapy. Furthermore, SVV injected mice show neither clinical symptoms nor recurrence of tumors for at least 200 days following injection. SVV can also be purified to high titer and can be produced at >200,000 virus particles per cell in permissive cell lines. SVV-based viral therapy therefore shows considerable promise as a safe, effective and new line of treatment for selected types of cancers. Further, SVV has a small and easily manipulatable genome, simple and fast lifecycle, and a well-understood biology of replication, and thus is amenable to modification. These properties, at least in part, allow for methods that generate modified SVVs that have new cell or tissue specific tropisms, such that SVV-based therapy can be directed to new tumor types resistant to infection by the original SVV isolate.
[0012] Accordingly, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion of any one of these sequences that is at least 50 nucleotides in length, or 95% identical to a contiguous portion of any one of these sequences that is at least 10, 15 or 20 nucleotides in length. The isolated nucleic acids of the invention can be RNA or DNA.
[0013] For all aspects of the invention, an isolated nucleic acid can comprise a nucleic acid sequence having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of any one of the SVV nucleic acid SEQ ID NO sequences herein, wherein the contiguous portion is at least about 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 2000 or 2500 nucleotides in length, for example. The SVV nucleic acid SEQ ID NO sequences include, for example, SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 168.
[0014] For all aspects of the invention, an isolated protein or peptide can comprise an amino acid sequence having at least about 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of any one of the SVV amino acid SEQ ID NO sequences herein, wherein the contiguous portion is at least at least about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 350 amino acids in length, for example. The SVV amino acid SEQ ID NO sequences include, for example, SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 169.
[0015] In another aspect, the invention provides an isolated nucleic acid comprising a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:168, or to a contiguous portion of SEQ ID NO:168 that is at least 20, 50, 100, or 200 nucleotides in length. The isolated nucleic acids can comprise specific portions of SEQ ID NO:168, including but not limited to: the 5' untranslated region (UTR) of SVV spanning nucleotides 1-666 of SEQ ID NO:168; the coding sequence for the SVV polyprotein spanning nucleotides 667-7209 of SEQ ID NO:168; the coding sequence for the leader peptide of SVV spanning nucleotides 667-903 of SEQ ID NO:168; the coding sequence for the SVV VP4 protein spanning nucleotides 904-1116 of SEQ ID NO:168; the coding sequence for the SVV VP2 protein spanning nucleotides 1117-1968 of SEQ ID NO:168; the coding sequence for the SVV VP3 protein spanning nucleotides 1969-2685 of SEQ ID NO:168; the coding sequence for the SVV VP1 protein spanning nucleotides 2686-3477 of SEQ ID NO:168; the coding sequence for the SVV 2A protein spanning nucleotides 3478-3504 of SEQ ID NO:168; the coding sequence for the SVV 2B protein spanning nucleotides 3505-3888 of SEQ ID NO:168; the coding sequence for the SVV 2C protein spanning nucleotides 3889-4854 of SEQ ID NO:168; the coding sequence for the SVV 3A protein spanning nucleotides 4855-5124 of SEQ ID NO:168; the coding sequence for the SVV 3B protein spanning nucleotides 5125-5190 of SEQ ID NO:168; the coding sequence for the SVV 3C protein spanning nucleotides 5191-5823 of SEQ ID NO:168; the coding sequence for the SVV 3D protein spanning nucleotides 5824-7209 of SEQ ID NO:168; and the 3'UTR of SVV spanning nucleotides 7210-7280 of SEQ ID NO:168.
[0016] In one aspect, the invention provides methods for using the SVV 2A, SVV leader peptide, or other SVV proteins or peptide portions thereof, to shut off host cell protein translation. In one aspect, such SVV proteins can be used to shut off host cell protein translation by interfering or inhibiting with the cap binding protein complex in the host cell.
[0017] In another aspect, the invention provides methods for using SVV 2A or other SVV proteins or peptide portions thereof in order to cleave a peptide or protein.
[0018] In other aspects, the invention provides an isolated nucleic acid that hybridizes under conditions of high, moderate stringency or low stringency to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length.
[0019] In another aspect, the invention provides a vector comprising a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length. Vector compositions can also comprise the nucleic acid regions of SEQ ID NO:168 that code for SVV proteins.
[0020] The present invention also provides an isolated polypeptide encoded by a nucleic acid having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to a nucleic acid sequence comprising SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length. The invention also provides an isolated polypeptide encoded by a nucleic acid having at least 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid region of SEQ ID NO:168 that encodes a SVV protein.
[0021] In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 169, or to a contiguous portion of any one of these sequences that is at least 10 amino acids in length.
[0022] In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of SEQ ID NO:169 that is at least 9, 10, 15, 20 or 50 amino acids in length. Exemplary contiguous portions of SEQ ID NO:169, include but are not limited to, regions that comprise a SVV protein, such as: the leader peptide spanning residues 1-79; VP4 spanning residues 80-150; VP2 spanning residues 151-434; VP3 spanning residues 435-673; VP1 spanning residues 674-937; 2A spanning residues 938-946; 2B spanning residues 947-1074; 2C spanning residues 1075-1396; 3A spanning residues 1397-1486; 3B spanning residues 1487-1508; 3C spanning residues 1509-1719; and 3D spanning residues 1720-2181.
[0023] In another aspect, the invention provides an isolated antibody which specifically binds a polypeptide comprising an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 169, or to a contiguous portion of any one of these sequences that is at least 9, 10, 15, or amino acids in length. The isolated antibody can be generated such that it binds to any protein epitope or antigen of SEQ ID NOS:2 or 169. Further, the antibody can be a polyclonal antibody, a monoclonal antibody or a chimeric antibody.
[0024] In one aspect, the invention provides an isolated SVV or derivative or relative thereof, having a genomic sequence comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1 or SEQ ID NO:168.
[0025] In another aspect, the invention provides an isolated virus having all the identifying characteristics and nucleic acid sequence of American Type Culture Collection (ATCC) Patent Deposit number PTA-5343. Some of the viruses of the present invention are directed to the PTA-5343 isolate, variants, homologues, relatives, derivatives and mutants of the PTA-5343 isolate, and variants, homologues, derivatives and mutants of other viruses that are modified in respect to sequences of SVV (both wild-type and mutant) that are determined to be responsible for its oncolytic properties.
[0026] The present invention further provides an isolated SVV comprising the following characteristics: a single stranded RNA genome (positive (+) sense strand) of ˜7.5 or of ˜7.3 kilobases (kb); a diameter of ˜27 nanometers (nm); a capsid comprising at least 3 proteins that have approximate molecular weights of about 31 kDa, 36 kDa and 27 kDa; a buoyant density of approximately 1.34 g/mL on cesium chloride (CsCl) gradients; and replication competence in tumor cells. In this aspect, the 31 kDa capsid protein (VP1) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:8 or residues 674-937 of SEQ ID NO:169; the 36 kDa capsid protein (VP2) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:4 or residues 151-434 of SEQ ID NO:169; and the 27 kDa capsid protein (VP3) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:6 or residues 435-673 of SEQ ID NO:169.
[0027] In another aspect, the invention provides an isolated SVV derivative or relative comprising the following characteristics: replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in normal cells. An SVV relative includes SVV-like picornaviruses, including viruses from the following USDA isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. If an SVV-like picornavirus does not naturally have the characteristics of replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in non-tumor cells, then the SVV-like picornavirus can be mutated such that these characteristics are obtained. Such mutations can be designed by comparing the sequence of the SVV-like picornavirus to SVV, and making mutations into the SVV-like picornavirus such that its amino acid sequence is identical or substantially identical (in a particular region) to SVV. In another aspect, the virus is replication competent in tumor cell types having neuroendocrine properties.
[0028] In other aspects, the present invention provides: a pharmaceutical composition comprising an effective amount of a virus of the invention and a pharmaceutically acceptable carrier; a cell comprising a virus of the invention; a viral lysate containing antigens of a virus of the invention; and an isolated and purified viral antigen obtained from a virus of the invention.
[0029] In yet another aspect, the invention provides a method of purifying a virus of the invention, comprising: infecting a cell with the virus; harvesting cell lysate; subjecting cell lysate to at least one round of gradient centrifugation; and isolating the virus from the gradient.
[0030] In another aspect, the invention provides a method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a portion of SEQ ID NO:1 or SEQ ID NO:168. In one aspect, the invention provides a method for treating cancer a method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1. The virus that has a genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 can be, for example, a SVV mutant, a SVV-like picornavirus, or a cardiovirus. The SVV-like picornavirus can be, for example, a virus from one of the following isolates MN 88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. The SVV-like picornaviruses can be wild-type or mutant.
[0031] In another aspect, the invention provides a method for treating cancer comprising administering an effective amount of a virus comprising a capsid encoding region that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS:3, 5, 7, nucleotides 904-3477 of SEQ ID NO:169, or to a contiguous portion thereof that is at least 75, 100, 200, or 500 nucleotides in length. The invention also provides a method for treating cancer comprising administering an effective amount of a virus comprising a capsid that comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, 6, 8, residues 80-937 of SEQ ID NO:169, or a contiguous portion thereof that is at least 25, 50, or 100 amino acids in length.
[0032] In one aspect, the present invention provides a method for inhibiting cancer progression comprising contacting a cancer cell with a virus or derivative thereof, wherein the virus or derivative thereof specifically binds to the cancerous cell, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168.
[0033] In another aspect, the invention provides a method for inhibiting cancer progression comprising contacting a cancer cell with a virus or derivative thereof, wherein the virus or derivative thereof specifically infects the cancerous cell, wherein the virus has a genomic sequence that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200, or 500 nucleotides in length.
[0034] In another aspect, the present invention provides a method for killing cancer cells comprising contacting a cancer cell with an effective amount of a virus or derivative thereof, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168.
[0035] In another aspect, the present invention provides a method for killing cancer cells comprising contacting a cancer cell with an effective amount of a virus or derivative thereof,
[0036] wherein the virus has a genomic sequence that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:168, or to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200 or 500 nucleotides in length.
[0037] In these methods directed to cancer, the virus can be a picornavirus. The picornavirus can be a cardiovirus, erbovirus, aphthovirus, kobuvirus, hepatovirus, parechovirus, teschovirus, enterovirus, rhinovirus, SVV, or an SVV-like picornavirus. The cardiovirus can be selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. The SVV can be a virus having the ATCC deposit number PTA-5343 or a virus comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. The SVV-like picornavirus can be a virus comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. The SVV-like picornavirus can be, for example, a virus from one of the following isolates MN 88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. The SVV-like picornaviruses can be wild-type or mutant.
[0038] The present invention also provides a method of killing an abnormally proliferative cell comprising contacting the cell with a virus of the invention. In one aspect, the abnormally proliferative cell is a tumor cell. In various aspects of this method, the tumor cell is selected from the group consisting of: human small cell lung cancer, human retinoblastoma, human neuroblastoma, human medulloblastoma, mouse neuroblastoma, Wilms' tumor, and human non-small cell lung cancer.
[0039] The present invention also provides a method of treating a neoplastic condition in a subject comprising administering to the subject an effective amount of a virus of the invention to the mammal. In one aspect, the neoplastic condition is a neuroendocrine cancer. In another aspect, the subject is a mammal. In another aspect, the mammal is a human.
[0040] The present invention also provides a method of producing a virus of the invention, comprising: culturing cells infected with the virus under conditions that allow for replication of the virus and recovering the virus from the cells or the supernatant. In one aspect of this method, the cells are PER.C6 cells. In another aspect of this method, the cells are H446 cells. In the various aspects of this method, the cells may produce over 200,000 virus particles per cell.
[0041] In another aspect, the present invention provides a method for detecting a virus of the invention, comprising: isolating RNA from test material suspected to contain the virus of the invention; labeling RNA corresponding to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:168; probing the test material with the labeled RNA; and detecting the binding of the labeled RNA with the RNA isolated from the test material, wherein binding indicates the presence of the virus. In another aspect, the present invention provides a nucleic acid probe comprising a nucleotide sequence corresponding to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:168, or its complement.
[0042] The present invention also provides a method for making an oncolytic virus, the method comprising: (a) comparing a SVV genomic sequence with a test virus genomic sequence; (b) identifying at least a first amino acid difference between a polypeptide encoded by the SVV genomic sequence and a polypeptide encoded by the test virus genomic sequence; (c) mutating the test virus genomic sequence such that the polypeptide encoded by the test virus genomic sequence has at least one less amino acid difference to the polypeptide encoded by the SVV genomic sequence; (d) transfecting the mutated test virus genomic sequence into a tumor cell; and (e) determining whether the tumor cell is lytically infected by the mutated test virus genomic sequence. In one aspect, the amino acid(s) mutated in the test virus are amino acids in a structural region, such as in the capsid encoding region. In another aspect, the amino acids mutated in the test virus are amino acids in a non-structural region.
[0043] In one aspect of the method for making an oncolytic virus, the SVV genomic sequence is obtained from the isolated SVV having the ATCC deposit number PTA-5343 or from a virus comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. In one aspect, the SVV genomic sequence is obtained from the isolated SVV having the ATCC deposit number PTA-5343 or from a virus comprising a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In another aspect of this method, the step of mutating the test virus genomic sequence comprises mutating a cDNA having the test virus genomic sequence. In another aspect of this method, the step of transfecting the mutated test virus genomic sequence comprises transfecting RNA, wherein the RNA is generated from the cDNA having the mutated test virus genomic sequence.
[0044] In another aspect of the method for making an oncolytic virus, the test virus is a picornavirus. The test picornavirus can be a teschovirus, enterovirus, rhinovirus, cardiovirus, erbovirus, apthovirus, kobuvirus, hepatovirus, parechovirus or teschovirus. In another aspect, the test virus is a cardiovirus. In another aspect, the test virus is a SVV-like picornavirus. The SVV-like picornavirus can be, for example, a virus from one of the following isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. In another aspect, the amino acid differences identified in the methods for making an oncolytic virus are between a SVV capsid protein and a test virus capsid protein sequence. In another aspect for making an oncolytic virus, the test virus genomic sequence is selected from the group consisting of: Vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. In another aspect, the test virus genomic sequence is selected from an encephalomyocarditis virus. In yet another aspect, the encephalomyocarditis virus, the SVV-like picornavirus, or any other test virus can be selected from an isolate having a nucleic acid sequence comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SVV of ATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length.
[0045] In another aspect of the method for making an oncolytic cardiovirus, the amino acid difference between the test virus and SVV is in a capsid protein region of SVV, wherein the amino acid difference is aligned within SVV SEQ ID NO:4, 6, 8, residues 80-937 of SEQ ID NO:169, residues 80-150 of SEQ ID NO:169, residues 151-434 of SEQ ID NO:169, residues 435-673 of SEQ ID NO:169, or residues 674-937 of SEQ ID NO:169.
[0046] The present invention also provides a method for making a mutant virus having an altered cell-type tropism, the method comprising: (a) creating a library of viral mutants comprising a plurality of nucleic acid sequences; (b) transfecting the library of viral mutants into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) incubating a non-permissive cell with the isolated plurality of mutant viruses; and (e) recovering a mutant virus that was produced in the non-permissive cell, thereby making a mutant virus having an altered tropism. In one aspect, this method further comprises the steps of: (f) incubating the recovered mutant virus in the non-permissive cell; and (g) recovering a mutant virus that that was produced in the non-permissive cell. In another aspect, the method further comprises iteratively repeating steps (f) and (g). In another aspect, the library of viral mutants is created from a parental sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof.
[0047] In one aspect of the method for making a mutant virus having an altered cell-type tropism, the incubating is conducted in a multi-well high-throughput platform wherein the platform comprises a different non-permissive cell-type in each well. In this aspect, the method can further comprise screening the platform to identify which wells contain a mutant virus that kills the cells. In another aspect, the screening is conducted by analyzing light absorbance in each well.
[0048] In another aspect of the method for making a mutant virus having an altered cell-type tropism, the non-permissive cell is a tumor cell.
[0049] In another aspect of the method for making a mutant virus having an altered cell-type tropism, the step of creating the library of viral mutants comprises: (i) providing a polynucleotide having a sequence identical to a portion of a genomic sequence of a virus; (ii) mutating the polynucleotide in order to generate a plurality of different mutant polynucleotide sequences; and (iii) ligating the plurality of mutated polynucleotides into a vector having the genomic sequence of the virus except for the portion of the genomic sequence of the virus that the polynucleotide in step (i) contains, thereby creating the library of viral mutants. In one aspect, the genomic sequence of a virus is from a picornavirus. In another aspect, the genomic sequence of a virus comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. In another aspect, the genomic sequence of a virus comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In one aspect, the virus that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200, or 500 nucleotides in length is a SVV-like picornavirus. In another aspect, in the step of creating the library of viral mutants, the mutating of step (ii) is conducted by random insertion of nucleotides into the polynucleotide. In one aspect, the random insertion of nucleotides is conducted by trinucleotide-mutagenesis (TRIM). In another aspect, at least a portion of the nucleotides inserted into the polynucleotide encodes an epitope tag. In another aspect, in the step of creating the library of viral mutants, the mutating of step (ii) is conducted in a capsid encoding region of the polynucleotide.
[0050] The present invention also provides a method for making a mutant virus having an altered cell-type tropism, the method comprising: (a) creating a library of mutant polynucleotide sequences of a virus, wherein the creating comprises: providing a polynucleotide encoding a capsid region of the virus; mutating the polynucleotide in order to generate a plurality of different mutant capsid-encoding polynucleotide sequences; and ligating the plurality of mutated capsid-encoding polynucleotides into a vector having the genomic sequence of the virus except for the capsid-encoding region, thereby creating the library of mutant polynucleotide sequences of the virus; (b) transfecting the library of mutant polynucleotide sequences into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) incubating a non-permissive cell with the isolated plurality of mutant viruses; and (e) recovering a mutant virus that that was produced in the non-permissive cell, thereby making a mutant virus having an altered tropism. In one aspect, the method further comprises the steps of: (f) incubating the recovered mutant virus in the non-permissive cell; and (g) recovering a mutant virus that that was produced in the non-permissive cell. In another aspect, the method further comprises iteratively repeating steps (f) and (g). In another aspect, the mutating is conducted by random insertion of nucleotides into the capsid-encoding polynucleotide. In another aspect, at least a portion of the nucleotides randomly inserted into the capsid-encoding polynucleotide encodes an epitope tag. In another aspect, the random insertion of nucleotides is conducted by TRIM. In another aspect, the plurality of different mutant capsid-encoding polynucleotide sequences comprises greater than 108 or 109 different capsid-encoding polynucleotide sequences. The library of mutant polynucleotide sequences can be from, for example, a cardiovirus or an SVV-like picornavirus.
[0051] In one aspect, a method for making a mutant SVV having an altered cell-type tropism comprises: (a) creating a cDNA library of SVV mutants; (b) generating SVV RNA from the cDNA library of SVV mutants; (c) transfecting the SVV RNA into a permissive cell, such that a plurality of mutant SVV is produced; (d) isolating the plurality of mutant SVV; (e) incubating a non-permissive tumor cell with the isolated plurality of mutant SVV; and (f) recovering a mutant SVV that lytically infects the non-permissive tumor cell, thereby making a mutant SVV having an altered tropism. In another aspect, the method further comprises the steps of: (g) incubating the recovered mutant SVV in the non-permissive cell; and (h) recovering a mutant SVV that lytically infects the non-permissive tumor cell. In another aspect, the method further comprises iteratively repeating steps (g) and (h). In one aspect, the incubating is conducted in a multi-well high-throughput platform wherein the platform comprises a different non-permissive tumor cell-type in each well. In another aspect, the method further comprises screening the platform to identify which wells contain a mutant SVV that lytically infects the cells. In another aspect, the screening is conducted by analyzing light absorbance in each well. In one aspect, the cDNA library of SVV mutants comprises a plurality of mutant SVV capsid polynucleotide sequences. In another aspect, the plurality of mutant SVV capsid polynucleotide sequences is generated by random insertion of nucleotides. In another aspect, at least a portion of the sequence of the nucleotides randomly inserted encodes an epitope tag. In another aspect, the random insertion of nucleotides is conducted by TRIM. In another aspect, the cDNA library of SVV mutants is generated from a SVV of ATCC deposit number PTA-5343. In another aspect, the cDNA library of SVV mutants is generated from a SVV comprising a sequence having at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In one aspect, the cDNA library of SVV mutants is generated from an SVV comprising a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In another aspect, the non-permissive tumor cell is a tumor cell-line or a tumor cell-type isolated from a patient.
[0052] The present invention also provides a method for making a mutant virus having a tumor cell-type tropism in vivo, the method comprising: (a) creating a library of viral mutants comprising a plurality of nucleic acid sequences; (b) transfecting the library of viral mutants into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) administering the isolated plurality of mutant viruses to a mammal with a tumor, wherein the mammal is not a natural host of the unmutated form of the mutant virus; and (e) recovering a virus that replicated in the tumor, thereby making a mutant virus having a tumor cell-type tropism in vivo. In one aspect, the step of creating a library of viral mutants comprises: providing a polynucleotide encoding a capsid region of a virus; mutating the polynucleotide in order to generate a plurality of different mutant capsid-encoding polynucleotide sequences; and ligating the plurality of mutated capsid-encoding polynucleotides into a vector having the genomic sequence of the virus except for the capsid-encoding region, thereby creating the library of viral mutants. In another aspect, the virus recovered in step (e) lytically infects cells of the tumor. In another aspect for a method for making a mutant virus having a tumor cell-type tropism in vivo, the tumor is a xenograft, a syngeneic tumor, an orthotopic tumor or a transgenic tumor. In another aspect, the mammal is a mouse.
[0053] For all the methods of the present invention, the virus can be a picornavirus. The picornavirus can be a cardiovirus, erbovirus, aphthovirus, kobuvirus, hepatovirus, parechovirus, teschovirus, entrovirus, rhinovirus, or a virus belonging to the genus to which SVV belongs. The virus can be a cardiovirus. The virus can be an SVV-like picornavirus. The virus can be SVV. The SVV can be a SVV having the ATCC Patent Deposit No. PTA-5343 or a SVV comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1, 3, 6, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. Further, the cardiovirus can be selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. In one aspect, the SVV-like picornavirus is selected from the group of isolates consisting of: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. In another aspect, the present invention encompasses any virus that is selected from an isolate having at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SVV of ATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof or is otherwise considered related to SVV to by sequence homology.
[0054] In another aspect, the present invention encompasses any virus having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity to SVV of ATCC deposit number PTA-5343, to SEQ ID NO:168, or to a contiguous portion of SEQ ID NOS: 1 or 168 that is at least 100, 200, 300, 400, 500, 750, 1000, 1500, or 2000 nucleotides in length.
[0055] The present invention also provides an oncolytic virus made by any of the methods for making a mutant virus disclosed herein. In one aspect, the present invention provides a method for treating a patient with an oncolytic virus, the method comprising: (a) inactivating an oncolytic virus made by any of the methods for making a mutant virus disclosed herein, such that the oncolytic virus is non-infectious and the tropism of the oncolytic virus is unaffected; and (b) administering the irradiated oncolytic virus to a patient afflicted with a tumor. In another aspect, the method for treating a patient further comprises attaching a toxin to the inactivated oncolytic virus.
[0056] In another aspect, the present invention provides a method for treating a patient with a tumor with SVV, the method comprising: (a) inactivating a SVV such that the virus is non-infectious and the tropism is unaffected; and (b) administering the inactivated SVV in a patient afflicted with a tumor. In another aspect, the method for treating a patient with a tumor with SVV further comprises attaching a toxin to the inactivated SVV.
[0057] In another aspect, the present invention provides a SVV composition comprising an inactivated SVV or attenuated SVV. In another aspect, the present invention provides a SVV comprising an epitope tag incorporated in the capsid region.
[0058] The present invention also provides a method for treating a patient with a tumor with SVV, the method comprising: (a) creating a mutant SVV comprising an epitope tag encoded in the capsid; (b) attaching a toxin to the epitope tag; and (c) administering the mutant SVV with the attached toxin to a patient afflicted with a tumor. In one aspect, the creating comprises: inserting an oligonucleotide encoding an epitope tag into a capsid-encoding region polynucleotide of SVV. In one aspect, the mutant SVV does not have an altered cell-type tropism. In another aspect, the method further comprises inactivating the mutant SVV such that the mutant SVV is not infectious or cannot replicate.
[0059] The present invention also provides a method for detecting a tumor cell in a sample comprising: (a) isolating a tumor sample from a patient; (b) incubating the tumor sample with an epitope-tagged SVV; and (c) screening the tumor sample for bound SVV by detecting the epitope tag.
[0060] In one aspect, the invention provides a method for detecting a tumor cell in vivo comprising: (a) administering to a patient an inactivated epitope-tagged SVV, wherein a label is conjugated to the epitope-tag; and (b) detecting the label in the patient. In the methods for detecting a tumor cell of the present invention, the SVV can be a mutant SVV generated by the methods disclosed herein.
[0061] In one aspect, the invention provides a method for detecting a tumor cell in a sample comprising: (a) isolating a cell sample from a subject; (b) incubating the cell sample with SVV (or an SVV-like picornavirus); (c) incubating the cell sample from step (b) with an antibody specific to SVV (or an antibody specific to an SVV-like picornavirus); and (d) screening the cell sample for bound antibody, wherein bound antibody indicates that the sample contains a tumor cell.
[0062] In one aspect, the invention provides a method for determining whether a subject is candidate for SVV therapy, the method comprising: (a) isolating a cell from the subject; (b) incubating the cell with SVV; (c) incubating the sample from step (b) with an anti-SVV antibody; and (d) detecting for the presence of the anti-SVV antibody on or in the cell, wherein a positive detection indicates that the subject is a candidate for SVV therapy.
[0063] Screening a cell sample for bound antibody or detecting for the presence of an anti-SVV antibody can be conducted by adding a secondary antibody that can bind to the constant regions or non-epitope binding regions of the anti-SVV antibody, wherein the secondary antibody is conjugated or labeled with a detectable marker. The detectable marker can be, for example, a fluorophore such as fluorescein. When a secondary antibody is labeled with a detectable marker, the detectable marker can be detected, for example, by fluorescent microscopy. The cell from the subject can be from a tissue biopsy from the subject. The tissue biopsy can be from a tumor in the subject or from a region in the subject that is suspected to contain tumor cells. SVV directly labeled with fluorophore can also be used in identification of tumor cells.
[0064] Further, the methods for treating neoplastic conditions, for detecting neoplastic conditions and for producing SVV, apply to wild-type SVV, mutant (including modified or variant) SVV, relatives of SVV, SVV-like picornaviruses, and other tumor-specific viruses of the invention.
[0065] The viruses of the present invention, and the compositions thereof, can be used in the manufacture of a medicament for treating the diseases mentioned herein. Further, the viruses and composition thereof of the invention can be used for the treatment of the diseases mentioned herein. Thus, in one aspect of the present invention, the present invention provides the use of SVV (or mutants, derivatives, relatives, and compositions thereof) for the treatment of cancer or in the manufacture of a medicament for treating cancer.
[0066] SVV and SVV-like viruses for gene therapy: Replication defective SVV expressing gene(s) of interest can be used to deliver genes to correct genetic disorders. SVV and SVV-like viruses can also be used as delivery vehicle for siRNA to prevent any specific gene expression. Replication defective viruses can be grown in complementing cell lines and/or in the presence of a helper virus to provide for missing functions in the recombinant virus.
[0067] IRES of picornaviruses known to play a role in expression of genes in a tissue specific manner. IRES of SVV and SVV-like viruses can be used to replace IRES of other picornaviruses. This strategy can be used to generate viruses with altered tissue tropism. In one aspect, the invention provides an IRES of SVV or an IRES from an SVV-related virus for the purpose of expressing two genes from a single promoter in a tissue specific manner.
[0068] Self-cleavage properties of 2A protease of SVV can be used to express more than one gene in equal amounts using single promoter and transcription termination signal sequences. In one aspect, the invention provides a self-cleaving 2A peptide of SVV or of an SVV-related virus for the purpose of expressing of two or more proteins in equal amounts under the control of single promoter and a single poly(A) signal. In another aspect, the invention provides the use of an SVV or an SVV-related virus 3C protease to cleave polypeptides for production of proteins from a eukaryotic cell. In another aspect, the invention provides for the use of an SVV or an SVV-like virus leader peptide to cause shut off of cell protein synthesis in tumor cells or another cell type of interest.
[0069] Virus like particles of SVV can be generated and used as vaccines and identify a particular cell type in a mixed population of cells.
Deposit Information
[0070] The following material has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va., 20110-2209, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent. Material: Seneca Valley Virus (SVV). ATCC Patent Deposit Number: PTA-5343. Date of Deposit: Jul. 25, 2003.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIG. 1 shows a schematic of virotherapy using oncolytic viruses. Oncolytic viruses have the properties to replicate, spread and kill tumor cells selectively through a tumor mass by locally injecting the virus or by systemically delivering the virus.
[0072] FIG. 2 shows purified SVV stained with uranyl acetate and examined by transmission electron microscopy. Spherical virus particles are about 27 nm in diameter.
[0073] FIG. 3 is an electron micrograph of an SVV-infected PER.C6 cell that has a large crystalline inclusion and large vesicular bodies.
[0074] FIG. 4A shows an analysis of SVV RNA. SVV genomic RNA is extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen Corp., Carlsbad, Calif.). RNA is resolved through a 1.25% denaturing agarose gel. The band is visualized by ethidium bromide (EtBr) staining and photographed. In lane 2, a predominant band of SVV genomic RNA is observed, indicating that the size of the full-length SVV genome is about 7.5 kilobases.
[0075] FIG. 4B is a schematic showing the genome structure and protein products generated from polyprotein processing for picornaviruses, including SVV.
[0076] FIGS. 5A-5E presents the nucleotide sequence of SVV (SEQ ID NO:1) and the encoded amino acid sequence (SEQ ID NO:2). The stop codon is depicted by a "*" at positions 5671-3. As a general note, in sequence disclosures that include positions where the exact nucleotide is being confirmed, these positions are represented by an "n". Therefore, in codons that possess an "n", the relevant amino acid is depicted by a "x".
[0077] FIGS. 6A-6D presents the nucleotide sequence (SEQ ID NO:1) of the majority of the full-length genome of SVV. The nucleotide sequence was derived from the SVV isolate having the ATCC Patent Deposit Number: PTA-5343. Date of Deposit: Jul. 25, 2003.
[0078] FIGS. 7A-7B presents the amino acid sequence (SEQ ID NO:2) encoded by SEQ ID NO:1.
[0079] FIG. 8 presents the nucleotide sequence (SEQ ID NO:3) of the partial 1B or VP2 encoding region of SVV. This sequence is identical to nucleotides 4-429 of SEQ ID NO:1.
[0080] FIG. 9 presents the amino acid sequence (SEQ ID NO:4) of the partial SVV VP2 protein that is encoded by SEQ ID NO:3. The sequence listed in SEQ ID NO:4 is identical to amino acids 2-143 of SEQ ID NO:2.
[0081] FIG. 10 presents the nucleotide sequence (SEQ ID NO:5) of the 1C or VP3 encoding region of SVV. This sequence is identical to nucleotides 430-1146 of SEQ ID NO:1.
[0082] FIG. 11 presents the amino acid sequence (SEQ ID NO:6) of the SVV VP3 protein that is encoded by SEQ ID NO:5. The sequence listed in SEQ ID NO:6 is identical to amino acids 144-382 of SEQ ID NO:2.
[0083] FIG. 12 presents the nucleotide sequence (SEQ ID NO:7) of the 1D or VP1 encoding region of SVV. This sequence is identical to nucleotides 1147-1923 of SEQ ID NO:1.
[0084] FIG. 13 presents the amino acid sequence (SEQ ID NO:8) of the SVV VP1 protein that is encoded by SEQ ID NO:7. The sequence listed in SEQ ID NO:8 is identical to amino acids 383-641 of SEQ ID NO:2.
[0085] FIG. 14 presents the nucleotide sequence (SEQ ID NO:9) of the 2A encoding region of SVV. This sequence is identical to nucleotides 1924-1965 of SEQ ID NO:1.
[0086] FIG. 15 presents the amino acid sequence (SEQ ID NO:10) of the SVV 2A protein that is encoded by SEQ ID NO:9. The sequence listed in SEQ ID NO:10 is identical to amino acids 642-655 of SEQ ID NO:2.
[0087] FIG. 16 presents the nucleotide sequence (SEQ ID NO:11) of the 2B encoding region of SVV. This sequence is identical to nucleotides 1966-2349 of SEQ ID NO:1.
[0088] FIG. 17 presents the amino acid sequence (SEQ ID NO:12) of the SVV 2B protein that is encoded by SEQ ID NO:11. The sequence listed in SEQ ID NO:12 is identical to amino acids 656-783 of SEQ ID NO:2.
[0089] FIG. 18 presents the nucleotide sequence (SEQ ID NO:13) of the 2C encoding region of SVV. This sequence is identical to nucleotides 2350-3315 of SEQ ID NO:1.
[0090] FIG. 19 presents the amino acid sequence (SEQ ID NO:14) of the SVV 2C protein that is encoded by SEQ ID NO:13. The sequence listed in SEQ ID NO:14 is identical to amino acids 784-1105 of SEQ ID NO:2.
[0091] FIG. 20 presents the nucleotide sequence (SEQ ID NO:15) of the 3A encoding region of SVV. This sequence is identical to nucleotides 3316-3585 of SEQ ID NO:1.
[0092] FIG. 21 presents the amino acid sequence (SEQ ID NO:16) of the SVV 3A protein that is encoded by SEQ ID NO:15. The sequence listed in SEQ ID NO:16 is identical to amino acids 1106-1195 of SEQ ID NO:2.
[0093] FIG. 22 presents the nucleotide sequence (SEQ ID NO:17) of the 3B encoding region of SVV. This sequence is identical to nucleotides 3586-3651 of SEQ ID NO:1.
[0094] FIG. 23 presents the amino acid sequence (SEQ ID NO:18) of the SVV 3B protein that is encoded by SEQ ID NO:17. The sequence listed in SEQ ID NO:18 is identical to amino acids 1196-1217 of SEQ ID NO:2.
[0095] FIG. 24 presents the nucleotide sequence (SEQ ID NO:19) of the 3C encoding region of SVV. This sequence is identical to nucleotides 3652-4284 of SEQ ID NO:1.
[0096] FIG. 25 presents the amino acid sequence (SEQ ID NO:20) of the SVV 3C protein that is encoded by SEQ ID NO:19. The sequence listed in SEQ ID NO:20 is identical to amino acids 1218-1428 of SEQ ID NO:2.
[0097] FIG. 26 presents the nucleotide sequence (SEQ ID NO:21) of the 3D encoding region of SVV. This sequence is identical to nucleotides 4285-5673 of SEQ ID NO:1.
[0098] FIG. 27 presents the amino acid sequence (SEQ ID NO:22) of the SVV 3D protein that is encoded by SEQ ID NO:21. The sequence listed in SEQ ID NO:22 is identical to amino acids 1429-1890 of SEQ ID NO:2.
[0099] FIGS. 28A-28H present an amino acid sequence alignment between SVV SEQ ID NO:2 and various members of the Cardiovirus genus, such as Encephalomyocarditis virus (EMCV; species Encephalomyocarditis virus), Theiler's murine encephalomyocarditis virus (TMEV; species Theilovirus), a rat TMEV-like agent (TLV; species Theilovirus), and Vilyuisk human encephalomyelitis virus (VHEV; species Theilovirus). The specific sequences of the various Cardioviruses are presented in: SEQ ID NOS: 23 (EMCV-R), 24 (EMCV-PV21), 25 (EMCV-B), 26 (EMCV-Da), 27 (EMCV-Db), 28 (EMCV-PV2), 29 (EMCV-Mengo), 30 (TMEV/DA), 31 (TMEV/GDVII), 32 (TMEV/BeAn8386), 33 (TLV-NGS910) and 34 (VHEV/Siberia-55).
[0100] Number positions in FIG. 28 do not correspond to the numbering of the sequence listings. The "I" symbol indicates cleavage sites where the polyprotein is cleaved into its final functional products. For example, the alignment between positions 1 and 157 is in the 1A (VP4) region. The alignment between positions: 159 and 428 is in the 1B (VP2) region; 430 and 668 is in the 1C (VP3) region; 670 and 967 is in the 1D (VP1) region; 969 and 1111 is in the 2A region; 1112 and 1276 is in the 2B region; 1278 and 1609 is in the 2C region; 1611 and 1700 is in the 3A region; 1702 and 1723 is in the 3B region; 1725 and 1946 is in the 3C region; 1948 and 2410 is in the 3D region. The alignment also shows regions of potential conservation or similarity between the viral sequences as can be determined by standard sequence analysis programs. The alignments were generated using BioEdit 5.0.9 and Clustal W 1.81.
[0101] FIG. 29 lists the final polypeptide products of SVV with respect to SEQ ID NO:2. Some conserved motifs are bolded and underlined: 2A/2B "cleavage" (NPGP (SEQ ID NO:111)); 2C ATP-binding (GxxGxGKS/T (SEQ ID NO:112) and hyhyhyxxD); 3B (VPg)/RNA attachment residue (Y); 3C (pro) active site residues (H, C, H); 3D (pol) motifs (KDEL/IR (SEQ ID NO:113), PSG, YGDD (SEQ ID NO:114), FLKR (SEQ ID NO:115)).
[0102] FIG. 30 lists the picornavirus species that were used in sequence analyses with SEQ ID NOS:1 and 2 to determine the phylogenetic relationship between SVV and these picornaviruses (see Example 4, Part I).
[0103] FIG. 31 shows the phylogenetic relationship between SVV (SEQ ID NO:4) and other picornaviruses in view of VP2 sequence analyses. The figure shows a bootstrapped neighbor-joining tree (see Example 4, Part I).
[0104] FIG. 32 shows a bootstrapped neighbor-joining tree for VP3 between SVV (SEQ ID NO:6) and other picornaviruses (see Example 4, Part I).
[0105] FIG. 33 shows a bootstrapped neighbor-joining tree for VP1 between SVV (SEQ ID NO:8) and other picornaviruses (see Example 4, Part I).
[0106] FIG. 34 shows a bootstrapped neighbor-joining tree for P1 (i.e., 1A, 1B, 1C and 1D) between SVV (i.e., partial P1--amino acids 2-641 of SEQ ID NO:2) and other picornaviruses (see Example 4, Part I).
[0107] FIG. 35 shows a bootstrapped neighbor-joining tree for 2C between SVV (SEQ ID NO:14) and other picornaviruses (see Example 4, Part I).
[0108] FIG. 36 shows a bootstrapped neighbor-joining tree for 3C (pro) between SVV (SEQ ID NO:20) and other picornaviruses (see Example 4, Part I).
[0109] FIG. 37 shows a bootstrapped neighbor-joining tree for 3D (pol) between SVV (SEQ ID NO:22) and other picornaviruses (see Example 4, Part I).
[0110] FIG. 38 presents the actual amino acid percent identities of VP2 between SVV (SEQ ID NO:4) and other picornaviruses (see Example 4, Part I).
[0111] FIG. 39 presents the actual amino acid percent identities of VP3 between SVV (SEQ ID NO:6) and other picornaviruses (see Example 4, Part I).
[0112] FIG. 40 presents the actual amino acid percent identities of VP1 between SVV (SEQ ID NO:8) and other picornaviruses (see Example 4, Part I).
[0113] FIG. 41 presents the actual amino acid percent identities of P1 between SVV (partial capsid or P1--amino acids 2-641 of SEQ ID NO:2) and other picornaviruses (see Example 4, Part I).
[0114] FIG. 42 presents the actual amino acid percent identities of 2C between SVV (SEQ ID NO:14) and other picornaviruses (see Example 4, Part I).
[0115] FIG. 43 presents the actual amino acid percent identities of 3C (pro) between SVV (SEQ ID NO:20) and other picornaviruses (see Example 4, Part I).
[0116] FIG. 44 presents the actual amino acid percent identities of 3D (pol) between SVV (SEQ ID NO:22) and other picornaviruses (see Example 4, Part I).
[0117] FIG. 45 shows the VP2 (˜36 kDa), VP1 (˜31 kDa) and VP3 (˜27 kDa) proteins of SVV as analyzed by SDS-PAGE. Purified SVV was subjected to SDS-PAGE and proteins were visualized by silver stain. Lane "MWt" is molecular weight markers; lane "SVV" contains structural proteins of SVV. The sizes of three molecular weight markers and the names of viral proteins are also given.
[0118] FIGS. 46A-46B show the amounts of SVV in blood and tumor following systemic administration (Example 7). H446 tumor bearing nude mice were treated with SVV at a dose of 1×1012/kg by tail vein injection. The mice were bled at 0, 1, 3, 6, 24, 48, 72 hours and at 7 days post-injection, and the plasma was separated from the blood immediately after collection, diluted in infection medium, and used to infect PER.C6 cells. The tumors were harvested at 6, 24, 48, 72 hours and at 7 days post-injection. The tumors were cut into small sections and suspended in one mL of medium and CVL was made.
[0119] FIGS. 46C-46D presents data relating to SVV clearance in vivo. The figures show that SVV exhibits a substantially longer resident time in the blood compared to similar doses of i.v. adenovirus (Example 7), and therefore SVV has a slower clearance rate than adenovirus in vivo. Following a single intravenous (i.v.) dose, SVV remains present in the blood for up to 6 hours (FIG. 46C; FIG. 46C is a duplication of FIG. 46A for comparison purposes to FIG. 46D), whereas adenovirus is cleared or depleted from the blood in about an hour (FIG. 46D).
[0120] FIG. 47 shows immunohistochemistry and hematoxylin and eosin (H&E) staining of H446 xenograft sections (Example 7). H446 tumor bearing nude mice were treated with Hank's balanced salt solution (HBSS) or SVV at a dose of 1×1012 vp/kg by tail vein injection. The mice were sacrificed at 3 days post-injection and the tumors were collected. The virus proteins in the tumor cells are visualized by immunohistochemistry using SVV-specific mouse antibodies (upper panels). The general morphology of H446 tumor cells collected from HBSS or SVV treated mice were stained by H&E stain (lower panels).
[0121] FIG. 48 shows SVV mediated cytotoxicity in primary human hepatocytes (Example 9). Primary human hepatocytes plated in collagen coated 12-well plates were infected with SVV at 1, 10 and 100 and 1000 particles per cell (ppc). Three days after infection, the cell associated lactate dehydrogenase (LDH) and LDH in the culture supernatant were measured separately. Percent cytotoxicity was determined as a ration of LDH units in supernatant over maximal cellular LDH plus supernatant LDH.
[0122] FIG. 49 shows virus production by SVV in selected cell lines. To assess the replicative abilities of SVV, selected normal cells and tumor cells were infected with SVV at one virus particle per cell (ppc) (Example 9). After 72 hours, cells were harvested and CVL was assayed for titer on PER.C6 cells. For each cell line, the efficiency of SVV replication was expressed as plaque forming units per milliliter (pfu/ml).
[0123] FIG. 50 shows toxicity in nude and CD1 mice according to body weights (Example 10). The mean body weight of mice in each treatment group were measured different days post virus administration. Mice were injected with a single dose of SVV or PBS by tail vein on day 1.
[0124] FIG. 51 shows efficacy in a H446 xenograft model. H446 tumors are established in nude mice and the mice are sorted into groups (n=10) and treated via tail vein injection with either HBSS or six different doses of SVV (Example 11). On study day 20, five mice from the HBSS group that bear large tumors (mean tumor volume=2000 mm3) were injected with 1×1011 vp/kg (indicated by an arrow). Data is expressed as mean tumor volume+standard deviation (SD).
[0125] FIG. 52 shows a picture of H446 xenograft nude mice that have been untreated or treated with SVV (Example 11). The efficacy of SVV is very robust in that 100% of large pre-established tumors were completely eradicated. SVV-treated mice show neither clinical symptoms nor recurrence of tumors for at least 200 days following injection.
[0126] FIG. 53 presents data relating to SVV tumor specificity and efficacy in vitro (Example 11). The graphs show cell survival following incubation of either H446 human small cell lung carcinoma (SCLC) tumor cells (top graph) or normal human H460 cells (bottom graph). SVV specifically killed the tumor cells with an EC50 of approximately 10-3 particles per cell. In contrast, normal human cells were not killed at any concentration of SVV.
[0127] FIG. 54 depicts a representative plasmid containing the complete genome of SVV (Example 15). The presence of the T7 promoter on the vector upstream of the SVV sequence allows for the in vitro transcription of the SVV sequence such that SVV RNA molecules can be generated.
[0128] FIG. 55 depicts a schematic for the construction of a full-length and functional genomic SVV plasmid and subsequent SVV virus production (Example 16). To obtain a functional genomic SVV clone, the complete genome of a SVV can be cloned into a vector with a T7 promoter. This can be accomplished by making cDNA clones of the virus, sequencing them and cloning contiguous pieces into one plasmid, resulting in the plasmid depicted "pSVV". The plasmid with the full genome of SVV can then be reverse-transcribed to generate SVV RNA. The SVV RNA is then transfected into permissive mammalian cells and SVV virus particles can then be recovered and purified.
[0129] FIG. 56 depicts a schematic for the construction of a vector ("pSVV capsid") containing the coding sequence (i.e., coding regions for 1A-1D) for the SVV capsid (Example 16). The pSVV capsid can then be used to generate a library of SVV capsid mutants.
[0130] FIG. 57 shows one method of mutating the SVV capsid for the generation of a library of SVV capsid mutants (Example 16). The figure illustrates the insertion of an oligonucleotide sequence at random sites of the plasmid. The oligonucleotides can be random oligonucleotides, oligonucleotides with known sequences, or an oligonucleotide encoding an epitope tag. In the figure, the restriction enzyme CviJI randomly cleaves the pSVV capsid DNA. Linearized pSVV capsid DNA that has been cut at a single site is isolated and purified from a gel, and ligated with oligonucleotides.
[0131] FIG. 58 presents a scheme to generate a library of full-length SVV mutants comprising sequence mutations in the capsid encoding region (Example 16). For example, the capsid encoding region from a pSVV capsid mutant library (generated according to the scheme depicted in FIG. 57, for example) is isolated by restriction digestion and gel purification. The vector containing the full-length SVV sequence is also digested such that the capsid encoding region is cut out. The capsid encoding region from the pSVV capsid mutant library is then ligated to the pSVV vector that is missing its wild-type capsid sequence, thereby generating a library of full-length SVV mutants (the "pSVVFL" vector) having a plurality of mutations in the capsid encoding region.
[0132] FIG. 59 presents a general method for producing the SVV virus particles comprising a library of capsid mutations (Example 16). The pSVVFL vector is reverse-transcribed to generate SVV RNA. The SVV RNA is transfected into permissive cells, wherein SVV mutant virus particles are produced. The virus particles lyse the cells and a population of SVV virus particles comprising a plurality of capsid variants, "SVV capsid library," are isolated.
[0133] FIG. 60 shows a general method for screening SVV capsid mutants that can specifically infect tumor cells while being unable to infect non-tumor cells. The SVV capsid library is incubated with a tumor cell line or tissue of interest. After an initial incubation period, the cells are washed such that SVV virus particles that were unable to gain entry into the cells are eliminated. The cells are then maintained in culture until viral lysis is observed. Culture supernatant is then collected to isolate SVV capsid mutants that were able to lytically infect the tumor cell. These viruses can then be grown-up by infecting a known permissive cell-line prior to a counter-screen. A counter-screen is performed by incubating the SVV capsid mutant viruses that were able to infect the tumor cell with normal cells. Only those viruses that remain unbound in the supernatant are collected, thereby isolating mutant SVV viruses that have tumor-specificity. This process can be repeated to further refine the isolation of SVV tumor-specific viruses.
[0134] FIG. 61 shows a traditional method for testing whether virus mutants can bind and/or infect cell lines. Traditional methods require what are often inefficient methods for growing cell-lines, i.e. flasks, such that a mass-screen of a library of virus mutants in relation to a number of different cell-lines becomes burdensome.
[0135] FIG. 62 shows a high-throughput method of the invention for screening virus mutants that have the ability to specifically infect different cell-lines (Example 16). In this figure, a number of different tumor cell-lines are grown in a 384 well-plate. To each well, a sample of a virus is added (for example, a sample of a SVV capsid library). From those wells which show cytopathic effects, the media is collected such that any viruses in the media can be amplified by infecting permissive cell lines (for example, for SVV, H446 or PER.C6) in flasks or large tissue culture plates. The viruses are grown such that the RNA can be isolated and the sequence analyzed to determine the encoded peptide sequence inserted by the oligonucleotide-insertion mutagenesis of the capsid. The peptide itself can then be tested to determine whether it can bind to a tumor cell-type specifically.
[0136] FIG. 63 shows another high-throughput screening schematic (Example 16). Tumor and normal cell lines are grown in multi-well plates. Viruses are added to each well to test whether the cells are killed by virus-mediated lysis. Cytopathic effects can be quickly assayed by reading the light-absorbance in each well. Viruses from the wells showing cytopathic effects are grown up and tested in further in vitro (re-testing of tumor and normal cell lines) and in vivo models (testing whether the virus can kill explanted tumors in mice).
[0137] FIG. 64 shows that SVV capsid mutants (SEQ ID NOS: 45-48, respectively, in order of appearance) having new tumor-specific tropisms can be analyzed to generate tumor-selective peptides. Those SVV capsid mutants that enable the specific infection of a tumor cell line are sequenced to determine the peptide encoded by the oligonucleotide insertion. An amino acid consensus sequence can thereby be determined from the successful capsid mutants. Peptides having the consensus sequence can then be tested to determine whether they can bind specifically to the tumor cell-type in question. Tumor-selective peptides can then be attached to toxins or drugs in order to serve as tumor-specific targeting vehicles.
[0138] FIG. 65 illustrates that an SVV capsid library can be first tested in vivo. Mice (including normal, athymic, nude, CD-1 transgenics, etc.) can be explanted with a specific tumor. These mice are then injected with a SVV derivative library, such as a SVV capsid library. At certain time points, tumor cells are recovered from the mice, such that in those mice that display the elimination of a tumor, viruses will be isolated from initial tumor samples and grown-up in permissive cell lines.
[0139] FIG. 66 shows a clinical testing program for the SVV derivatives of the present invention.
[0140] FIG. 67 illustrates that SVV derivatives (with new tumor tropisms) encoding epitope tags in their capsid can be used for a variety of purposes. They can be used as a screening reagent to detect whether a specific tumor cell is present in tissue samples by assaying for the presence of the epitope. Alternatively, toxins or other therapeutics can be attached to the epitope tag, and the virus then administered to patients. Further, wild-type or derivative SVV can be irradiated or inactivated such that the virus particle itself is used as a therapeutic device. Either the virus particle induces cellular apoptosis due to the presence of apoptosis-inducing peptides, or the particle can have a toxin or some other therapeutic attached such that the virus is used a specific-targeting delivery device.
[0141] FIG. 68 shows the basic life-cycle of the picornavirus.
[0142] FIG. 69 shows a comparison of the polypeptide lengths of SVV compared to other picornaviruses.
[0143] FIG. 70 lists an amino acid comparison of the picornavirus 2A-like NPG/P proteins (SEQ ID NOS: 49-110, respectively, in order of appearance). The sequence for SVV is listed at residues 635-656 of SEQ ID NO:2.
[0144] FIGS. 71A and 71B list the amino acid sequence (SEQ ID NO:23) for EMCV-R.
[0145] FIGS. 72A and 72B list the amino acid sequence (SEQ ID NO:24) for EMCV-PV21 (Accession CAA52361).
[0146] FIGS. 73A and 73B list the amino acid sequence (SEQ ID NO:25) for EMCV-B (Accession P17593).
[0147] FIGS. 74A and 74B list the amino acid sequence (SEQ ID NO:26) for EMCV-Da (Accession P17594).
[0148] FIGS. 75A and 75B list the amino acid sequence (SEQ ID NO:27) for EMCV-Db.
[0149] FIGS. 76A and 76B list the amino acid sequence (SEQ ID NO:28) for EMCV-PV2 (Accession CAA60776).
[0150] FIGS. 77A and 77B list the amino acid sequence (SEQ ID NO:29) for EMCV-mengo (Accession AAA46547).
[0151] FIGS. 78A and 78B list the amino acid sequence (SEQ ID NO:30) for TMEV/DA (Accession AAA47928).
[0152] FIGS. 79A and 79B list the amino acid sequence (SEQ ID NO:31) for TMEV/GDVII (Accession AAA47929).
[0153] FIGS. 80A and 80B list the amino acid sequence (SEQ ID NO:32) for TMEV/BeAn8386 (Accession AAA47930).
[0154] FIGS. 81A and 81B list the amino acid sequence (SEQ ID NO:33) for TLV-NGS910 (Accession BAC58035).
[0155] FIG. 82 lists the amino acid sequence (SEQ ID NO:34) for VHEV/Siberia-55 (Accession AAA47931).
[0156] FIGS. 83A-83H present the full-length genomic sequence of SVV (SEQ ID NO:168) and the encoded polyprotein amino acid sequence (SEQ ID NO:169), where this full-length genomic sequence was obtained from SVV viruses grown from the SVV isolate having ATCC Patent Deposit Number PTA-5343. Specific features of the SVV genomic sequence, such as the specific coding regions for proteins cleaved from the polyprotein sequence are described herein.
[0157] FIGS. 84A-84D present the full-length genomic sequence of SVV (SEQ ID NO:168). The sequence was obtained from SVV grown from the SVV isolate having ATCC Patent Deposit Number PTA-5343.
[0158] FIGS. 85A-85B present the amino acid sequence of the full-length polyprotein of SVV (SEQ ID NO:169) encoded by the nucleotides 667-7209 of SEQ ID NO:168.
[0159] FIG. 86 provides a phylogenetic analysis or epidemiology of SVV with respect to the full-length genome and polyprotein sequence of SVV from SEQ ID NOS:168 and 169. SVV is a unique virus, phylogenetically similar to cardioviruses, but in a separate tree. The SVV-like picornaviruses are most likely in the same tree or genus as SVV due to the high level of sequence identity between SVV and the SVV-like picornaviruses (see FIGS. 87-89) and due to the ability of antibodies raised against SVV-like picornaviruses to bind SVV (and vice versa) (see Example 4, Part III, Serum Studies).
[0160] FIGS. 87A-87D show a nucleic acid sequence comparison between SVV and some SVV-like picornaviruses in the areas of the P1 structural region and 2A. In particular, the comparison is in the VP2(partial)-VP3-VP1-2A (partial) regions. The listed SVV sequence is SEQ ID NO:170; the listed sequence for isolate IA 89-47752 is SEQ ID NO:171; the listed sequence for isolate CA 131395 is SEQ ID NO:172; the listed sequence for isolate NC 88-23626 is SEQ ID NO:173; the listed sequence for isolate MN 88-36695 is SEQ ID NO:174; the listed sequence for isolate NJ 90-10324 is SEQ ID NO:175; the listed sequence for isolate IL 92-48963 is SEQ ID NO:176; the listed sequence for isolate LA 1278 (97-1278) is SEQ ID NO:177; and the listed consensus sequence is SEQ ID NO:178.
[0161] FIG. 88 shows a nucleic acid sequence comparison between SVV and isolates IA 89-47752 and CA 131395 in the 2C coding region (partial). The listed SVV sequence is SEQ ID NO:179; the listed sequence for isolate IA 89-47752 is SEQ ID NO:180; the listed sequence for isolate CA 131395 is SEQ ID NO:181; and the listed consensus sequence is SEQ ID NO:182.
[0162] FIGS. 89A-89B show a nucleic acid sequence comparison between SVV and isolates NC 88-23626, MN 88-36695, IA 89-47752, NJ 90-10324, IL 92-48963, LA 97-1278, and CA 131395 in the 3D polymerase coding region (partial) and 3' UTR region. The listed sequences are SVV (SEQ ID NO:183), NC 88-23626 (SEQ ID NO:184), MN 88-36695 (SEQ ID NO:185), IA 89-47752 (SEQ ID NO:186), NJ 90-10324 (SEQ ID NO:187), IL 92-48963 (SEQ ID NO:188), LA 97-1278 (SEQ ID NO:189), CA 131395 (SEQ ID NO:190), and consensus sequence (SEQ ID NO:191).
[0163] FIGS. 90A-90E show that a single dose of SVV is efficacious in reducing the size and preventing the growth of explanted tumors in mice. FIG. 90A shows that SVV can reduce the size and prevent the growth of explanted H446 human SCLC tumors (ED50=0.0007). FIG. 90B shows that SVV can reduce the size and prevent the growth of explanted Y79 human retinoblastoma tumors (ED50=0.0007). FIG. 90C shows that SVV can reduce the size and prevent the growth of explanted H69AR human SCLC-MDR (multi drug resistant) tumors (ED50=0.05). FIG. 90D shows that SVV can reduce the size and prevent the growth of explanted H1299 human HSCLC tumors (ED50=4.8). FIG. 90E shows that SVV can reduce the size and prevent the growth of explanted N1E-115 murine neuroblastoma tumors in A/J mice (normal immunocompetent mice) (ED50=0.001).
[0164] FIG. 91 show a molecular model of the EMCV and TMEV capsid structures in comparison with the sequence of SVV. A molecular model in conjunction with the use of algorithms for antigenic prediction allows for peptide sequences to be chosen for polyclonal antibody generation. β-sheets are shown in brown, α-helices are shown in green, and a 12-mer peptide sequence chosen for polyclonal generation is shown in yellow. The particular sequence (in the VP2 region) was chosen because it presents good surface exposure according to the model.
[0165] FIGS. 92A-92D show the specificity of polyclonal antibodies against SVV. FIG. 92A is a negative control, and presents an immunofluorescence image of cells infected with SVV that are stained with non-specific anti-mouse sera and secondary antibody. FIGS. 92B and 92C show immunofluorescence images of cells infected with SVV that are stained with mouse anti-SVV sera diluted 1:50 and secondary antibody (anti-mouse Ig conjugated to fluorescein). FIG. 92D shows that polyclonal anti-SVV antibodies can be used in viral binding assays; the image shows an immunofluorescence image of SVV concentrated in an outline around a cell because the cell was put on ice to prevent SVV internalization.
[0166] FIG. 93 shows the results of a neutralization assay of GP102 sera on SVV (see Example 18). The neutralization titer (calculated as the highest dilution that neutralizes the virus is 100%) is 1:100.
[0167] FIG. 94 shows the results of a neutralization assay of anti-SVV antisera on MN 88-36695 (see Example 18). The neutralization titer is 1:560.
[0168] FIG. 95A and FIG. 95B depict neighbor-joining trees. These trees were constructed using PHYLIP (Phylogeny Inference Package Computer Programs for Inferring Phylogenies) and show the relationship between SVV and seven SVV-like picornaviruses when comparing sequences from regions in P1 and partial 2A (FIG. 95A) and in the 3' end of the genome (FIG. 95B).
DETAILED DESCRIPTION OF THE INVENTION
[0169] The terms "virus," "viral particle," "virus particle," and "virion" are used interchangeably.
[0170] The terms "vector particle" and "viral vector particle" are interchangeable and are to be understood broadly--for example--as meaning infectious viral particles that are formed when, e.g., a viral vector of the invention is transduced or transfected into an appropriate cell or cell line for the generation of infectious particles.
[0171] The terms "derivative," "mutant," "variant" and "modified" are used interchangeably to generally indicate that a derivative, mutant, variant or modified virus can have a nucleic acid or amino acid sequence difference in respect to a template viral nucleic acid or amino acid sequence. For example, a SVV derivative, mutant, variant or modified SVV may refer to a SVV that has a nucleic acid or amino acid sequence difference with respect to the wild-type SVV nucleic acid or amino acid sequence of ATCC Patent Deposit Number PTA-5343.
[0172] An "SVV-like picornavirus" as used herein can have at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SVV at the nucleotide level (see SEQ ID NO:168, FIG. 84, and FIG. 83 for the SVV full-length genomic sequence), where the sequence comparison is not limited to a whole-genome analysis, but can be focused on a particular region of the genome, such as the 5'UTR, structural encoding regions, non-structural encoding regions, 3'UTR, and portions thereof. The particular length of the genome for sequence comparison that is adequate to determine relatedness/likeness to SVV is known to one skilled in the art, and the adequate length can vary with respect to the percentage of identity that is present. The length for sequence comparison can be, for example, at least 20, 50, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, or 2500 nucleotides. Where the length is shorter, one skilled in the art understands, for example, that the identity between sequences can be higher in order to consider the two sequences to be related. However, such guidance is qualified at least with respect to considerations of sequence conservation, in that certain regions of the genome are more conserved than others between related species. Additionally, if an antiserum generated from a virus can neutralize SVV infection of an SVV permissive cell line, then the virus is considered to be an SVV-like picornavirus. Additionally, if an antiserum generated from a virus can neutralize SVV infection of an SVV permissive cell line, and that antiserum can also bind to other viruses (for example, if the antiserum can be used in indirect immunofluorescence assays to detect virus), then the other viruses that can be bound by the antiserum are considered to be SVV-like picornaviruses. For purposes of the invention, SVV-like picornaviruses can include cardioviruses. Exemplary SVV permissive cells or cell lines include, but are not limited to, Y79, NCI-H446, N1E-115, NCI-H1770, NCI-H82, PER.C6®, NCI-H69AR, SK-NEP-1, IMR-32, NCI-H187, NCI-H209, HCC33, NCI-H1184, D283 Med, SK-N-AS, BEK PCB3E1, ST, NCI-H1299, DMS 153, NCI-H378, NCI-H295R, BEK, PPASMC, PCASMC, PAoSMC, NCI-H526, OVCAR-3, NCI-H207, ESK-4, SW-13, 293, Hs 578T, HS 1.Tes, and LOX IMVI.
[0173] As used herein, the terms "cancer," "cancer cells," "neoplastic cells," "neoplasia," "tumor," and "tumor cells," are used interchangeably, and refer to cells that exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. Neoplastic cells can be malignant or benign. According to the present invention, one type of preferred tumor cells are those with neurotropic properties.
[0174] The terms "identical" or percent "identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as Protein-Protein BLAST (Protein-Protein BLAST of GenBank databases (Altschul, S.F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410)) or by visual inspection. The BLAST algorithm is described in Altschul et al., J. Mol. Biol., 215:403-410 (1990), and publicly available BLAST software is provided through the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/).
[0175] For example, as used herein, the term "at least 90% identical to" refers to percent identities from 90 to 100 relative to the reference polypeptides (or polynucleotides). Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between a test and reference polynucleotide. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g., 10 out of 100 amino acid differences (90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.
[0176] The concepts of "high stringency," "intermediate stringency" or "low stringency" refer to nucleic acid hybridization conditions. High stringency conditions refers to conditions that require a greater identity between a target's nucleic acid sequence and a probe's nucleic acid sequence in order for annealing or hybridization to occur between the target and the probe. Low stringency conditions refer to conditions that require a lower identity between a target's nucleic acid sequence and a probe's nucleic acid sequence in order for annealing or hybridization to occur between the target and the probe. Stringency conditions can be controlled by the salt concentration of the buffer or by the temperature at which the hybridization is carried out, where higher salt concentrations result in less stringent conditions and where higher temperatures result in more stringent conditions. Although stringency conditions will vary based on the length and nucleic acid content of the sequences undergoing hybridization, representative conditions of high, intermediate and low stringency are described in the following exemplary conditions. A commonly used hybridization buffer is SSC (sodium chloride sodium citrate) with a 20× stock concentration corresponding to 0.3 M trisodium citrate and 3 M NaCl. For high stringency conditions, the working concentration of SSC can be 0.1×-0.5×(1.5-7.5 mM trisodium citrate, 15-75 mM NaCl) with the hybridization temperature set at 65° C. Intermediate conditions typically utilize a 0.5×-2×SSC concentration (7.5-30 mM trisodium citrate, 75-300 mM NaCl) at a temperature of 55-62° C. Hybridizations conducted under low stringency conditions can use a 2×-5×SSC concentration (30-75 mM trisodium citrate, 300-750 mM NaCl) at a temperature of 50-55° C. Note that these conditions are merely exemplary and are not to be considered limitations.
Seneca Valley Virus (SVV):
[0177] SVV is a novel, heretofore undiscovered RNA virus, and with respect to previously characterized picornaviruses, SVV is most closely related to members from the genus Cardiovirus in the family Picornaviridae (see International Application No. PCT/US2004/031594). The results of sequence analyses between SVV and other cardioviruses are discussed in PCT/US2004/031594, which is hereby incorporated by reference in its entirety. Since the time of the sequence analysis of SVV described in PCT/US2004/031594, the Picornavirus Study Group has initiated discussion as to whether SVV will be a member of a new genus. FIG. 86 presents a genetic relationship tree between members of the family Picornaviridae.
[0178] From initial sequence comparisons to known picornaviruses (see International Application No. PCT/US2004/031504), there were two phylogenetic classification options: (1) to include SVV as a new species in the genus Cardiovirus; or (2) assign SVV to a new genus. At that time and for the International application, SVV was designated to be a novel member of the genus Cardiovirus. After further analyses however, it has been found that several characteristics of SVV differ with that of cardioviruses. For example, some cardiovirus genomes contain an extended internal poly(C) tract in their 5' UTRs. SVV does not contain a poly(C) tract. From the additional 5' sequence information, the Internal Ribosome Entry Sequence (IRES) of SVV has been mapped and compared to other picornaviruses, and it has been determined that the SVV IRES is Type IV, whereas cardiovirus IRES's are Type II. The cardioviruses have a long (150 amino acid (aa)) 2A protease while SVV has a short (9 aa) 2A protease. The size of this protein as well as others (Leader peptide, 3A) differs significantly between SVV and cardioviruses. From the study of other picornaviruses, it is know that these proteins are likely involved in host cell interactions including tropism and virulence. Lastly, it is now thought that the overall sequences differ too much in a number of genome regions and SVV should therefore be considered to form a new genus. Additionally, multiple unique picornaviruses have been discovered at the USDA that are more similar to SVV than SVV is to other cardioviruses. Therefore, it has been decided by the Executive Committee of the International Committee for the Taxonomy of Viruses (ICVT) based on recommendations made by the Picornavirus Study Group that SVV will make up a new species of picornavirus, named Seneca Valley virus. However, currently, SVV and these unique USDA picornaviruses (herein referred to as being members of the group of SVV-like picornaviruses) are currently unassigned to any genus.
[0179] Several of the SVV-like picornaviruses discovered at the USDA are about 95-98% identical to SVV at the nucleotide level (for example, see FIGS. 87-89). Antisera against one virus (MN 88-36695) neutralizes SVV, and this virus is reactive to other antisera that can neutralize SVV. The SVV-like picornaviruses were isolated from pigs, and thus, pigs are likely a permissive host for SVV and other SVV-like viruses. Examples of SVV-like picornaviruses isolated from pigs include, but are not limited to, the following USDA isolates MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. SVV-like picornaviruses may also include cardioviruses closely related to SVV (as determined by sequence analysis or by cross-reactivity to antibodies raised against SVV antigens). Thus, for purposes of the present invention, SVV can be considered: (1) to be closely related to (or to be a member of) the genus Cardiovirus of the family Picornaviridae, and (2) to be a member of a new genus of the family Picornaviridae, where members of the new genus can include SVV and SVV-like picornaviruses not classified to be members of other genuses.
[0180] SVV, like cardioviruses, can be distinguished from other picornaviruses by special features of their genome organization, common pathological properties, and the dissociability of their virions at pHs between 5 and 7 in 0.1M NaCl (Scraba, D. et al., "Cardioviruses (Picornaviridae)," in Encyclopedia of Virology, 2nd Edition, R. G. Webster and A. Granoff, Editors, 1999). The genome of SVV consists of one single-stranded positive (+) sense strand RNA molecule having a size of 7,310 nucleotides including a poly(A) tail of 30 nucleotides in length (see FIGS. 83A-83H; FIGS. 84A-84D; SEQ ID NO:168). As SVV is a picornavirus, it has a number of features that are conserved in all picornaviruses: (i) genomic RNA is infectious, and thus can be transfected into cells to bypass the virus-receptor binding and entry steps in the viral life cycle; (ii) a long (about 600-1200 bp) untranslated region (UTR) at the 5' end of the genome (for SVV, nucleotides 1-666 of SEQ ID NO:168), and a shorter 3' untranslated region (about 50-100 bp; for SVV, nucleotides 7210-7280 of SEQ ID NO:168; (iii) the 5' UTR contains a clover-leaf secondary structure known as the internal ribosome entry site (IRES) (which can be, for example, from about nucleotide 300 to about nucleotide 366 of SEQ ID NO:168); cardioviruses have a Type II IRES and SVV has a Type IV IRES; (iv) the rest of the genome encodes a single polyprotein (for SVV, nucleotides 667-7209 of SEQ ID NO:168 encode the polyprotein (SEQ ID NO:169)) and (v) both ends of the genome are modified, the 5' end by a covalently attached small, basic protein, "Vpg," and the 3' end by polyadenylation (for SVV, nucleotides 7281-7310 of SEQ ID NO:168).
[0181] The invention provides the isolated SVV virus (ATCC Patent Deposit number PTA-5343) and the complete genomic content of SVV therefrom. At first, the largest SVV genomic fragment that was sequenced is an isolated SVV nucleic acid, derived from the PTA-5343 isolate, that comprises the majority of the SVV genomic sequence, and is listed in FIGS. 5A-5E and FIGS. 6A-6D, and has the designation of SEQ ID NO:1 herein. Translation of this nucleotide sequence shows that the majority of the single polyprotein of SVV is encoded by SEQ ID NO:1. The amino acid sequence encoded by nucleotides 1 to 5673 of SEQ ID NO:1 is listed in FIGS. 5A-E and FIGS. 7A-7B has the designation of SEQ ID NO:2 herein. The full-length genome or what appears to be the full-length genome has since been obtained, and is listed in FIGS. 83A-83H and SEQ ID NO:168. Nucleotides 667-7209 encode the full-length polyprotein of SVV, and the amino acid sequence of the polyprotein is listed in FIGS. 83A-83H and SEQ ID NO:169.
[0182] The invention provides isolated (or purified) portions of SEQ ID NO:1, including SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and isolated portions of SEQ ID NO:168, including the 5'UTR region (1-666), coding region for the leader peptide (667-903), coding region for the VP4 protein (904-1116), coding region for the VP2 protein (1117-1968), coding region for the VP3 protein (1969-2685), coding region for the VP1 protein (2686-3474), coding region for the coding region for the 2A protein (3478-3504), coding region for the 2B protein (3505-3888), coding region for the 2C protein (3889-4854), coding region for the 3A protein (4855-5124), coding region for the 3B protein (5125-5190), coding region for the 3C protein (5191-5823), coding region for the 3D protein (5824-7209), and the 3'UTR region including the poly(A) tail (7210-7310). The invention also provides isolated nucleic acids that are portions of the specified portions listed above. The invention also provides mutants or derivatives of such isolated portions. The isolated portions of SEQ ID NOS:1 and 168 can be subcloned into expression vectors such that polypeptides encoded by these portions can be isolated. Further encompassed by the invention are isolated nucleic acids that can hybridize to SEQ ID NO:1 or SEQ ID NO:168, or any portion thereof, under high, moderate or low stringency conditions. The following table lists the nucleotides of SEQ ID NO:168 that encode the SVV proteins. The invention provides isolated (or purified) SVV proteins or portions thereof. The table also lists the amino acid sequences of the SVV proteins with respect to the polyprotein sequence listed in SEQ ID NO:169.
TABLE-US-00001 TABLE A SVV Genome and Protein Features SVV Location in SEQ ID feature Location in SEQ ID NO: 168 NO: 169 5'UTR 1-666 N/A (not allowed) Leader 667-903 (coding sequence for Leader 1-79 peptide) VP4 904-1116 (coding sequence for VP4) 80-150 VP2 1117-1968 (coding sequence for VP2) 151-434 VP3 1969-2685 (coding sequence for VP3) 435-673 VP1 2686-3474 or 3477 (coding sequence for 674-936 or 937 VP1) 2A 3478-3504 (coding sequence for 2A) 938-946 2B 3505-3888 (coding sequence for 2B) 947-1074 2C 3889-4854 (coding sequence for 2C) 1075-1396 3A 4855-5124 (coding sequence for 3A) 1397-1486 3B 5125-5190 (coding sequence for 3B) 1487-1508 3C 5191-5823 (coding sequence for 3C) 1509-1719 3D 5824-7209 (coding sequence for 3D) 1720-2181 3'UTR 7210-7310 N/A
[0183] The invention provides an isolated SVV leader sequence peptide with the amino acid sequence of residues 1-79 of SEQ ID NO:169, which is encoded by nucleotides 667-903 of SEQ ID NO:168.
[0184] The invention provides an isolated SVV VP4 (1A) protein with the amino acid sequence of residues 80-150 of SEQ ID NO:169, which is encoded by nucleotides 904-1116 of SEQ ID NO:168.
[0185] The invention provides an isolated SVV VP2 (1B) protein with the amino acid sequence of residues 151-434 of SEQ ID NO:169, which is encoded by nucleotides 1117-1968 of SEQ ID NO:168. The invention also provides an isolated partial SVV VP2 (1B) protein with the amino acid sequence of SEQ ID NO:4, as listed in FIG. 9 (which corresponds to amino acids 2-143 of SEQ ID NO:2). The amino acid sequence of the partial SVV VP2 protein is encoded by the nucleic acid sequence of SEQ ID NO:3, as listed in FIG. 8 (which corresponds to nucleotides 4-429 of SEQ ID NO:1).
[0186] The invention provides an isolated SVV VP3 (1C) protein with the amino acid sequence of residues 435-673 of SEQ ID NO:169, which is encoded by nucleotides 1969-2685 of SEQ ID NO:168. The invention also provides an isolated SVV VP3 (1C) protein with the amino acid sequence of SEQ ID NO:6, as listed in FIG. 11 (which corresponds to amino acids 144-382 of SEQ ID NO:2). The amino acid sequence of the SVV VP3 protein is encoded by the nucleic acid sequence of SEQ ID NO:5, as listed in FIG. 10 (which corresponds to nucleotides 430-1146 of SEQ ID NO:1).
[0187] The invention provides an isolated SVV VP1 (1D) protein with the amino acid sequence of residues 674-937 of SEQ ID NO:169, which is encoded by nucleotides 2686-3477 of SEQ ID NO:168. The invention also provides an isolated SVV VP1 (1D) protein with the amino acid sequence of SEQ ID NO:8, as listed in FIG. 13 (which corresponds to amino acids 383-641 of SEQ ID NO:2). The amino acid sequence of the SVV VP1 protein is encoded by the nucleic acid sequence of SEQ ID NO:7, as listed in FIG. 12 (which corresponds to nucleotides 1147-1923 of SEQ ID NO:1).
[0188] The invention provides an isolated SVV 2A protein with the amino acid sequence of residues 938-946 of SEQ ID NO:169, which is encoded by nucleotides 3478-3504 of SEQ ID NO:168. The invention also provides an isolated SVV 2A protein with the amino acid sequence of SEQ ID NO:10, as listed in FIG. 15 (which corresponds to amino acids 642-655 of SEQ ID NO:2). The amino acid sequence of the SVV 2A protein is encoded by the nucleic acid sequence of SEQ ID NO:9, as listed in FIG. 14 (which corresponds to nucleotides 1924-1965 of SEQ ID NO:1).
[0189] The invention provides an isolated SVV 2B protein with the amino acid sequence of residues 947-1074 of SEQ ID NO:169, which is encoded by nucleotides 3505-3888 of SEQ ID NO:168. The present invention also provides an isolated SVV 2B protein with the amino acid sequence of SEQ ID NO:12, as listed in FIG. 17 (which corresponds to amino acids 656-783 of SEQ ID NO:2). The amino acid sequence of the SVV 2B protein is encoded by the nucleic acid sequence of SEQ ID NO:11, as listed in FIG. 16 (which corresponds to nucleotides 1966-2349 of SEQ ID NO:1).
[0190] The invention provides an isolated SVV 2C protein with the amino acid sequence of residues 1075-1396 of SEQ ID NO:169, which is encoded by nucleotides 3889-4854 of SEQ ID NO:168. The invention also provides an isolated SVV 2C protein with the amino acid sequence of SEQ ID NO:14, as listed in FIG. 19 (which corresponds to amino acids 784-1105 of SEQ ID NO:2). The amino acid sequence of the SVV 2B protein is encoded by the nucleic acid sequence of SEQ ID NO:13, as listed in FIG. 18 (which corresponds to nucleotides 2350-3315 of SEQ ID NO:1).
[0191] The invention provides an isolated SVV 3A protein with the amino acid sequence of residues 1397-1486 of SEQ ID NO:169, which is encoded by nucleotides 4855-5124 of SEQ ID NO:168. The invention also provides an isolated SVV 3A protein with the amino acid sequence of SEQ ID NO:16, as listed in FIG. 21 (which corresponds to amino acids 1106-1195 of SEQ ID NO:2). The amino acid sequence of the SVV 3A protein is encoded by the nucleic acid sequence of SEQ ID NO:15, as listed in FIG. 20 (which corresponds to nucleotides 3316-3585 of SEQ ID NO:1).
[0192] The invention provides an isolated SVV 3B (VPg) protein with the amino acid sequence of residues 1487-1508 of SEQ ID NO:169, which is encoded by nucleotides 5125-5190 of SEQ ID NO:168. The invention also provides an isolated SVV 3B protein with the amino acid sequence of SEQ ID NO:18, as listed in FIG. 23 (which corresponds to amino acids 1196-1217 of SEQ ID NO:2). The amino acid sequence of the SVV 3B protein is encoded by the nucleic acid sequence of SEQ ID NO:17, as listed in FIG. 22 (which corresponds to nucleotides 3586-3651 of SEQ ID NO:1).
[0193] The invention provides an isolated SVV 3C ("pro" or "protease") protein with the amino acid sequence of residues 1509-1719 of SEQ ID NO:169, which is encoded by nucleotides 5191-5823 of SEQ ID NO:168. The invention also provides an isolated SVV 3C protein with the amino acid sequence of SEQ ID NO:20, as listed in FIG. 25 (which corresponds to amino acids 1218-1428 of SEQ ID NO:2). The amino acid sequence of the SVV 3C protein is encoded by the nucleic acid sequence of SEQ ID NO:19, as listed in FIG. 24 (which corresponds to nucleotides 3652-4284 of SEQ ID NO:1).
[0194] The invention provides an isolated SVV 3D ("pol" or "polymerase") protein with the amino acid sequence of residues 1720-2181 of SEQ ID NO:169, which is encoded by nucleotides 5824-7209 of SEQ ID NO:168. The invention also provides an isolated SVV 3D protein with the amino acid sequence of SEQ ID NO:22, as listed in FIG. 27 (which corresponds to amino acids 1429-1890 of SEQ ID NO:2). The amino acid sequence of the SVV 3C protein is encoded by the nucleic acid sequence of SEQ ID NO:19, as listed in FIG. 24 (which corresponds to nucleotides 4285-5673 of SEQ ID NO:1; nucleotides 5671-5673, "tga," code for a stop-codon, which is depicted in the amino acid sequence listings as an asterisk "*").
[0195] The nucleic acids of the present invention include both RNA and DNA forms, and implicitly, the complementary sequences of the provided listings.
[0196] Thus, the isolated SVV nucleic acid depicted by SEQ ID NO:168 has a length of 7,310 nucleotides that encodes a polyprotein with the amino acid sequence depicted by SEQ ID NO:169. The isolated SVV nucleic acid depicted by SEQ ID NO:1 has a length of 5,752 nucleotides that encodes a polypeptide with the amino acid sequence depicted by SEQ ID NO:2. The SVV genomic sequence is translated as a single polyprotein that is cleaved into various downstream "translation products." The present invention encompasses all nucleic acid fragments of SEQ ID NO: 168 and SEQ ID NO:1, and all polypeptides encoded by such fragments.
[0197] The full-length SVV polyprotein amino acid sequence is depicted by SEQ ID NO:169 and is encoded by nucleotides 667-7209 of SEQ ID NO:168. The majority of the full-length SVV polyprotein amino acid sequence is encoded by nucleotides 1-5673 of SEQ ID NO:1. The polyprotein is cleaved into three precursor proteins, P1, P2 and P3 (see FIG. 4B). P1, P2 and P3 are further cleaved into smaller products. The cleavage products of the structural region P1 (1ABCD; or the capsid region) are 1ABC, VP0, VP4, VP2, VP3 and VP 1. The cleavage products of the non-structural protein P2 (2ABC) are 2A, 2BC, 2B and 2C. The cleavage products of the non-structural region P3 polyprotein (3ABCD) are 3AB, 3CD, 3A, 3C, 3D, 3C', and 3D'.
[0198] In certain embodiments, the invention provides isolated nucleic acids that comprise: (i) the coding sequence of 1ABCD or the capsid region (nucleotides 904-3477 of SEQ ID NO:168); (ii) the coding sequence of 1ABC (nucleotides 904-2685 of SEQ ID NO:168); (iii) the coding sequence of VP0 (nucleotides 904-1968 of SEQ ID NO:168); (iv) the coding sequence of 2ABC (nucleotides 3478-4854 of SEQ ID NO:168; nucleotides 1924-3315 of SEQ ID NO:1); (v) the coding sequence of 2BC (nucleotides 3505-4854 of SEQ ID NO:168; nucleotides 1966-3315 of SEQ ID NO:1); (iii) the coding sequence of 3ABCD (nucleotides 4855-7209 of SEQ ID NO:168; nucleotides 3316-5673 of SEQ ID NO:1); (iv) the coding sequence of 3AB (nucleotides 4855-5190 of SEQ ID NO:168; nucleotides 3316-3651 of SEQ ID NO:1); and (v) the coding sequence of 3CD (nucleotides 5191-7209 of SEQ ID NO:168; nucleotides 3652-5673 of SEQ ID NO:1). The invention also provides isolated proteins or peptides encoded by the coding sequences described above, including fragments thereof.
[0199] The basic capsid structure of picornaviruses consists of a densely packed icosahedral arrangement of 60 protomers, each consisting of 4 polypeptides, VP1, VP2, VP3 and VP4, all of which are derived from the cleavage of the original protomer, VP0. The SVV virus particle is about 27 nm in diameter (see FIG. 2), which is consistent with the size of other picornavirus particles, which are about 27-30 nm in diameter.
[0200] The kinetics of picornavirus replication is rapid, the cycle being completed in about 5-10 hours (typically by about 8 hours) (see FIG. 68 for a schematic of the picornavirus replication cycle). Upon receptor binding, the genomic RNA is released from the particle into the cytoplasm. Genomic RNA is then translated directly by polysomes, but in about 30 minutes after infection, cellular protein synthesis declines sharply, almost to zero. This phenomenon is called "shutoff," and is a primary cause of cytopathic effects (cpe). Shutoff appears to be due to cleavage of the host cell's 220 kDa cap-binding complex (CBC) that is involved in binding the m7G cap structure at the 5' end of all eukaryotic mRNA during initiation of translation. The cleavage of the CBC appears to be caused by the 2A protease.
[0201] The 5' UTR contains the IRES. Normally, eukaryotic translation is initiated when ribosomes bind to the 5' methylated cap and then scans along the mRNA to find the first AUG initiation codon. The IRES overcomes this process and allows Picornavirus RNA's to continue to be translated after degradation of CBC. In one embodiment, the invention provides for an isolated nucleic acid comprising the SVV IRES, wherein the IRES is contained within the 5'UTR. In one embodiment, the SVV IRES can be from nucleotides 300-366 of SEQ ID NO:168. The 5'UTR of SVV is present at nucleotides 1-666 of SEQ ID NO:168.
[0202] The virus polyprotein is initially cleaved by the 2A protease into polyproteins P1, P2 and P3 (see FIG. 4B). Further cleavage events are then carried out by 3C, the main picornavirus protease. One of the cleavage products made by 3C is the virus RNA-dependent RNA polymerase (3D), which copies the genomic RNA to produce a negative (-) sense strand. The (-) sense strand forms the template for the (+) strand (genomic) RNA synthesis. Some of the (+) strands are translated to produce additional viral proteins are some (+) strands are packaged into capsids to form new virus particles.
[0203] The (+) strand RNA genome is believed to be packaged into preformed capsids, although the molecular interactions between the genome and the capsid are not clear. Empty capsids are common in all picornavirus infections. The capsid is assembled by cleavage of the P1 polyprotein precursor into a protomer consisting of VP0, VP3, and VP1, which join together enclosing the genome. Maturation and infectivity of the virus particle relies on an internal autocatalytic cleavage of VP0 into VP2 and VP4. Release of newly formed virus particles occurs when the cell lyses.
[0204] The present invention also provides an isolated virus having all the identifying characteristics and nucleic acid sequence of ATCC Patent Deposit number PTA-5343. Viruses of the present invention can be directed to the PTA-5343 isolate, variants, homologues, derivatives and mutants of the PTA-5343 isolate, and variants, homologues, derivatives and mutants of other picornaviruses that are modified in respect to sequences of SVV (both wild-type as disclosed herein and mutant) that are determined to be responsible for its oncolytic properties.
[0205] The present invention further provides antibodies that are specific against: the isolated SVV having the ATTC Patent Deposit number PTA-5343, and epitopes from the isolated SVV proteins having the amino acid sequences SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 169 (including entire polyprotein, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, 3D, and portions thereof; see Table A supra for amino acids in SEQ ID NO:169 that make up these proteins). The invention also includes antibodies that are specific against epitopes from the proteins that are encoded by fragments or portions of SEQ ID NO:1 or SEQ ID NO:168.
[0206] Comparative analyses of the RNA sequences from a variety of cardiovirus isolates have shown >45% nucleotide identity between genomes. Cardioviruses can be subclassified into the EMC-like viruses ("EMCV"--such as, Mengo, B, R; and also MM, ME, Columbia-SK), the Theiler's-like viruses ("TMEV"--such as, BeAn, DA and GD VII strains), and the Vilyuisk viruses.
[0207] In analyzing the SVV sequence to other viruses, it appears that SVV is a cardiovirus (see Example 4 and Figures referenced therein). If EMCV and TMEV are taken as the standard cardioviruses, SVV is clearly not a typical cardiovirus. However, even these two viruses have their differences, notably in the 5' UTR (Pevear et al., 1987, J. Virol., 61: 1507-1516). Phylogenetically SVV clusters with EMCV and TMEV in much of its polyprotein (P1, 2C, 3Cpro and 3Dpol regions; see FIGS. 31-37), indicating that SVV is most likely a cardiovirus.
[0208] SVV is phylogenetically similar to cardioviruses, but it has now been determined to be in a separate tree (see FIG. 86). SVV can be in a separate genus because: (1) SVV IRES is Type IV (cardiovirus IRES are Type II); (2) multiple unique viruses ("SVV-like picornaviruses") are more similar to SVV than SVV is to other cardioviruses (see Example 18 and FIGS. 87-89); and antibodies that can neutralize SVV infection of permissive cell lines or were raised against SVV are able to bind to the SVV-like picornaviruses. Thus, an SVV-like picornavirus can be used in any of the present methods, including the methods to treat cancer, where it is determined that the SVV-like picornavirus is naturally oncolytic or is made to be oncolytic (for example, by designing mutations in the SVV-like picornavirus genome based on the SVV sequence). In one embodiment, MN 88-36696 is used in the present methods to treat cancer.
Methods for Treating Cancer:
[0209] The present invention provides methods for cancer therapy using viruses modified in view of the oncolytic properties of SVV, including picornaviruses (including SVV-like picornaviruses), derivatives, variants, mutants or homologues thereof. The present invention shows that wild-type SVV (i.e., ATTC Patent Deposit number PTA-5343) has the ability to selectively kill some types of tumors. For example, SVV can selectively kill tumor cells that have neurotropic or neuroendocrine properties, including small cell lung cancer (SCLC) and neuroblastomas. Other examples of neuroendocrine tumors that are contemplated to be treated by the viruses of the present invention include, but are not limited to: adrenal pheochromocytomas, gastrinomas (causing Zollinger-Ellison syndrome), glucagonomas, insulinomas, medullary carcinomas (including medullary thyroid carcinoma), multiple endocrine neoplasia syndromes, pancreatic endocrine tumors, paragangliomas, VIPomas (vasoactive intestinal polypeptide tumor), islet cell tumors, and pheochromocytoma.
[0210] In one embodiment, the invention provides methods for treating or reducing neuroendocrine tumors by administering to a subject SVV or an SVV-like picornavirus, where the neuroendocrine tumor expresses (or overexpresses) one or more neuroendocrine tumor markers, including but not limited to, NTR (Neurotensin receptor), ATOH (, GL11, Myc, GRP receptors, GRP, Neuronal enolase (neuron specific enolase (NSE)), carcinoembryonic antigen (CEA), chromoganin A, NCAM, IgF2, BCL-2, sonic hedgehog pathway, and a chemokine receptor.
[0211] Also encompassed in the present invention are the four types of neuroendocrine lung tumors. The most serious type, small cell lung cancer (SCLC), is among the most rapidly growing and spreading of all cancers. Large cell neuroendocrine carcinoma is a rare cancer that, with the exception of the size of the cells forming the cancer, is very similar to SCLC in its prognosis and in how patients are treated. Carcinoid tumors, also known as carcinoids, comprise the other 2 types of lung neuroendocrine cancer. These two types are typical carcinoid and atypical carcinoid.
[0212] Not being bound by theory, the ability of SVV to specifically kill tumor cells may include, but is not limited to: selective replication, cell protein synthesis shut-off, apoptosis, lysis via tumor-selective cell entry, tumor-selective translation, tumor-selective proteolysis, tumor-selective RNA replication, and combinations thereof.
[0213] SVV has many advantageous characteristics over other oncolytic viruses, including modified adenoviruses, for example: (i) SVV has a very high selectivity for cancers with neural properties, including SCLC, Wilms' tumor, retinoblastoma, and neuroblastoma--for example, SVV shows a greater than 10,000-fold selectivity toward neuroendocrine tumor cells; (ii) SVV has been shown to have a 1,000 fold better cell-killing specificity than chemotherapy treatments; (iii) SVV exhibits no overt toxicity in mice following systemic administration with as high as 1014 viral particles per kilogram; (iv) the efficacy of SVV is very robust in that 100% of large pre-established tumors can be eradicated in mice, with no recurrence of tumor growth; (v) SVV can be purified to high titer and can be produced at more than 200,000 particles per cell in permissive cell lines; (vi) SVV has a small size (the SVV virus particle is less than 30 nm in diameter) enabling better penetration and spread in tumors than other oncolytic viruses, (vii) SVV replicates quickly (less than 12 hours) and (viii) no modification of SVV is necessary for its use as a specific anti-tumor agent.
[0214] Further, initial studies (see Example 6) indicate some additional factors that make SVV an advantageous tool for oncolytic viral therapy: (i) human serum samples do not contain neutralizing antibodies directed against SVV; (ii) SVV is not inhibited by complement; and (iii) SVV does not produce hemagglutination of human erythrocytes. All of these factors contribute to the fact that SVV exhibits a longer circulation time in vivo than other oncolytic viruses (for example, see Example 7).
[0215] The present invention provides methods for selectively killing a neoplastic cell in a cell population that comprises contacting an effective amount of SVV with said cell population under conditions where the virus can transduce the neoplastic cells in the cell population, replicate and kill the neoplastic cells. Besides methods where SVV kills tumor cells in vivo, the present methods encompass embodiments where the tumors can be: (1) cultured in vitro when infected by SVV; (2) cultured in the presence of non-tumor cells; and (3) the cells are mammalian (both tumor and non-tumor cells), including where the cells are human cells. The in vitro culturing of cells and infection by SVV can have various applications. For example, in vitro infection be used as a method to produce large amounts of SVV, as method for determining or detecting whether neoplastic cells are present in a cell population, or as a method for screening whether a mutant SVV can specifically target and kill various tumor cell or tissue types.
[0216] The present invention further provides an ex vivo method of treating cancer wherein cells are isolated from a human cancer patient, cultured in vitro, infected with a SVV which selectively kills the cancer cells, and the non-tumor cells are introduced back to the patient. Alternatively, cells isolated form a patient can be infected with SVV and immediately introduced back to the patient as a method for administering SVV to a patient. In one embodiment, the cancer cells are of a hematopoietic origin. Optionally, the patient may receive treatment (e.g., chemotherapy or radiation) to destroy the patient's tumor cell in vivo before the cultured cells are introduced back to the patient. In one embodiment, the treatment may be used to destroy the patient's bone marrow cells.
[0217] Polymer coated SVV can be used to target the SVV to any specific cell type. This coating strategy can also be used to overcome antibodies to SVV.
[0218] SVV possesses potent antitumor activity against tumor cell-types with neural characteristics. SVV does not exhibit cytolytic activity against tested normal human. Further SVV is not cytotoxic to primary human hepatocytes. Table 1 below summarizes initial studies that have been conducted to determine the in vitro cytolytic potency of SVV against selected tumor cell types.
TABLE-US-00002 TABLE 1 SVV Cytolytic Potency Against Selected Tumor Cell-Types Cell Line Cell Type EC50 (VP/cell) H446 Human SCLC 0.0012 PER.C6 Human Embryonic Retinoblast 0.02 H69AR SCLC-Multidrug Resistant 0.035 293 AD5 DNA Transformed Human 0.036 Kidney Y79 Human Retinoblastoma 0.00035 IMR32 Human Brain Neuroblastoma 0.035 D283 Med Human Brain Cerebellar 0.25 Medulloblastoma SK-N-AS Human Brain Neuroblastoma 0.474 N1E-115 Mouse Neuroblastoma 0.0028 BEKPCB3E1 Bovine embryonic Kidney cells 0.99 transformed with Ad5E1 H1299 Human non-SCLC 7.66 ST Porcine Testis 5.9 DMS153 Human SCLC 9.2 BEK Bovine Embryonic Kidney 17.55 M059K Human Brain Malignant 1,061 Glioblastoma PK15 Porcine Kidney 1,144 FBRC Fetal Bovine Retina 10,170 HCN-1A Human Brain 23,708 H460 Human LCLC >30,000 (inactive) Neuro 2A Mouse Neuroblastoma >30,000 (inactive) DMS79 Human SCLC >30,000 (inactive) H69 Human SCLC >30,000 (inactive) C8D30 Mouse Brain >30,000 (inactive) MRC-5 Human Fetal Lung Fibroblast >30,000 (inactive) HMVEC Neonatal vascular endothelial cells >30,000 (inactive) HMVEC Adult vascular endothelial cells >30,000 (inactive) A375-S2 Human Melanoma >30,000 (inactive) SK-MEL-28 Melanoma >30,000 (inactive) PC3 Human prostate cancer >30,000 (inactive) PC3M2AC6 Human prostate cancer >30,000 (inactive) LnCap Human Prostate cancer >30,000 (inactive) DU145 Human prostate cancer >30,000 (inactive)
[0219] Table 1-A below provides a list of cell lines that are permissive are non-permissive to SVV infection. The Table shows the cytolytic potency and selectivity of SVV.
TABLE-US-00003 TABLE 1 In Vitro Cytolytic Potency and Selectivity of SVV Cell Line Species Stage State Organ Type Metastatic Site EC50* PERMISSIVE Y79 Human Adult Cancer Eye, Retina Retinoblastoma 0.00035, 0.0007 NCI-H446 Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0.0012, 0.002, Cancer Lung Carcinoma 0.0007 (SCLC) N1E-115 Murine Adult Cancer Brain Neuroblastoma 0.0028, 0.001 NCI-H1770 Human Adult Metastatic Lung Non-Small Cell Lymph Node 0.00724 Cancer Lung Carcinoma (NSCLC) NCI-H82 Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0.015 Cancer Lung Carcinoma (SCLC) PER.C6 ® Human Fetal Cancer Eye, Retina Retinoblast 0.02, 0.0049 NCI-H69AR Human Adult Cancer Lung Small Cell Lung 0.035, 0.05 Carcinoma, multi-drug resistant (SCLC) SK-NEP-1 Human Adult Metastatic Kidney Wilms' Tumor Pleural effusion 0.03 Cancer IMR-32 Human Adult Cancer Brain Neuroblastoma 0.035, 0.0059, 0.05 NCI-H187 Human Adult Metastatic Lung Classic Small Cell Pleural effusion 0.00343 Cancer Lung Carcinoma (SCLC) NCI-H209 Human Adult Metastatic Lung Small Cell Lung Bone Marrow 0.04 Cancer Carcinoma (SCLC) NCI-H1184 Human Adult Metastatic Lung Small Cell Lung Lymph Node 0.155 Cancer Carcinoma (SCLC) D283 Med Human Adult Metastatic Brain, Medulloblastoma Peritoneum 0.25 Cancer Cerebellum SK-N-AS Human Adult Metastatic Brain Neuroblastoma Bone Marrow 0.474 Cancer BEK PCB3E1 Bovine Fetal Normal, Ad5 Kidney Ad5E1 transformed 0.99 transformed ST Porcine Fetal Normal, Testis 5.9 immortalized NCI-H1299 Human Adult Metastatic Lung Large Cell Lung Lymph Node 7.66, 4.8 Cancer Carcinoma DMS 153 Human Adult Metastatic Lung Small Cell Lung Liver 9.2 Cancer Carcinoma (SCLC) NCI-H295R Human Adult Cancer Adrenal Gland, Adrenocortical 16.5 Cortex Carcinoma BEK Bovine Fetal Normal, Kidney 17.55 immortalized PPASMC Porcine Adult Normal, Lung, Smooth Muscle Cells 18.4 Primary Pulmonary Artery PCASMC Porcine Adult Normal, Heart, Coronary Smooth Muscle Cells 11.9 Primary Artery PAoSMC Porcine Adult Normal, Heart, Aorta Smooth Muscle Cells 88 Primary NCI-H526 Human Adult Metastatic Lung Variant Small Cell Bone Marrow 46.4 Cancer Lung Carcinoma (SCLC) OVCAR-3 Human Adult Cancer Ovary Adenocarcinoma 39 ESK-4 Porcine Fetal Normal, Kidney Fibroblast 60 Immortalized SW-13 Human Adult Cancer Adrenal Gland, Small Cell <100 Cortex Adenocarcinoma 293 Human Fetal Normal, Ad5 Kidney Ad5 transformed 0.036, 184.8 transformed Hs 578T Human Adult Cancer Breast Carcinoma 273 Hs 1.Tes Human Fetal Normal, Testis 416 Immortalized LOX IMVI Human Adult Cancer Skin Melanoma 569 PK(15) Porcine Adult Normal, Kidney 1144, 129 Immortalized NON PERMISSIVE WI-38 Human Fetal Normal, Lung Fibroblast >10,000 Immortalized IMR-90 Human Fetal Normal, Lung Fibroblast >10,000 Immortalized MRC-5 Human Fetal Normal, Lung Fibroblast >10,000 Immortalized HCN-1A Human Adult Normal, Brain, Cortical >10,000 Immortalized Neuron HMVEC Human Adult Normal, Skin Microvascular >10,000 (neonatal) Primary Endothelial Cells HMVEC Human Adult Normal, Skin Microvascular >10,000 Primary Endothelial Cells HUVEC Human Adult Normal, Umbilical Vein Endothelial Cells >10,000 Primary HRE Human Adult Normal, Kidney Epithelial Cells >10,000 Primary HRCE Human Adult Normal, Kidney Cortical Epithelial Cells >10,000 Primary PHH Human Adult Normal, Liver Hepatocyte >10,000 Primary HCASMC-c Human Adult Normal, Heart, Coronary Smooth Muscle Cells >10,000 Primary Artery HCAEC Human Adult Normal, Heart, Coronary Endothelial Cells >10,000 Primary Artery HAEC Human Adult Normal, Heart, Aorta Endothelial Cells >10,000 Primary HAoSMC-c Human Adult Normal, Heart, Aorta Smooth Muscle Cells >10,000 Primary NHA Human Adult Normal, Brain Astrocytes 1713 Primary HPASMC Human Adult Normal, Lung Smooth Muscle Cells >10,000 Primary PBMC Human Adult Normal, Peripheral Blood Mononuclear Cells >10,000 Primary SF-295 Human Adult Cancer Brain Glioblastoma >10,000 U251 Human Adult Cancer Brain Glioblastoma >10,000 SF-539 Human Adult Cancer Brain Glioblastoma >10,000 SNB-19 Human Adult Cancer Brain Glioblastoma >10,000 SF-268 Human Adult Cancer Brain Glioblastoma 3103 U-118MG Human Adult Cancer Brain Glioblastoma, >10,000 Astrocytoma SNB-75 Human Adult Cancer Brain Astrocytoma >10,000 M059K Human Adult Cancer Brain, Glial Cell Malignant 1061 Glioblastoma KK Human Adult Cancer Brain, Glial Cell Glioblastoma >10,000 HCC-2998 Human Adult Cancer Colon Carcinoma >10,000 KM12 Human Adult Cancer Colon Carcinoma >10,000 HT-29 Human Adult Cancer Colon Adenocarcinoma >10,000 HCT 116 Human Adult Cancer Colon Carcinoma >10,000 HCT-15 Human Adult Cancer Colon Carcinoma >10,000 COLO 205 Human Adult Metastatic Colon Adenocarcinoma Ascites >10,000 Cancer SW620 Human Adult Metastatic Colon Colorectal Carcinoma Lymph Node 6503, >10,000 Cancer PC3M-2AC6 Human Adult Cancer Prostate >10,000 PC3M-2AC6 + Human Adult Cancer Prostate ND 2-AP PC-3 Human Adult Metastatic Prostate Adenocarcinoma Bone >10,000 Cancer LNCaP.FGC Human Adult Metastatic Prostate Adenocarcinoma Lymph Node >10,000 Cancer DU 145 Human Adult Metastatic Prostate Adenocarcinoma Brain >10,000 Cancer Hep3B Human Adult Cancer Liver Hepatocellular >10,000 Carcinoma Hep G2 Human Adult Cancer Liver Hepatocellular >10,000 Carcinoma 786-O Human Adult Cancer Kidney Clear Cell >10,000 Adenocarcinoma TK-10 Human Adult Cancer Kidney Carcinoma >10,000 RXF 393 Human Adult Cancer Kidney Carcinoma >10,000 UO-31 Human Adult Cancer Kidney Carcinoma >10,000 SN12C Human Adult Cancer Kidney Carcinoma >10,000 A-498 Human Adult Cancer Kidney Carcinoma >10,000 ACHN Human Adult Cancer Kidney Carcinoma >10,000 SW839 Human Adult Cancer Kidney Renal Clear Cell >10,000 Adenocarcinoma Caki-1 Human Adult Metastatic Kidney Clear Cell Skin >10,000 Cancer Adenocarcinoma 5637 Human Adult Cancer Bladder Carcinoma >10,000 NCI-H1339 Human Adult Cancer Lung >10,000 NCI-H1514 Human Adult Cancer Lung >10,000 A549 Human Adult Cancer Lung Carcinoma >10,000 S8 Human Adult Cancer Lung Carcinoma >10,000 NCI-H727 Human Adult Cancer Lung Carcinoid >10,000 NCI-H835 Human Adult Cancer Lung Carcinoid >10,000 UMC-11 Human Adult Cancer Lung Carcinoid >10,000 DMS 114 Human Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) DMS 53 Human Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) NCI-H69 Human Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) NCI-H2195 Human Adult Metastatic Lung Small Cell Lung Bone Marrow >10,000 Cancer Carcinoma (SCLC) DMS 79 Human Adult Metastatic Lung Small Cell Lung Pleural effusion >10,000 Cancer Carcinoma (SCLC) NCI-H146 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer Lung Carcinoma (SCLC) NCI-H1618 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer Lung Carcinoma (SCLC) NCI-H345 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer Lung Carcinoma (SCLC) HOP-62 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) EKVX Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) HOP-92 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H522 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H23 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H322M Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H226 Human Adult Metastatic Lung Squamous Cell Pleural effusion >10,000 Cancer Carcinoma, Mesothelioma (NSCLC) NCI-H460 Human Adult Metastatic Lung Large Cell Lung Pleural effusion >10,000 Cancer Carcinoma HeLa, HeLa Human Adult Cancer Cervix Adenocarcinoma >10,000 S3 CCRF-CEM Human Adult Cancer Peripheral Acute Lymphoblastic >10,000 Blood, T Leukemia (ALL) lymphoblast MOLT-4 Human Adult Cancer Peripheral Acute Lymphoblastic >10,000 Blood, T Leukemia (ALL) lymphoblast RPMI 8226 Human Adult Cancer Peripheral Plasmacytoma, >10,000 Blood, B Myeloma lymphocyte SR Human Adult Metastatic Lymphoblast Large Cell Pleural effusion >10,000 Cancer Lymphoblastic Lymphoma HL-60(TB) Human Adult Cancer Peripheral Acute Promyelocytic >10,000 Blood, Leukemia (APL) Promyleoblast K-562 Human Adult Metastatic Bone Marrow Chronic Myelogenous Pleural effusion >10,000 Cancer Leukemia (CML) UACC-257 Human Adult Cancer Skin Melanoma >10,000 M14 Human Adult Cancer Skin Melanoma >10,000 UACC-62 Human Adult Cancer Skin Melanoma 6614 SK-MEL-2 Human Adult Cancer Skin Malignant Melanoma >10,000 SK-MEL-28 Human Adult Cancer Skin Malignant Melanoma >10,000 A375.S2 Human Adult Cancer Skin Malignant Melanoma >10,000 SK-MEL-28 Human Adult Cancer Skin Malignant Melanoma >10,000 SK-MEL-5 Human Adult Metastatic Skin Malignant Melanoma Lymph Node >10,000 Cancer MALME-3M Human Adult Metastatic Skin Malignant Melanoma Lung >10,000 Cancer
BT-549 Human Adult Cancer Breast Ductal Carcinoma >10,000 NCI/ Human Adult Cancer Breast Carcinoma >10,000 ADR-RES MCF7 Human Adult Metastatic Breast Adenocarcinoma Pleural effusion >10,000 Cancer MDA-MB-231 Human Adult Metastatic Breast Adenocarcinoma Pleural effusion >10,000 Cancer T-47D Human Adult Metastatic Breast Ductal Carcinoma Pleural effusion >10,000 Cancer MDA-MB-435 Human Adult Metastatic Breast Ductal Pleural effusion >10,000 Cancer Adenocarcinoma IGR-OV1 Human Adult Cancer Ovary Carcinoma >10,000 OVCAR-4 Human Adult Cancer Ovary Adenocarcinoma >10,000 OVCAR-5 Human Adult Cancer Ovary Adenocarcinoma >10,000 OVCAR-8 Human Adult Cancer Ovary Adenocarcinoma >10,000 SK-OV-3 Human Adult Metastatic Ovary Adenocarcinoma Ascites >10,000 Cancer BxPC-3 Human Adult Cancer Pancreas Adenocarcinoma >10,000 AsPC-1 Human Adult Metastatic Pancreas Adenocarcinoma Ascites >1000 Cancer NCI-H295 Human Adult Cancer Adrenal Gland, Adrenocortical >10,000 Cortex Carcinoma TT Human Adult Cancer Thyroid Medullary Carcinoma >10,000 C8-D30 Murine Adult Normal Brain, >10,000 Cerebellum LLC1 Murine Adult Cancer Lung Lewis Lung Carcinoma >10,000 RM-1 Murine Adult Cancer Prostate >10,000 MLTC-1 Murine Adult Cancer Testis Leydig Cell Tumor >10,000 KLN 205 Murine Adult Cancer Lung Squamous Cell >10,000 Carcinoma CMT-64 Murine Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) CMT-93 Murine Adult Cancer Rectum Polyploid Carcinoma >10,000 B16-F0 Murine Adult Cancer Skin Melanoma >10,000 RM-2 Murine Adult Cancer Prostate >10,000 RM-9 Murine Adult Cancer Prostate >10,000 Neuro-2A Murine Adult Cancer Brain Neuroblastoma >10,000 FBRC Bovine Fetal Eye, Retina >10,000 MDBK Bovine Adult Normal, Kidney >10,000 Immortalized CSL 503 Ovine Adult Normal, Lung Ad5E1 >10,000 Immortalized transformed OFRC Ovine Adult Normal, Eye, Retina Ad5E1 >10,000 Immortalized transformed PC-12 Rat Adult Cancer Adrenal Gland Pheochromocytoma >10,000 Vero Monkey Adult Normal, Kidney >10,000 Immortalized PAOEC Porcine Adult Normal, Heart, Aorta Endothelial Cells >10,000 Primary PCAEC Porcine Adult Normal, Heart, Coronary Endothelial Cells >10,000 Primary Artery PPAEC Porcine Adult Normal, Lung, Endothelial Cells >10,000 Primary Pulmonary Artery TBD NCI-H289 Human Adult Cancer Lung TBD NCI-H1963 Human Adult Cancer Lung Small Cell Lung TBD Carcinoma (SCLC) NCI-H2227 Human Adult Cancer Lung Small Cell Lung TBD Carcinoma (SCLC) NCI-H378 Human Adult Metastatic Lung Classic Small Cell Pleural effusion TBD Cancer Lung Carcinoma (SCLC) NCI-H2107 Human Adult Metastatic Lung Small Cell Lung Bone Marrow TBD Cancer Carcinoma (SCLC) HCC970 Human Adult Metastatic Lung Small Cell Lung Bone Marrow TBD Cancer Carcinoma (SCLC) HCC33 Human Adult Metastatic Lung Small Cell Lung Pleural effusion <1000/TBD Cancer Carcinoma (SCLC) BON Human Adult Cancer Pancreas Carcinoid TBD H1T-T15 Hamster Adult Normal, Pancreas Islets of Langerhans, TBD Immortalized b-cell *EC50 determined after 3 days except where noted
[0220] Table 1-A lists the results of SVV permissivity experiments on 165 primary cells and cell lines, representing 22 tissues from 8 different species. The results indicate that virtually all adult normal are nonpermissive for SVV. Thirteen primary adult human cell cultures tested were nonpermissive. Of the twelve bovine, ovine, porcine and primate normal cell cultures tested, only three cell cultures were permissive, which were porcine smooth muscle cells. This result is consistent with the hypothesis that the natural host for SVV may be pigs. Besides the porcine smooth muscle cells, only neuroendocrine cancer cell lines or most fetal lines were permissive.
[0221] Murine studies (see Examples) show that SVV can specifically kill tumors with great efficacy and specificity in vivo. These in vivo studies show that SVV has a number of advantages over other oncolytic viruses. For example, one important factor affecting the ability of an oncolytic tumor virus to eradicate established tumors is viral penetration. In studies with adenoviral vectors, Ad5 based vectors had no effect on SCLC tumor development in athymic mice. Based on immunohistochemical results, adenovirus did not appear to penetrate the established tumors. In contrast, SVV was able to eliminate H446 SCLC tumors in athymic nude mice following a single systemic administration. SVV has a small size (<30 nm in diameter) enabling better penetration and spread in tumor tissue than other viruses, and thus, the small size of SVV may contribute to its ability to successfully penetrate and eradicate established tumors.
[0222] Additional in vivo tests demonstrate the efficacy of a single intravenous dose of SVV in murine tumor models using athymic nude mice and immunocompetent mice. The tumor models tested were: (1) H446 (human SCLC); (2) Y79 (human retinoblastoma); (3) H69AR (human multi-drug resistant SCLC); (4) H1299 (human NSCLC); and (5) N1E-115 (murine neuroblastoma). The results of these tests are shown in FIGS. 90A-E and Example 11. The tests demonstrate efficacy of a single intravenous dose of SVV in all models and show an agreement between relative ranks of in vitro ED50 and in vivo efficacy in human xenograft models. The results in the N1E-115 immunocompetent murine neuroblastoma model shows that SVV can be efficacious against tumors in subjects with normal immune systems.
[0223] Chemoresistance is a major issue facing any patient that receives chemotherapy as a facet of cancer therapy. Patients that become chemoresistant often, if not always, have a much poorer prognosis and may be left with no alternative therapy. It is well known that one of the major causes of chemoresistance is the expression, over expression, or increased activity of a family of proteins called Multiple Drug Resistant proteins (MRPs). Applicants have found that a sensitivity of certain tumor cells for SVV is also correlated with the chemoresistant state of cancer cells and MRP expression. H69 is a chemosensitive (adriamycin) cell line that is resistant to SVV in vitro, whereas H69AR is a chemoresistant cell line that overexpresses MRPs and is sensitive to SVV (see Table 1). Evidence indicates that overexpression of MRPs, including MDR, correlates with sensitivity of cells to SVV killing. Thus, in one embodiment, the present invention provides a method for treating cancer wherein SVV kills cells overexpressing an MRP.
[0224] The invention also provides methods for treating diseases that are a result of abnormal cells, such as abnormally proliferative cells. The method comprises contacting said abnormal cells with SVV in a manner that results in the destruction of a portion or all of the abnormal cells. SVV can be used to treat a variety of diseases that are a result of abnormal cells. Examples of these diseases include, but are not limited to, cancers wherein the tumor cells display neuroendocrine features and neurofibromatosis.
[0225] Neuroendocrine tumors can be identified by a variety of methods. For example, neuroendocrine tumors produce and secrete a multitude of peptide hormones and amines. Some of these substances cause a specific clinical syndrome: carcinoid, Zollinger-Ellison, hyperglycemic, glucagonoma and WDHA syndrome. Specific markers for these syndromes are basal and/or stimulated levels of urinary 5-HIAA, serum or plasma gastrin, insulin, glucagon and vasoactive intestinal polypeptide, respectively. Some carcinoid tumors and about one third of endocrine pancreatic tumors do not present any clinical symptoms and are called `nonfunctioning` tumors. Therefore, general tumor markers such as chromogranin A, pancreatic polypeptide, serum neuron-specific enolase and subunits of glycoprotein hormones have been used for screening purposes in patients without distinct clinical hormone-related symptoms. Among these general tumor markers chromogranin A, although its precise function is not yet established, has been shown to be a very sensitive and specific serum marker for various types of neuroendocrine tumors. This is because it may also be elevated in many cases of less well-differentiated tumors of neuroendocrine origin that do not secrete known hormones. At the moment, chromogranin A is considered the best general neuroendocrine serum or plasma marker available both for diagnosis and therapeutic evaluation and is increased in 50-100% of patients with various neuroendocrine tumors. Chromogranin A serum or plasma levels reflect tumor load, and it may be an independent marker of prognosis in patients with midgut carcinoids.
[0226] The invention also provides a pharmaceutical composition comprising SVV and a pharmaceutically acceptable carrier. Such compositions, which can comprise an effective amount of SVV in a pharmaceutically acceptable carrier, are suitable for local or systemic administration to individuals in unit dosage forms, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or oral solutions or suspensions, oil in water or water in oil emulsions, and the like. Formulations for parenteral and non-parenteral drug delivery are known in the art. Compositions also include lyophilized and/or reconstituted forms of SVV. Acceptable pharmaceutical carriers are, for example, saline solution, protamine sulfate (Elkins-Sinn, Inc., Chemy Hill, N.J.), water, aqueous buffers, such as phosphate buffers and Tris buffers, or Polybrene (Sigma Chemical, St. Louis, Mo.) and phosphate-buffered saline and sucrose. The selection of a suitable pharmaceutical carrier is deemed to be apparent to those skilled in the art from the teachings contained herein. These solutions are sterile and generally free particulate matter other than SVV. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. Excipients that enhance infection of cells by SVV may be included.
[0227] SVV is administered to a host or subject in an amount that is effective to inhibit, prevent or destroy the growth of the tumor cells through replication of the virus in the tumor cells. Methods that utilize SVV for cancer therapy include systemic, regional or local delivery of the virus at safe, developable, and tolerable doses to elicit therapeutically useful destruction of tumor cells. Even following systemic administration, the therapeutic index for SVV is at least 10, preferably at least 100 or more preferably at least 1000. In general, SVV is administered in an amount of between 1×108 and 1×1014 vp/kg. The exact dosage to be administered is dependent upon a variety of factors including the age, weight, and sex of the patient, and the size and severity of the tumor being treated. The viruses may be administered one or more times, which may be dependent upon the immune response potential of the host. Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. If necessary, the immune response may be diminished by employing a variety of immunosuppressants, so as to permit repetitive administration and/or enhance replication by reducing the immune response to the viruses. Anti-neoplastic viral therapy of the present invention may be combined with other anti-neoplastic protocols. Delivery can be achieved in a variety of ways, employing liposomes, direct injection, catheters, topical application, inhalation, etc. Further, a DNA copy of the SVV genomic RNA, or portions thereof, can also be a method of delivery, where the DNA is subsequently transcribed by cells to produce SVV virus particles or particular SVV polypeptides.
[0228] A therapeutically effective dose refers to that amount of the virus that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of viruses can be determined by standard procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population of animals or cells; for viruses, the dose is in units of vp/kg) and the ED50 (the dose--vp/kg--therapeutically effective in 50% of the population of animals or cells) or the EC50 (the effective concentration--vp/cell (see Table 1 for example)--in 50% of the population of animals or cells). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50 or EC50. Viruses which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of viruses lies preferably within a range of circulating concentrations that include the ED50 or EC50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
[0229] In yet another aspect, a method for treating a host organism having a neoplastic condition is provided, comprising administering a therapeutically effective amount of a viral composition of the invention to said host organism. In one embodiment, the neoplastic tissue is abnormally proliferating, and the neoplastic tissue can be malignant tumor tissue. Preferably, the virus is distributed throughout the tissue or tumor mass due to its capacity for selective replication in the tumor tissue. Neoplastic conditions potentially amenable to treatment with the methods of the invention include those with neurotropic properties.
Methods for Producing the Viruses of the Present Invention:
[0230] Methods for producing the present viruses to very high titers and yields are additional aspects of the invention. As stated, SVV can be purified to high titer and can be produced at more than 200,000 particles per cell in permissive cell lines. Cells that are capable of producing high quantities of viruses include, but are not limited to, PER.C6 (Fallaux et al., Human Gene Therapy, 9:1909-1917, 1998), H446 (ATCC#HTB-171) and the other cell lines listed in Table 1 where the EC50 value is less than 10.
[0231] For example, the cultivation of picornaviruses can be conducted as follows. The virus of interest is plaque purified and a well-isolated plaque is picked and amplified in a permissive cell line, such as PER.C6. A crude virus lysate (CVL) from the infected cells can be made by multiple cycles of freeze and thaw, and used to infect large numbers of permissive cells. The permissive cells can be grown in various tissue culture flasks, for example, 50×150 cm2 flasks using various media, such as Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, Calif.)) containing 10% fetal bovine serum (Biowhitaker, Walkersvile, Md.) and 10 mM magnesium chloride (Sigma, St Louis, Mo.). The infected cells can be harvested between 12 and 48 hours after infection or when complete cytopathic effects (CPE) are noticed, and are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellet is resuspended in the cell culture supernatant and is subjected to multiple cycles of freeze and thaw. The resulting CVL is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus can be purified by gradient centrifugation. For example, two rounds of CsCl gradients can suffice for SVV purification: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A260=9.5×1012 particles (Scraba D. G., and Palmenberg, A. C. 1999. Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Second edition, R. G. Webster and A Granoff, Eds). Infectivity titers of purified virus are also determined by a standard plaque and/or tissue culture infective dose 50 (TCID50) assay using PER.C6 or any other suitable cell type. The yield of SVV from PER.C6 cells are greater than 200,000 particles per cell with particles to PFU ratio of about 100. The yields of SVV from other permissive cells (H446-ATCC#HTB-171) may be at least this high or higher. SVV can also be purified by column chromatography.
[0232] In addition, several steps in a commercially attractive large scale Good Manufacturing Processes (GMP) are applicable to the purification of SVV. The invention also contemplates methods for purifying SVV that are based on methods for purifying adenoviruses. These methods include isolating SVV based on its density, since SVV has a very similar density to adenovirus and can be co-purified with adenovirus.
Methods for Detecting and Studying Tumors:
[0233] The present invention provides methods for detecting tumor or neoplastic cells in a patient using the viruses of the present invention. Cellular samples can be obtained from a patient and screened by incubating the sample with an epitope-tagged SVV (or other tumor-specific viruses provided by the invention, i.e., tumor-specific mutant cardioviruses), and then screening the sample for bound SVV by detecting the epitope tag. Alternatively, the sample can be screened by detecting whether the SVV causes any cellular lysis. If SVV does cause cellular lysis, or if SVV can bind specifically to cells in the sample, this would indicate the possibility that the sample contains neoplastic or tumor cells known to be capable of being bound and/or infected by SVV.
[0234] Additionally, SVV can be used in a method for detecting a tumor cell in vivo. In such a method, epitope-tagged SVV can first be inactivated in a manner where SVV can still bind to tumor cells specifically but cannot replicate. Tumor cells that have bound SVV can be detected by assaying for the epitope tag. Detection of the epitope tag can be accomplished by antibodies that specifically bind the epitope, where the antibodies are either labeled (for example, fluorescently) or where the antibodies can then be detected by labeled secondary antibodies.
[0235] The present methods of detection encompass detecting any type of tumor or neoplastic cell that is specifically targeted by any virus of the present invention. Specific tumor types include, for example, neuroendocrine-type tumors, such as retinoblastomas, SCLC, neuroblastomas glioblastomas and medulloblastomas.
[0236] The present invention also provides the use of SVV as a tool to study tumor cells. SVV selectively destroys some tumor cell types, and has very little, if any, toxic effects on non-tumor cells. Because of these characteristics, SVV can be used to study tumors and possibly discover a new tumor specific gene and/or pathway. In other words, there is some characteristic of the tumor cells that allows replication of SVV, wherein normal cells do not exhibit said characteristic. Upon identification of a new tumor specific gene and/or pathway, therapeutic antibodies or small molecules can then be designed or screened to determine whether these reagents are anti-tumor agents.
[0237] The present invention also provides a method for identifying all types of cancers that respond to SVV. In one embodiment, the method for identifying SVV-responsive cells comprises obtaining cells, contacting said cells with SVV and detecting cell killing or detecting viral replication. Cell killing can be detected using various methods known to one skilled in the art (e.g., MTS assay, see High-Throughput section herein). Methods of detecting virus replication are also known to one skilled in the art (e.g., observance of CPE, plaque assay, DNA quantification methods, FACS to detect quantity of virus in the tumor cells, RT-PCR assays to detect viral RNA, etc.). In one embodiment, the cells are cancer cells. Examples of cancer cells include, but are not limited to, established tumor cell lines and tumor cells obtained from a mammal. In one embodiment, the mammal is a human. In a further embodiment, the cells are cancer cells obtained from a human cancer patient.
[0238] The method for identifying SVV-responsive cancer cells may be used to discover tumor cell lines or tumor tissues that are permissive for SVV replication. Also, by determining the characteristics of permissive tumor cells, one may be able to identify characteristics of tumor cells that cause the cells to be selectively killed by SVV. The discovery of these characteristics could lead to new targets for cancer drugs. Also, the methods for identifying SVV responsive cancer cells could be used as a screen for human cancer patients who would benefit from treatment with SVV.
[0239] For example, antibodies against SVV or an SVV-like picornavirus (polyclonal, monoclonal, etc.) can be used in a viral binding assay to pre-screen patients prior to SVV or SVV-like picornavirus therapy. The pre-screening can be conducted generally as follows: (1) cells from a patient are isolated, the cells can be from a tumor biopsy for example, (2) the cells are stained with anti-SVV or anti-SVV-like picornavirus antibodies, (3) a secondary antibody conjugated with a marker (such as fluoroscein or some other detectable dye or fluorophore) that is specific to the anti-SVV or anti-SVV-like picornavirus antibodies is added (for example, if the antibodies were raised in a rabbit, then the secondary antibody would be specific for rabbit immunoglobulins), and (4) detection for the marker is conducted--for example, fluorescence microscopy can be conducted where the marker is fluorescein. (Step 3 is optional if the anti-SVV or anti-SVV-like picornavirus antibodies are directly conjugated, i.e., where the antibodies are monoclonal. If the antibodies are polyclonal, indirect immunofluorescence--use of a secondary antibody--is suggested.) If the patient's tumor cells are permissive for SVV or SVV-like picornavirus infection, then the patient is a candidate for SVV or SVV-like picornavirus therapy. In a virus binding assay, the patient's tumor cells can be determined to be permissive for SVV if the cells are positive for antibody staining. For example, FIGS. 92B-92C shows immunofluorescent images of cells permissive for SVV and have been infected with SVV.
[0240] In pre-screening patients with a viral binding assay, the cell sample from the patient can also be a tissue section of a tissue suspected to contain tumor cells. The tissue section can then be prepared into sections and incubated with SVV prior to histochemistry with anti-SVV or anti-SVV-like picornavirus antibodies.
[0241] The invention also provides methods of detecting SVV. In one embodiment, the detection assay is based on antibodies specific to SVV polypeptide epitopes. In another embodiment, the detection assay is based on the hybridization of nucleic acids. In one embodiment, RNA is isolated from SVV, labeled (e.g., radioactive, chemiluminsecence, fluorescence, etc.) to make a probe. RNA is then isolated from test material, bound to nitrocellulose (or a similar or functionally equivalent substrate), probed with the labeled SVV RNA, and the amount of bound probe detected. Also, the RNA of the virus may be directly or indirectly sequenced and a PCR assay developed based on the sequences. In one embodiment, the PCR assay is a real time PCR assay.
Methods for Making Viruses with Altered Tropism:
[0242] The present invention provides methods for constructing SVV mutants (or variants or derivatives) where these mutants have an altered cell-type tropism. SVV-like picornaviruses may also be mutated in order to provide a particular cell-type tropism. Specifically, SVV and SVV-like picornavirus mutants are selected for their ability to specifically bind and/or kill tumor or neoplastic cells that are known to be resistant to wild-type SVV or wild-type SVV-like picornavirus binding.
[0243] The native or wild-type SVV has a simple genome and structure that allow the modification of the native virus to target other cancer indications. These new derivatives have an expanded tropism toward non-neural cancers and still maintain the high therapeutic index found in the native SVV. One possible means of targeting is the inclusion of tissue-specific peptides or ligands onto the virus.
[0244] To select cancer-targeting viral candidates, the present invention provides methods to construct and screen an oncolytic virus library with a genetic insertion that encodes a random peptide sequence in the capsid region of the native SVV. A random peptide library with a diversity of 108 is believed to be sufficient and should yield peptides that specifically direct the virus to tumor tissue.
[0245] Various studies have shown that tumor cells exhibit different characteristics from normal cells such as: (1) tumor cells have more permeable cell membranes; (2) tumors have specific stromal cell types such as angiogenic endothelial cells which have previously been shown to express cell type specific receptors; and (3) tumor cells differentially express certain receptors, antigens and extracellular matrix proteins (Arap, W. et al., Nat. Med., 2002, 8(2): 121-127; Kolonin, M. et al., Curr. Opin. Chem. Biol., 2001, 5(3): 308-313; St. Croix, B. et al., Science, 2000, 289(5482): 1997-1202). These studies demonstrated that tumor and normal tissues are distinct at the molecular level and targeted drug delivery and treatment of cancer is feasible. Specifically, several peptides selected by homing to blood vessels in mouse models have been used for targeted delivery of cytotoxic drugs (Arap, W. et al., Science, 1998, 279(5349): 377-380), pro-apoptotic peptides (Ellerby, H. M. et al., Nat. Med., 1999, 17(8): 768-774), metalloprotease inhibitor (Koivunen, E. et al., Nat. Biotechnol, 1999, 17(8): 768-774), cytokine (Curnis, F. et al., Nat. Biotechnol., 2000, 18(11): 1185-1190), fluorophores (Hong. F. D. and Clayman, G. L., Cancer Res., 2000, 60(23): 6551-6556) and genes (Trepel, M. et al., Hum. Gene Ther., 2000, 11(14): 1971-1981). The tumor-targeting peptides have proven to increase the efficacy and lower the toxicity of the parental drugs.
[0246] A library of SVV derivatives can be generated by the insertion of a random peptide sequence into the capsid region of the virus. As shown in FIG. 57, a vector is first generated that contains the SVV capsid region, i.e., "pSVV capsid." This capsid vector can then be mutagenized, for example, by cutting the vector with a restriction enzyme that cuts DNA at random positions, i.e., CviJI (a blunt cutter). The vector is cut at numerous positions, and DNA that has been cut only once by CviJI can be isolated by gel-purification (see FIG. 57). This isolated population of DNA contains a plurality of species that have been cut in the capsid region at different locations. This population is then incubated with oligonucleotides and ligase, such that a percentage of the oligonucleotides will be ligated into the capsid region of the vector at a number of different positions. In this manner, a library of mutant SVV capsids can be generated.
[0247] The oligonucleotides that are inserted into the capsid encoding region can be either random oligonucleotides, non-random oligonucleotides (i.e., the sequence of the oligonucleotide is pre-determined), or semi-random (i.e., a portion of the oligonucleotide is pre-determined and a portion has a random sequence). The non-random aspect of the contemplated oligonucleotides can comprise an epitope-encoding region. Contemplated epitopes include, but are not limited to, c-myc--a 10 amino acid segment of the human protooncogene myc (EQKLISEEDL (SEQ ID NO: 35); HA--haemoglutinin protein from human influenza hemagglutinin protein (YPYDVPDYA (SEQ ID NO: 36)); and His6 (SEQ ID NO:116)--a sequence encoding for six consecutive histidines.
[0248] The library of mutant capsid polynucleotides (for example, "pSVV capsid library" in FIG. 57) can then be digested with restriction enzymes such that only the mutant capsid encoding region is excised. This mutant capsid encoding region is then ligated into a vector containing the full-length genomic sequence minus the capsid encoding region (see FIG. 58, for example). This ligation generates a vector having a full-length genomic sequence, where the population (or library) of vectors comprise a plurality of mutant capsids. In FIG. 58, this library of SVV mutants comprising different capsids is denoted as "pSVVFL capsid." The pSVVFL capsid vector library is then linearized and in vitro transcribed in order to generate mutant SVV RNA (see FIG. 59). The mutant SVV RNA is then transfected into a permissive cell line such that those SVV sequences that do not possess a debilitating mutation in its capsid are translated by the host cells to produce a plurality of mutant SVV particles. In FIG. 59, the plurality of mutant SVV particles are denoted as a "SVV capsid library."
[0249] The peptide encoded by the oligonucleotide inserted into the capsid encoding region can serve as a targeting moiety for specific viral infection. The viruses that target a specific type of cancer would selectively infect only those cancer cells that have a receptor to the peptide, replicate in those cells, kill those cells, and spread to only those same types of cells. This methodology enables the identification of novel tumor-targeting peptides and ligands, tumor-selective receptors, therapeutic SVV derivatives and other virus derivatives, including picornavirus derivatives.
[0250] In vitro and in vivo screening of SVV mutant libraries have several advantages over other technologies such as peptide bead libraries and phage display. Unlike these other technologies, the desirable candidate here, i.e. an SVV derivative that selectively binds to a cancer cell, will replicate in situ. This replication-based library approach has numerous advantages over prior methods of discovering new cell binding moieties, such as phage display. First, the screening of a SVV library is based on replication. Only the desired viral derivatives can replicate in the target tissue, in this case certain cancer cells. The screening/selection process will yield very specific viral candidates that have both the targeting peptide moiety and may be a cancer therapeutic itself. On the contrary, phage display screens will only result in binding events and yields only the targeting peptide candidates. Thus, SVV library screening offers a much faster and selective approach. Second, during in vitro or in vivo phage display screening, phages recovered from the target cells have to be amplified in bacteria, while SVV derivatives can be directly recovered and purified from infected cells (or from the culture supernatant of lytically infected cells). Third, SVV has a smaller genome that renders easier manipulability; thus it is possible to randomly insert the genetic information into the capsid region to ensure an optimized insertion. Therefore, construction and screening of the SVV library has a high possibility to produce highly effective viral derivatives. These derivatives are designed and screened to specifically infect cancers with non-neural properties.
[0251] The insertion of oligonucleotides into the capsid encoding region will result in the generation of some defective mutants. Mutants may be defective in the sense that the insertion of an oligonucleotide sequence can result in a stop codon, such that the viral polyprotein will not be produced. Also, mutants may be defective in the sense that the insertion of an oligonucleotide sequence may result in the alteration of the capsid structure such that capsid can no longer be assembled. To decrease the probability that the insertion of oligonucleotide sequences may result in stop codon or untenable capsid structure, random oligonucleotides can be designed such that they do not encode for stop codons or for certain amino acids using methods such as TRIM.
[0252] To determine whether there is an optimal insertion point in the capsid region for oligonucleotides, one can generate an RGD-SVV library (see Example 16). The polynucleotide encoding the SVV capsid is randomly cut, for example, with CviJI. The randomly linearized capsid polynucleotides are then ligated to oligonucleotides encoding at least the RGD amino acid sequence (arginine-glycine-aspartic acid). These RGD-capsid sequences are then ligated into SVV full-length sequence vectors that are missing the capsid sequence. RGD-SVV derivatives viruses are produced and tested for their ability to infect and replicate in certain integrin-expressing cell lines (as the RGD peptide has been shown to target entities to integrin receptors). The RGD-SVV derivatives that are successful in infecting the integrin-expressing cell lines are then analyzed to determine whether there is a predominant insertion site for the RGD oligonucleotide. This site can then be used for site-directed insertion of random, non-random or semi-random oligonucleotides.
[0253] Further, in comparing portions of the capsid encoding region between SVV and other picornaviruses (see FIG. 28), there are various non-boxed regions between the viruses where the sequence similarity is at its lowest. These regions may be important in contributing to the different tropisms between the viruses. Thus, these regions may be candidate locations for oligonucleotide insertion mutagenesis of the SVV capsid (and for other viral capsids).
Inactivated SVV as a Tumor-Specific Therapeutic:
[0254] Since SVV and SVV-capsid derivatives can target specific tumor cell-types and/or tissues, the SVV particle itself can be used as a delivery vehicle for therapeutics. In such a method, the need for the oncolytic abilities of SVV becomes optional, as the delivered therapeutic can kill the targeted tumor cell.
[0255] For example, the wild-type SVV can be inactivated such that the virus no longer lyses infected cells, but where the virus can still specifically bind and enter targeted tumor cell-types. There are many standard methods known in the art to inactivate the replicative functions of viruses. For example, whole virus vaccines are inactivated by formalin or β-propiolactone such that the viruses cannot replicate. The wild-type SVV may itself contain peptides that cause the apoptosis of cells. Alternatively, SVV can be irradiated. However, irradiated viruses should first be tested to ensure that they are still capable of specifically targeting tumor cells, as certain irradiation conditions may cause protein, and thus capsid, alterations. Further, mutant SVVs can be generated where the packaging signal sequence is deleted. These SVV mutants are able to specifically bind and enter target cells, but replicated SVV genomic RNA will not be packaged and assembled into capsids. However, this method may prove to be useful as initial entry of these mutant SVVs will cause host-protein synthesis shut-off such that tumor-cell death is still achieved.
[0256] Derivative SVVs having mutant capsids can also be inactivated and used to kill cancer cells. Derivative SVVs with oligonucleotides encoding epitope tags inserted into the capsid region can be used as vehicles to deliver toxins to tumor cells. As described herein, derivative SVVs can be randomly mutagenized and screened for tumor-specific tropisms. Toxins can be attached to the epitope tags, such that the virus delivers the toxin to tumor cells. Alternatively, therapeutic antibodies that specifically bind to the epitope tag can be used, such that the virus delivers the therapeutic antibody to the tumor cell.
High-Throughput Screening:
[0257] The present invention encompasses high-throughput methods for screening viruses that have the ability to specifically infect different cell-lines. The specificity of infection can be detected by assaying for cytopathic effects. For example, a number of different tumor cell-lines can be grown in different wells of a multi-well plate that is amenable for high-throughput screening, for example a 384 well-plate. To each well, a sample of virus is added to test whether the cells are killed by virus-mediated lysis. From those wells that show cytopathic effects, the media is collected such that any viruses in the media can be amplified by infecting permissive cell lines in flasks or large tissue culture plates. The viruses are grown such that the RNA can be isolated and the sequence analyzed to determine sequence mutations that may be responsible for providing a tumor cell-type specific tropism for a virus.
[0258] Various colorimetric and fluorometric methods can quickly assay cytopathic effects, including fluorescent-dye based assays, ATP-based assays, MTS assays and LDH assays. Fluorescent-dye based assays can include nucleic acid stains to detect dead-cell populations, as cell-impermeant nucleic acid stains can specifically detect dead-cell populations. If it is desired to simultaneously detect both live-cell and dead-cell populations, nucleic acid stains can be used in combination with intracellular esterase substrates, membrane-permeant nucleic acid stains, membrane potential-sensitive probes, organelle probes or other cell-permeant indicators to detect the live-cell population. For example, Invitrogen (Carlsbad, Calif.) offers various SYTOX® nucleic acid stains that only penetrate cells with compromised plasma membranes. Ethidium bromide and propidium iodide can also be used to detect dead or dying cells. These stains are high-affinity nucleic acid stains that can be detected by any light-absorbance reader
[0259] For example, lysis can be based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant. To detect the presence of LDH in cell culture supernatants, a substrate mixture can be added such that LDH will reduce the tetrazolium salt INT to formazan by a coupled enzymatic reaction. The formazan dye can then be detected by a light-absorbance reader. Alternatively, an MTS assay [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- -2H-tetrazolium, inner salt] using phenzine methosulfate (PMS) as the electron coupling reagent can also be used to detect cytotoxicity. Promega (Madison, Wis.) offers a CellTiter 96® AQueous One Solution Cell Proliferation Assay kit where the solution reagent is added directly to culture wells, incubated for 1-4 hours and then absorbance is recorded at 490 nm. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.
[0260] There are numerous high-throughput devices for reading light-absorbance. For example, SpectraMax Plus 384 Absorbance Platereader (Molecular Devices) can detect wavelengths from 190-1000 nm in 1 nm increments. The device can read 96-well microplates in 5 seconds and 384-well microplates in 16 seconds for ultra fast sample throughput.
[0261] Virus replication can also be assayed as an indication of successful infection, and such detection methods can be used in a high-throughput manner. For example, real-time RT-PCR methods can be used to detect the presence of virus transcripts in cell-culture supernatants. Upon reverse-transcription of viral RNA into cDNA, the cDNA can be amplified and detected by PCR with the use of double-stranded DNA-binding dyes (for example, SYBR® Green, Qiagen GmbH, Germany). The amount of PCR product can then be directly measured using a fluorimeter.
[0262] Viruses from the wells showing cytopathic effects are grown up and tested in further in vitro (re-testing of tumor and normal cell lines) and in vivo models (testing whether the virus can kill explanted tumors in mice).
Antibodies:
[0263] The present invention is also directed to antibodies that specifically bind to the viruses of the present invention, including the proteins of the viruses. Antibodies of the present invention include naturally occurring as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (Huse et al., Science 246:1275-1281, 1989). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual, Cold Spring Harbor Laboratory Press, 1988); Hilyard et al., Protein Engineering: A practical approach, IRL Press 1992; Borrabeck, Antibody Engineering, 2d ed., Oxford University Press 1995). Antibodies of the invention include intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding to an epitopic determinant present in a polypeptide of the present invention.
[0264] Where a peptide portion of a SVV polypeptide of the invention (i.e., any peptide fragment from SEQ ID NO:2 or SEQ ID NO:169) or peptide portion of another viral polypeptide of the invention used as an immunogen for antibody generation is non-immunogenic, it can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein. Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (for example, by Harlow and Lane, supra, 1988). Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (see, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols, Manson, ed., Humana Press 1992, pages 1-5; Coligan et al., "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters," in Curr. Protocols Immunol. (1992), section 2.4.1).
[0265] Monoclonal antibodies also can be obtained using methods that are well known and routine in the art (Kohler and Milstein, Nature 256:495, 1975; Coligan et al., supra, 1992, sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988). For example, spleen cells from a mouse immunized with a virus, viral polypeptide or fragment thereof, can be fused to an appropriate myeloma cell line to produce hybridoma cells. Cloned hybridoma cell lines can be screened using, for example, labeled SVV polypeptide to identify clones that secrete monoclonal antibodies having the appropriate specificity, and hybridomas expressing antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies. Polyclonal antibodies similarly can be isolated, for example, from serum of an immunized animal. Such antibodies, in addition to being useful for performing a method of the invention, also are useful, for example, for preparing standardized kits. A recombinant phage that expresses, for example, a single chain antibody also provides an antibody that can be used for preparing standardized kits. Monoclonal antibodies, for example, can be isolated and purified from hybridoma cultures by a variety of well established techniques, including, for example, affinity chromatography with Protein-A SEPHAROSE gel, size exclusion chromatography, and ion exchange chromatography (Barnes et al., in Meth. Mol. Biol. 10:79-104, Humana Press 1992); Coligan et al., supra, 1992, see sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3).
[0266] An antigen-binding fragment of an antibody can be prepared by proteolytic hydrolysis of a particular antibody, or by expression of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol-reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see, for example, Goldenberg, U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647; Nisonhoff et al., Arch. Biochem. Biophys. 89:230. 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Meth. Enzymol., 1:422 (Academic Press 1967); Coligan et al., supra, 1992, see sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
[0267] Another example of an antigen binding fragment of an antibody is a peptide coding for a single complementarity determining region (CDR). CDR peptides can be obtained by constructing polynucleotides encoding the CDR of an antibody of interest. Such polynucleotides can be prepared, for example, using the polymerase chain reaction to synthesize a variable region encoded by RNA obtained from antibody-producing cells (for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991).
[0268] The antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
[0269] There are many different labels and methods of labeling antibodies known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or alternatively to the antigen, or will be able to ascertain such, using routine experimentation.
[0270] As various changes can be made in the above methods and compositions without departing from the scope and spirit of the invention as described, it is intended that all subject matter contained in the above description, shown in the accompanying drawings, or defined in the appended claims be interpreted as illustrative, and not in a limiting sense.
EXAMPLES
[0271] The examples described below are provided to illustrate the present invention and are not included for the purpose of limiting the invention.
Example 1
Amplification and Purification of Virus
[0272] Cultivation of SVV in PER.C6 cells: SVV is plaque purified once and a well isolated plaque is picked and amplified in PER.C6 cells (Fallaux et al., 1998). A crude virus lysate (CVL) from SVV infected PER.C6 cells is made by three cycles of freeze and thaw and used to infect PER.C6 cells. PER.C6 cells are grown in 50×150 cm2 T.C. flasks using Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, Calif., USA)) containing 10% fetal bovine serum (Biowhitaker, Walkersvile, Md., USA) and 10 mM magnesium chloride (Sigma, St Louis, Mo., USA). The infected cells harvested 30 hr after infection when complete CPE is noticed and are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellet is resuspended in the cell culture supernatant (30 ml) and is subjected to three cycles of freeze and thaw. The resulting CVL is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus is purified by two rounds of CsCl gradients: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A260=9.5×1012 particles (Scraba D. G., and Palmenberg, A. C. 1999. Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Second edition, R. G. Webster and A Granoff, Eds). Titers of purified virus are also determined by a standard plaque assay using PER.C6 cells. The yield of SVV from PER.C6 cells are greater than 200, 000 particles per cell with particles to PFU ratio of about 100. The yields of SVV from other permissive cells (H446-ATCC# HTB-171) may be at least this high or higher.
Example 2
Electron Microscopy
[0273] SVV is mounted onto formvar carbon-coated grids using the direct application method, stained with uranyl acetate, and examined in a transmission electron microscope. Representative micrographs of the virus are taken at high magnification. For the transmission electron microscope, ultra-thin sections of SVV-infected PER.C6 cells are cut from the embedded blocks, and the resulting sections are examined in the transmission electron microscope.
[0274] The purified SVV particles are spherical and about 27 nm in diameter, appearing singly or in small aggregates on the grid. A representative picture of SVV is shown in FIG. 2. In some places, broken viral particles and empty capsids with stain penetration are also seen. Ultrastructural studies of infected PER.C6 cells revealed crystalline inclusions in the cytoplasm. A representative picture of PER.C6 cells infected with SVV is shown in FIG. 3. The virus infected cells revealed a few large vesicular bodies (empty vesicles).
Example 3
Nucleic Acid Isolation of SVV
[0275] RNA Isolation: SVV genomic RNA was extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen). Isolation was performed according to the supplier's recommendations. Briefly, 250 μl of the purified SVV was mixed with 3 volumes TRIZOL and 240 μl of chloroform. The aqueous phase containing RNA was precipitated with 600 μl isopropanol. The RNA pellet was washed twice with 70% ethanol, dried and dissolved in DEPC-treated water. The quantity of RNA extracted was estimated by optical density measurements at 260 nm. An aliquot of RNA was resolved through a 1.25% denaturing agarose gel (Cambrex Bio Sciences Rockland Inc., Rockland, Me. USA) and the band was visualized by ethidium bromide staining and photographed (FIG. 4).
[0276] cDNA synthesis: cDNA of the SVV genome was synthesized by RT-PCR. Synthesis of cDNA was performed under standard conditions using 1 μg of RNA, AMV reverse transcriptase, and random 14-mer oligonucleotide or oligo-dT. Fragments of the cDNA were amplified, cloned into plasmids and the clones are sequenced.
Example 4
SVV Sequence Analysis and Epidemiology
[0277] Part I: SVV SEQ ID NO:1
[0278] The nucleotide sequence of SVV SEQ ID NO:1 was analyzed to determine its evolutionary relationship to other viruses. The translated product (SEQ ID NO:2) for this ORF was picornavirus-like and reached from the middle of VP2 to the termination codon at the end of the 3D polymerase and was 1890 amino acids in length (FIG. 5A-5E and 7A-7B). The 3' untranslated region (UTR), nucleotides 5671-5734, which follows the ORF is 64 nucleotides (nt) in length, including the termination codon and excluding the poly(A) tail of which 18 residues are provided (FIG. 5E).
[0279] Preliminary comparisons (not shown) of three partial genome segments of SVV had revealed that SVV was most closely related members of the genus Cardiovirus (family Picornaviridae). Therefore an alignment of the polyprotein sequences of SVV, encephalomyocarditis virus (EMCV; species Encephalomyocarditis virus, Theiler's murine encephalomyelitis virus (TMEV; species Theilovirus), Vilyuisk human encephalomyelitis virus (VHEV; species Theilovirus) and a rat TMEV-like agent (TLV; species Theilovirus) was constructed (FIG. 28). From this alignment, the SVV polyprotein processing was compared to the polyprotein processing of the most closely related members of the Cardiovirus genus. Cleavage sites between the individual polypeptides is demarcated by the "I" character in FIG. 28.
[0280] In picornaviruses, most polyprotein cleavages are carried out by one or more virus-encoded proteases, although in cardio-, aphtho-, erbo- and teschoviruses the cleavage between P1-2A and 2B is carried out by a poorly understood cis-acting mechanism related to the 2A sequence itself and critically involving the sequence "NPG/P", where "I" represents the break between the 2A and 2B polypeptides (Donnelly et al., 1997, J. Gen. Virol. 78: 13-21). One of the parechoviruses, Ljungan virus, has this sequence (NPGP) present upstream of a typical parechovirus 2A and is either an additional 2A or is the C-terminal end of the P1 capsid region. In all nine currently recognised picornavirus genera, 3Cpro carries out all but the cis-acting self-cleaving reactions (i.e. 2A cleaves at its N-terminus in entero- and rhinoviruses and L cleaves at its C-terminus inaphthoviruses and erboviruses). The post-assembly cleavage of the capsid polypeptide VP0 to VP4 and VP2 is not carried out by 3Cpro, but by an unknown mechanism which may involve the virus RNA. The VP0 cleavage does not occur in parechoviruses and kobuviruses. The normal cardiovirus 3Cpro cleavage site has either a glutamine (Q) or glutamate (E) at the -1 position and glycine (G), serine (S), adenine (A) or asparagine (N) at the +1 position (Table 2). The cleavages of the SVV polyprotein conform to this pattern except for the VP3/VP1 site which is histidine (H)/serine (S) (Table 2); however, H/S is probably present as the cleavage site between 3A and 3B.sup.VPg in at least one strain of equine rhinitis A virus (ERAV; genus Aphthovirus) (Wutz et al., 1996, J. Gen. Virol. 77:1719-1730).
TABLE-US-00004 TABLE 2 Cleavage sites of SVV and cardioviruses Between SVV EMCV TMEV Rat TLV VHEV L VP4 Not LQ/GN PQ/GN PQ/GN PQ/GN known (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 125) 138) 152) 163) VP4 VP2 Not LA/DQ LL/DQ LL/DQ LL/DE known (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 126) 139) 153) 164) LM/DQ (SEQ ID NO: 140) VP2 VP3 EQ/GP RQ/SP AQ/SP PQ/SP PQ/SP (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: ID NO: 117) 127) 141) 154) 165) VP3 VP1 FH/ST PQ/GV PQ/GV PQ/GV PQ/GV (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: ID NO: 118) 128) 142) 155) 166) PQ/GI (SEQ ID NO: 143) PQ/GS (SEQ ID NO: 144) VP1 2A KQ/KM LE/SP LE/NP LQ/NP LE/NP (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: ID NO: 119) 129) 145) 156) 167) 2A 2B NPG/P* NPG/P* NPG/P* NPG/P* Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 111) 130) 146) 157) 2B 2C MQ/GP QQ/SP PQ/GP AQ/SP Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 120) 131) 147) 158) 2C 3A LQ/SP AQ/GP AQ/SP AQ/SP Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 121) 132) 148) 159) AQ/AP (SEQ ID NO: 133) 3A 3B SE/NA EQ/GP EQ/AA EQ/AA Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 122) 134) 149) 160) 3B 3C MQ/QP IQ/GP IQ/GG IQ/GG Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 123) 135) 150) 161) VQ/GP (SEQ ID NO: 136) 3C 3D MQ/GL PQ/GA PQ/GA PQ/GA Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 124) 137) 151) 162) *,the break between 2A and 2B is not a cleavage event
[0281] Primary cleavages (P1/P2 and P2/P3) of SVV: These primary cleavage events are predicted to occur in a similar fashion to cardio-, aphtho-, erbo- and teschoviruses, involving separation of P1-2A from 2B by a novel mechanism involving the sequence NPG/P (SEQ ID NO:111) and a traditional cleavage event by 3Cpro between 2BC and P3 (Table 2).
[0282] P1 cleavages: Cleavages within the SVV P1 capsid coding region were relatively easy to predict by alignment with sequence with EMCV and TMEV (Table 2).
[0283] P2 cleavages: The 2C protein is involved in RNA synthesis. The 2C polypeptide of SVV contains NTP-binding motifs GxxGxGKS/T (SEQ ID NO:112) (domain A) and hyhyhyxxD (in which by is any hydrophobic residue; domain B) present in putative helicases and all picornavirus 2Cs (FIG. 29).
[0284] P3 cleavages: Prediction of the P3 cleavage sites was also relatively straightforward. Little is known about the function of the 3A polypeptide. However, all picornavirus 3A proteins contain a putative transmembrane alpha-helix. Primary sequence identity is low in this protein between SVV and cardioviruses (See FIG. 28 between positions 1612 to 1701).
[0285] The genome-linked polypeptide, VPg, which is encoded by the 3B region, shares few amino acids in common with the other cardioviruses, however, the third residue is a tyrosine, consistent with its linkage to the 5' end of the virus genome (Rothberg et al., 1978). See FIG. 28 between positions 1703 and 1724.
[0286] The three-dimensional structure of four picornavirus 3C cysteine proteases have been solved and the active-site residues identified (HAV, Allaire et al., 1994, Nature, 369: 72-76; Bergmann et al., 1997, J. Virol., 71: 2436-2448; PV-1, Mosimann et al., 1997, J. Mol. Biol., 273: 1032-1047; HRV-14, Matthews et al., 1994, Cell, 77: 761-771; and HRV-2, Matthews et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 11000-11007). The cysteine bolded in FIG. 29 is the nucleophile, while the first bolded histidine is the general base and the specificity for glutamine residues is defined mainly by the second bolded histidine; all three residues are conserved in the SVV sequence (FIG. 29) and all other known picornaviruses (FIG. 28; for 3C sequence comparison see between positions 1726 and 1946).
[0287] The 3D polypeptide is the major component of the RNA-dependent RNA polymerase and SVV contains motifs conserved in picorna-like virus RNA-dependent RNA polymerases, i.e. KDEL/IR (SEQ ID NO:113), PSG, YGDD (SEQ ID NO:114) and FLKR (SEQ ID NO:115) (FIG. 3; FIG. 28 between positions 1948 and 2410).
[0288] Myristoylation of the N-terminus of P1: In most picornaviruses the P1 precursor polypeptide is covalently bound by its N-terminal glycine residue (when present the N-terminal methionine is removed) to a molecule of myristic acid via an amide linkage (Chow et al., 1987, Nature, 327: 482-486). Consequently the cleavage products VP0 and VP4 which contain the P1 N-terminus are also myristoylated. This myristoylation is carried out by myristoyl transferase which recognises an eight amino acid signal beginning with glycine. In picornaviruses, a five residue consensus sequence motif, G-x-x-x-T/S, has been identified (Palmenberg, 1989, In Molecular Aspects of Picornavirus Infection and Detection, pp. 211-241, Ed. Semler & Ehrenfeld, Washington D.C., Amer. Soc. for Micro.). Parechoviruses (Human parechovirus and Ljungan virus) as well as not having a maturation cleavage of VP0 are apparently not myristoylated, however, there appears to be some type of molecule blocking the N-terminus of VP0 for these viruses.
Comparisons of the Individual SVV Polypeptides with the Public Sequence Databases
[0289] Each of the SVV polypeptides (SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22) were compared to the public protein sequence databases using the FASTA online program at the European Bioinformatics Institute (EBI; http://www.ebi.ac.uk/). The results (best matches) of these comparisons are shown in Table 3. The capsid polypeptides (VP2, VP3 and VP1) taken as a whole, along with 2C, 3Cpro and 3Dpol are most closely related to members of the cardiovirus genus, however, the short predicted 2A sequence is closer to that of Ljungan virus (genus Parechovirus). A more detailed comparison of the SVV 2A nucleotide sequence with similar sequences is shown in FIG. 28 (see also FIG. 70 for 2A-like NPG/P protein comparison).
TABLE-US-00005 TABLE 3 Database matches of individual predicted polypeptides of Seneca Valley virus SVV Length % % identity Matched polypeptide (aa) identity ungapped aa overlap Organism protein L (Leader) No data -- -- -- -- -- VP4 (1A) No data -- -- -- -- -- VP2 (1B) >142 42.857 44.037 112 TMEV WW VP2 ~51 -- ~80 EMCV BEL-2887A/91 VP2 VP3 (1C) 239 44.068 46.637 236 EMCV ATCC VR-129B VP3 VP1 (1D) 259 31.086 36.404 267 EMCV M100/1/02 VP1 2A 14 71.429 71.429 14 Ljungan virus 174F 2A1 2B 128 39.286 41.509 56 Ureaplasma urealyticum Multiple banded antigen 2C 322 38.602 40.190 329 EMCV PV21 2C 3A 90 37.838 41.791 74 Chlorobium tepidum TLS* Enolase 2† 3B.sup.VPg 22 No -- -- -- -- matches 3Cpro 211 37.089 38.537 213 EMCV-R 3C protease 3Dpol 462 58.009 58.515 462 EMCV-PV21 3D polymerase *a photosynthetic, anaerobic, green-sulfur bacterium †2-phosphoglycerate dehydratase 2) (2-phospho-D-glycerate hydro-lyase 2
[0290] The significance of the matches of SVV 2B with Ureaplasma urealyticum multiple banded antigen or 3A with Chlorobium tepidum endolase 2 is not clear, however, these relationships maybe worthy of further investigation.
Phylogenetic Comparison of SVV Polypeptides with Other Picornaviruses
[0291] Those SVV polypeptides which could be aligned with the cardioviruses (VP2, VP3, VP1, 2C, 3C and 3D) were compared with the same proteins of representative members of each of the picornavirus species (Table 4). The programs BioEdit v5.0.9 (Hall, 1999, Nucl. Acids. Symp. Ser., 41: 95-98) and Clustal X v1.83 (Thompson et al., 1997, Nucl. Acids Res., 25:4876-4882) were used to make the alignments and to construct distance matrices and unrooted Neighbor-joining trees according to the algorithm of Saitou and Nei (Satiou and Nei, 1987, Mol. Biol. Evol., 4: 406-425). Confidence limits on branches were accessed by bootstrap resampling (1000 pseudo-replicates). The trees were drawn using TreeView 1.6.6 (Page, 1996) (FIGS. 31 to 37). The distance matrices used to construct the trees used values corrected for multiple substitutions, while FIGS. 38-44 show the actual percentage amino acid identities. Table 4 shows the current classification of the family Picornaviridae and the representative virus sequences used in these comparisons.
TABLE-US-00006 TABLE 4 The taxonomic classification of the picornaviruses used in the comparisons with SVV. Genus Species Representative virus Abbrev. Acc. No. Enterovirus Poliovirus Poliovirus 1 PV-1 V01149 Human enterovirus A Coxsackievirus A16 CV-A16 U05876 Human enterovirus B Coxsackievirus B5 CV-B5 X67706 Human enterovirus C Coxsackievirus A21 CV-A21 D00538 Human enterovirus D Enterovirus 70 EV-70 D00820 Simian enterovirus A Simian enterovirus A1 SEV-A AF201894 Bovine enterovirus Bovine enterovirus 1 BEV-1 D00214 Porcine enterovirus B Porcine enterovirus 9 PEV-9 AF363453 New genus? Not yet designated Simian virus 2* SV2 AY064708 Porcine enterovirus A Porcine enterovirus 8* PEV-8 AF406813 Rhinovirus Human rhinovirus A Human rhinovirus 2 HRV-2 X02316 Human rhinovirus B Human rhinovirus 14 HRV-14 K02121 Cardiovirus Encephalomyocarditis virus Encephalomyocarditis virus EMCV M81861 Theilovirus Theiler's murine encephalomyelitis TMEV M20562 virus Aphthovirus Foot-and-mouth disease virus Foot-and-mouth disease virus O FMDV-O X00871 Equine rhinitis A virus Equine rhinitis A virus ERAV X96870 Hepatovirus Hepatitis A virus Hepatitis A virus HAV M14707 Avian encephalomyelitis-like Avian encephalomyelitis virus AEV AJ225173 viruses Parechovirus Human parechovirus Human parechovirus 1 HPeV-1 L02971 Ljungan virus Ljungan virus LV AF327920 Kobuvirus Aichi virus Aichi virus AiV AB040749 Bovine kobuvirus Bovine kobuvirus BKV AB084788 Erbovirus Equine rhinitis B virus Equine rhinitis B virus 1 ERBV-1 X96871 Teschovirus Porcine teschovirus Porcine teschovirus 1 PTV-1 AJ011380 *the current taxonomic status of SV2 and PEV-8 places them in the enterovirus genus, however, it has been suggested that they may be reclassified in a new genus (Krumbholz et al., 2002; Oberste et al., 2003).
[0292] The trees of the individual capsid proteins (FIGS. 31 to 33) are not all representative of the tree produced when the data from all tree polypeptides is combined (FIG. 34). This is probably the result of difficulties in aligning the capsid polypeptides, particularly when they are not full length as is the case for VP2 (FIG. 31). However, the P1, 2C, 3Cpro and 3Dpol trees are all in agreement and show that SVV clusters with EMCV and TMEV.
Seneca Valley Virus as a Member of the Cardiovirus Genus
[0293] Clearly the 3Dpol of SVV is related to the cardioviruses, almost as closely as EMCV and TMEV are to each other (FIG. 37; FIG. 44). In the other polypeptides which are generally considered as being relatively conserved in picornaviruses, 2C and 3C, SVV is also most closely related to the cardioviruses although it is not as closely related to EMCV and TMEV as they are to each other (FIG. 42 and FIG. 43, respectively). In the outer capsid proteins (taken as a whole), SVV is also most closely related to the cardioviruses and has approximately the same relationship as the two aphthovirus species, Foot-and-mouth disease virus and Equine rhinitis A virus (˜33%). SVV diverges greatly from the cardioviruses in the 2B and 3A polypeptides and has no detectable relationship with any known picornavirus. However, this is not without precedent; avian encephalomyelitis virus differs considerably from hepatitis A virus (HAV) in 2A, 2B and 3A (Marvil et al., 1999, J. Gen. Virol., 80:653-662) but is tentatively classified within the genus Hepatovirus along with HAV.
[0294] Seneca Valley virus is clearly not a typical cardiovirus if EMCV and TMEV are taken as the standard. However, even these two viruses have their differences, notably in the 5' UTR (Pevear et al., 1987, J. Gen. Virol., 61: 1507-1516). However, phylogenetically SVV clusters with EMCV and TMEV in much of its polyprotein (P1, 2C, 3Cpro and 3Dpol regions). Ultimately, the taxonomic position of SVV within the Picornaviridae will be decided by the Executive Committee (EC) of the International Committee for the Taxonomy of Viruses (ICTV) following recommendations by the Picornaviridae Study Group and supporting published material. There are two options: i) include SVV as a new species in the cardiovirus genus; or ii) assign SVV to a new genus.
[0295] Part II: SVV SEQ ID NO:168
[0296] The full-length genome of SVV (FIGS. 83A-83H; SEQ ID NO:168; Example 15) allowed further epidemiological studies. The results of the further epidemiological studies are shown in FIG. 86, where SVV is shown to be genetically related to cardioviruses such as EMCV and TMEV, but in a separate tree.
[0297] The features of the SVV full-length genome with respect to its untranslated and coding regions are listed at Table A supra. The features of the full-length SVV in comparison to EMCV and TMEV-GDVII are listed in the table below.
TABLE-US-00007 EMCV EMCV TMEV-GDVII TMEV-GDVII SVV SVV [M81861] [M81861] [M20562] [M20562] Feature nt length aa length nt length aa length nt length aa length 5 `UTR 666 -- 833 -- 1068 -- Leader 237 79 201 67 228 76 VP4 213 71 210 70 213 71 VP2 852 284 768 256 801 267 VP3 717 239 693 231 696 232 VP1 792 264 831 277 828 276 2A 27 9 429 143 426 142 2B 384 128 450 150 381 127 2C 966 399 975 325 978 326 3A 270 90 264 88 264 88 3B 66 22 60 20 60 20 3D 1386 462 1380 460 1383 461 3` UTR 71 -- 126 -- 128 --
[0298] The cleavage sites of SVV (based on full-length sequence, see also bolded amino acids between at protein boundaries in FIGS. 83A-83H) are compared to the cleavage sites of other cardioviruses in the table below.
TABLE-US-00008 Between SVV EMCV TMEV Rat TLV VHEV L VP4 LQ/GN LQ/GN PQ/GN PQ/GN PQ/GN (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 192) NO: 192) NO: l93) NO: 193) NO: 193) VP4 VP2 LK/DH LA/DQ LL/DQ LL/DQ LL/DE (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 194) NO: 195) NO: 196) NO: 196) NO: 198) LM/DQ (SEQ ID NO: 197) VP2 VP3 EQ/GP RQ/SP AQ/SP PQ/SP PQ/SP (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 117) NO: 199) NO: 200) NO: 201) NO :201) VP3 VP1 FH/ST PQ/GV PQ/GV PQ/GV PQ/GV (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 118) NO: 202) NO: 202) NO: 202) NO: 202) PQ/GI (SEQ ID NO: 203) PQ/GS (SEQ ID NO: 204) VP1 2A MQ/SG LE/SP LE/NP LQ/NP LE/NP (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 205) NO: 206) NO: 207) NO: 208) NO: 207) 2A 2B NPG/P* NPG/P* NPG/P* NPG/P* unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 111) NO: 111) NO: 111) NO: 111) 2B 2C MQ/GP QQ/SP PQ/GP AQ/SP unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 120) NO: 209) NO: 210) NO: 200) 2C 3A LQ/SP AQ/GP AQ/SP AQ/SP unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 121) NO: 211) NO: 200) NO: 200) AQ/AP (SEQ ID NO: 212) 3A 3B SE/NA EQ/GP EQ/AA EQ/AA unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 122) NO: 213) NO: 214) NO: 214) 3B 3C MQ/QP IQ/GP IQ/GG IQ/GG unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 123) NO: 215) NO: 217) NO: 217) VQ/GP (SEQ ID NO: 216) 3C 3D MQ/GL PQ/GA PQ/GA PQ/GA unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 124) NO: 218) NO: 218) NO: 218) *ribosome skipping sequence
[0299] Multiple unique viruses were discovered at the USDA that are more similar to SVV than SVV is to other cardioviruses. These USDA virus isolates, herein considered to be members of the group called "SVV-like picornaviruses," are: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. These SVV-like picornaviruses and SVV are considered to comprise a new picornavirus genus.
[0300] Each of these SVV-like picornaviruses are unique, and are about 95%-98% identical to SVV at the nucleotide level (see FIGS. 87-89 for nucleotide sequence comparisons between SVV and these USDA isolates).
[0301] Part III: Serum Studies
[0302] Pigs are a permissive host for the USDA virus isolates identified above. The isolate MN 88-36695 was inoculated into a gnobiotic pig and antisera generated (GP102). The antisera binds to all of the other USDA isolates listed above and to SVV. The antisera does not react with 24 common porcine virus pathogens indicating its specificity. Porcine sera was also tested for neutralizing antibodies to 1278 (Plum Island virus). Sera were collected in the US and 8/29 sera were positive with titers ranging from 1:57 to 1:36,500.
[0303] To test whether the pig is the natural source for SVV, serum samples from various animals were obtained and tested for their ability to act as neutralizing antibodies against SVV infection of permissive cells. The Serum Neutralization Assay is conducted as follows: (1) Dilute various serums 1:2 and 1:4; (2) Mix with 100 TCID50 of virus (SVV; but any virus can be tested to determine whether a serum can neutralize its infection); (3) Incubate at 37° C. for 1 hour; (4) Add to 1×104 PER.C6 cells (or other permissive cell type); (5) Incubate at 37° C. for 3 days; and (6) Measure CPE using MTS assay. The neutralization titer is defined as the highest dilution of sera that neutralizes SVV (or other virus in question) at 100%.
[0304] The serum neutralization results showed that there is a minimal or no presence of neutralizing antibodies in human and primate populations. In one experiment, 0/22 human sera contained neutralizing antibodies to SVV. In another experiment, only 1/28 human sera contained neutralizing antibodies. In a third experiment, 0/50 human sera from Amish farmers were neutralizing. In another experiment, 0/52 primate sera from four species were neutralizing.
[0305] The serum neutralization results showed that there is a prevalence of neutralizing antibodies in farm animal populations. In one experiment, 27/71 porcine sera from farms were neutralizing. In another experiment, 4/30 porcine sera from a disease-free farm were neutralizing. In another experiment, 10/50 bovine sera were neutralizing. In yet another experiment, 5/35 wild mouse sera were neutralizing. Because antibodies cross-reactive to SVV and/or SVV-like picornaviruses have been found in pigs, cows, and mice, these data indicate that SVV and/or SVV-like picornaviruses may be prevalent in a wide-variety of non-primate animals.
[0306] A crude viral lysate of MN 88-36695 was tested to assess its cytotoxicity ability on two cell lines permissive (NCI-H446; HEK293) for SVV and on two cell lines non-permissive (NCI-H460 and S8) for SVV. The cytotoxicity profile for MN 88-36695 was identical to SVV: the TCID50 for NCI-H446 was 1.6×10-6; the TCID50 for HEK293 was 1.3×10-2; and NCI-H460 and S8 were non-permissive for MN 88-36695. This data indicates that SVV-like picornaviruses have the potential to be used in the present methods directed to cancer therapy. In one embodiment, the invention provides for the use of the MN 88-36695 SVV-like picornavirus in any of the methods directed to cancer therapy, diagnosis, or screening.
[0307] Antisera to MN 88-36694 and SVV were tested in serum neutralization assays on each virus. Anti-SVV mouse serum was able to neutralize infection by both MN 88-36695 and SVV (neutralization titers on infection were 1:640 for MN 88-36695 and 1:1000 for SVV). Anti-MN 88-36695 gnobiotic pig serum was able to neutralize infection by both MN 88-36695 and SVV (neutralization titers on infection were 1:5120 for MN 88-36695 and 1:100 for SVV).
[0308] These data indicate that SVV is genetically and serologically linked to the porcine USDA virus isolates.
Example 4
SDS-PAGE and N-Terminal Sequence Analysis of SVV Capsid Proteins
[0309] Purified SVV is subjected to electrophoresis using NuPAGE pre-cast Bis-Tris polyacrylamide mini-gel electrophoresis system (Novex, San Diego, Calif., USA). One half of the gel is visualized by silver stain while the other half is used to prepare samples for amino acid sequencing of the N-termini of the capsid proteins. Prior to transfer of proteins to membrane, the gel is soaked in 10 mM CAPS buffer, pH 11, for 1 hour, and a PVDF membrane (Amersham) is wetted in methanol. Proteins are transferred to the PVDF membrane. After transfer, proteins are visualized by staining with Amido black for approximately 1 minute, and bands of interest are excised with a scalpel and air dried. The proteins can be subjected to automated N-terminal sequence determination by Edman degradation using a pulsed phase sequencer.
[0310] Three major structural proteins of the purified SVV are shown in FIG. 45 (approximately 36 kDa, 31 kDa, and 27 kDa).
Example 5
Assay for Neutralization Antibodies to SVV in Human Serum Samples
[0311] Preexisting antibodies to particular viral vectors may limit the use of such vectors for systemic delivery applications such as for treatment of metastatic cancer, because preexisting antibodies may bind to systemically delivered vectors and neutralize them before the vectors have a chance to transduce the targeted tissue or organ. Therefore, it is desirable to ensure that humans do not carry neutralization antibodies to viral vectors selected for systemic delivery. To determine whether human sera samples contain SVV-specific neutralizing antibodies, neutralization assays are carried out using randomly collected human sera samples.
[0312] Tissue culture infective dose 50: One day before the experiment, 180 μl of PER.C6 cell suspension containing 1×104 cells are plated in 96-well tissue culture dish. The crude virus lysate (CVL) of SVV is diluted in log steps from 10-0 to 10-11 in DMEM medium (Dulbecco's Modified Eagle's Medium) and 20 μl of each dilution is transferred to three wells of a Falcon 96-well tissue culture plate containing PER.C6 cells. The plates are incubated at 37° C. in 5% CO2 and read at 3 days for microscopic evidence of cytopathic effect (CPE), and the tissue culture infective dose 50 (TCID50) is calculated.
[0313] Neutralization assay: First, 40 μl of medium is placed in all the wells and then 40 μl of heat-inactivated serum is added to the first well and mixed by pipeting, making a 1:4 dilution used for screening purposes. 40 μl is then transferred to the next well to perform a two-fold dilution of the serum samples. 40 μl of SVV virus, containing 100 TCID50, is added to wells containing diluted serum samples. Plates are incubated at 37° C. for 1 hour. 40 μl of the mix is taken and transferred to a plate containing PER.C6 cells (1×104 cells/160 μl/well). The plates are incubated at 37° C. for 3 days. After this time, the cultures are read microscopically for CPE.
[0314] In a representative neutralization assay performed as described above, twenty-two human sera samples randomly collected from USA, Europe and Japan were examined for SVV specific neutralizing antibodies. The serum samples were serially diluted and mixed with a fixed amount of SVV containing 100 TCID50. Serum-virus mixtures were then used to infect PER.C6 cells and incubated for 24 hours. Neutralizing antibody titer was determined as the reciprocal of the highest dilution of serum able to block CPE formation. In this experiment, no dilution of serum blocked CPE formation indicating that the human serum samples did not contain SVV neutralizing antibodies.
[0315] Further SVV infection of PER.C6 was not inhibited by incubation with human blood (see Example 6), indicating that SVV infection was not inhibited by complement or by hemagglutination. As a result, SVV exhibits a longer circulation time in vivo than other oncolytic viruses, which is a significant problem with the use of oncolytic adenoviruses.
Example 6
Binding of SVV to Human Erythrocytes and Hemagglutination
[0316] Various viral serotypes have been shown to cause in vitro hemagglutination of erythrocytes isolated from blood of various animal species. Hemagglutination or binding to erythrocytes may cause toxicity in vivo and may also affect in vivo biodistribution and the efficacy of a viral vector. Therefore, it is desirable to analyze the erythrocyte agglutination properties of a viral vector selected for systemic administration to treat metastatic cancers.
[0317] Hemagglutination assay: To determine whether SVV causes agglutination of human erythrocytes, hemagglutination assays are carried out in U-bottom 96-well plates. Purified SVV is serially diluted in 25 μl PBS (Phosphate Buffered Saline) in duplicates, and an equal volume of 1% erythrocyte suspension is added to each well. Blood samples used for isolation of erythrocytes are obtained from healthy individuals with heparin as an anticoagulant. Erythrocytes are prepared by washing the blood three times in cold PBS to remove the plasma and the white blood cells. After the last wash, erythrocytes are suspended in PBS to make a 1% (V/V) cell suspension. The virus and erythrocytes are gently mixed and the plates are incubated at room temperature for 1 hour and monitored for a hemagglutination pattern.
[0318] Whole blood inactivation assay: To rule out direct inactivation of SVV by blood components, aliquots of virus are incubated with heparinized human blood belonging to A, B, AB and O blood groups or PBS for 30 minutes or 1 hour at room temperature prior to separation of plasma, after which PER.C6 cells are infected and titers are calculated.
[0319] In representative assays performed as described above, no hemagglutination of human erythrocytes of different blood groups (A, B, AB and O) was seen at any tested dilutions of SVV. A slight increase in the virus titer is noticed when SVV is mixed with blood human samples and incubated for 30 minutes and 1 hour, indicating that the virus is not inactivated by blood components but becomes more infectious under tested conditions.
Example 7
In Vivo Clearance
[0320] Blood circulation time: To determine the blood circulation time and the amount of the virus in the tumor, H446 tumor bearing nude mice were treated with SVV at a dose of 1×1012 vp/kg by tail vein injection. The mice were bled at 0, 1, 3, 6, 24, 48, 72 hours and 7 days (189 hours) post-injection and the plasma was separated from the blood immediately after collection, diluted in infection medium, and used to infect PER.C6 cells. The injected mice were sacrificed at 6, 24, 48, 72 hours and 7 days post-injection and the tumors were collected. The tumors were cut into small sections and suspended in one ml of medium and subjected to three cycles of freeze and thaw to release the virus from the infected cells. Serial log dilutions of supernatants were made and assayed for titer on PER.C6 cells. SVV titers were expressed as pfu/ml. The tumor sections were also subjected to H&E staining and immunohistochemistry to detect the virus capsid proteins in the tumor.
[0321] The circulating levels of virus particles in the blood were determined based on the assumption that 7.3% of mouse body weight is blood. In representative assays performed as essentially as described above, within 6 hours of virus administration, the circulating levels of SVV reduced to zero particles and SVV was not detectable at later time points (FIG. 46A). In the tumor, SVV was detectable at 6 hours post-injection, after which the amount of the virus increased steadily by two logs (FIG. 46B). The virus was detectable in the tumor as late as 7 days postinjection (FIG. 46B). The tumor sections when subjected to immunohistochemistry, revealed SVV proteins in the tumor cells (FIG. 47, top panels). When stained by H&E, the tumor sections revealed several rounded tumor cells (FIG. 47, bottom panels).
[0322] SVV also exhibits a substantially longer resident time in the blood compared to similar doses of i.v. adenovirus. Following a single i.v. dose, SVV remains present in the blood for up to 6 hours (FIG. 46C; FIG. 46C is a duplication of FIG. 46A for comparison purposes to FIG. 46D), whereas adenovirus is cleared from the blood in about an hour (FIG. 46D).
Example 8
Tumor Cell Selectivity
[0323] In vitro cell killing activity of SVV: To determine the susceptibility of human, bovine, porcine, and mouse cells, normal and tumor cells were obtained from various sources and infected with SVV. All cell types were cultured in media and under the conditions recommended by the supplier. Primary human hepatocytes may be purchased from In Vitro Technologies (Baltimore, Md.) and cultured in Hepatocyte Culture Media (HCM®, BioWhittaker/Clonetics Inc., San Diego, Calif.).
[0324] In vitro cytopathic assay: To determine which types of cells are susceptible to SVV infection, monolayers of proliferating normal cells and tumor cells were infected with serial dilutions of purified SVV. The cells were monitored for CPE and compared with uninfected cells. Three days following infection, a MTS cytotoxic assay is performed and effective concentration 50 (Ec50) values in particles per cell are calculated. See Tables 5 and 6 below and Table 1A supra.
TABLE-US-00009 TABLE 5 Cell lines with EC50 values less than 100 EC50 number Cell lines with EC50 <1 H446 (human sclc) 0.001197 PERC6 0.01996 H69AR (sclc-multidrug resistant) 0.03477 293 (human kidney transformed with ad5E1) 0.03615 Y79 (human retinoblastoma) 0.0003505 IMR32 (human brain; neuroblastoma) 0.03509 D283med (human brain; cerebellum; 0.2503 medulloblastoma) SK-N-AS (human brain; neuroblastoma) 0.474 N1E-115 (mouse neuroblastoma) 0.002846 SK-NEP-1 (kidney, wilms' tumor, pleural 0.03434 effusion, human) BEKPCB3E1 (bovine embryonic kidney cells 0.99 transformed with ad5E1 Cell Lines with EC50 <10 (1-10) H1299 (human-non sclc) 7.656 ST (pig testes) 5.929 DMS 153 (human sclc) 9.233 Cell lines with EC50 <100 (10-100) BEK (bovine embryonic kidney) 17.55
TABLE-US-00010 TABLE 6 Cell lines with EC50 values more than 1000 M059K (human brain; HUVEC (human vein endothelial CMT-64 (mouse-sclc) malignant glioblastoma) cells) KK (human glioblastoma) HAEC (human aortic endothelial LLC-1 (mouse-LCLC)) cells) U-118MG (human WI38 (human lung fibroblast) RM-1 (mouse-prostate) glioblastoma) DMS 79 (human sclc) MRC-5 (human lung fibroblast) RM-2 (mouse-prostate) H69 (human sclc) IMR90 (human lung fibroblast) RM-9 (mouse-prostate) DMS 114 (human sclc) HMVEC (human microvascular MLTC-1 (mouse-testes) endothelial cells-adult) DMS 53 (human sclc) HMVEC (human microvascular KLN-205 (mouse-sqcc) endothelial cells-neonatal) H460 (human-LCLC) HCN-1A (human brain) CMT-93 (mouse-rectal) A375-S2 (human HRCE (human renal cortical B16F0 (mouse melanoma) epithelial cells) melanoma) SK-MEL-28 (human Neuro-2A (mouse melanoma) neuroblastoma) PC3 (human prostate) C8D30 (mouse brain) PC3M2AC6 (human PK15 (pig-kidney) prostate) LNCaP (human prostate) FBRC (fetal bovine retina) DU145 (human prostate) MDBK (bovine kidney) Hep3B (human liver CSL 503 (sheep lung cells carcinoma) transformed with ad5E1) Hep2G (human liver OFRC (ovine fetal retina carcinoma) cells) SW620 (human-colon) SW839 (human kidney) 5637 (human bladder) HeLa S3 S8
[0325] The MTS assay was performed according to the manufacturer's instructions (CellTiter 96® AQueous Assay by Promega, Madison, Wis.). The CellTiter 96® AQueous Assay preferably uses the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl- )-2H-tetrazolium, inner salt; MTS) and an electron coupling reagent, phenazine methosulfate (PMS). Contact-inhibited normal human cells evaluated in the study include: HUVEC (human umbilical vein endothelial cells), HAEC (human aortic endothelial cells, Clonetics/BioWhittaker #CC-2535), Wi38 (normal human embryo lung fibroblasts, ATCC #CCL-75), IMR90 (human normal lung fibroblasts, ATCC CCL-186), MRC-5 (human normal lung fibroblasts, ATCC, #CCL-171) and HRCE (human renal cortical epithelial cells, Clonetics/BioWhittaker #CC-2554).
[0326] SVV does not produce CPE in any of the above contact-inhibited normal cells. No virus-induced CPE was seen in the following human tumor cell lines: Hep3B (ATCC #HB-8064), HepG2 (human hepatocellular carcinoma, ATCC #HB-8065), LNCaP (human prostate carcinoma, ATCC #CRL-10995), PC3M-2AC6, SW620 (human colorectal adenocarcinoma, ATCC #CCL-227), SW 839 (human kidney adenocarcinoma, ATCC #HTB-49), 5637 (human urinary bladder carcinoma, ATCC #HTB-9), DMS-114 (small cell lung cancer, ATCC #CRL-2066), DMS 153 (human small cell lung cancer, ATCC #CRL-2064), A549 (human lung carcinoma, ATCC #CCL-185), HeLa S3 (human cervical adenocarcinoma, ATCC #CCL-2.2), NCI-H460 (human large cell lung cancer, ATCC #HTB-177), KK (glioblastoma), and U-118 MG (human glioblastoma, ATCC #HTB-15). Note--the cell lines in Table 6 with EC50 values greater than 1000 are most likely not permissive for SVV replication and/or virion production; although the possibility remains that SVV can bind and enter into these cells but CPE is not observed because SVV replication cannot occur inside the cell or that replication does occur but CPE is not observed because there is some other post-entry block (i.e., no packaging of replicated SVV genomes into virions). However, considering the absence of CPE in these cell lines, these cell-lines, and potentially tumor-types thereof, are good candidates to test which cell and tumor-types are permissive or non-permissive for SVV replication. Although wild-type SVV is tumor-specific, and has been shown to target neuroendocrine tumors, including small cell lung cancer and neuroblastomas, there may be individual patients that have types of etiologies such that SVV is not permissive in their form of neuroendocrine tumor. Therefore, the invention does contemplate the generation of SVV derivatives that can kill tumor cell-types isolated from individual patients where the tumors are non-permissive to the wild-type SVV, and the tumor-types isolated from these individuals can include, for example, glioblastoma, lymphoma, small cell lung cancer, large cell lung cancer, melanoma, prostate cancer, liver carcinoma, colon cancer, kidney cancer, colon cancer, bladder cancer, rectal cancer and squamous cell lung cancer.
[0327] SVV-mediated cytotoxicity on primary human hepatocytes (In Vitro Technologies) was determined by LDH release assay (CytoTox® 96 Non-Radioactive Cytotoxicity Assay, Promega, #G1780). Primary human hepatocytes plated in collagen coated 12-well plates were infected with SVV at 1, 10 and 100 and 1000 particles per cell (ppc). After 3 hours of infection, the infection medium was replaced with 2 ml of growth medium and incubated for 3 days in a CO2 incubator. The cell associated lactate dehydrogenase (LDH) and LDH in the culture supernatant was measured separately. Percent cytotoxicity is determined as a ratio of LDH units in supernatant over maximal cellular LDH plus supernatant LDH.
Percent cytotoxicity = LDH units in culture supernatant × 100 Sum of LDH units in supernatant and cell lysate ##EQU00001##
The data shown in FIG. 48 illustrates the absence of SVV mediated hepatoxicity at all tested multiplicity of infections.
Example 10
Virus Production Assay
[0328] To assess the replicative abilities of SVV, several selected contact-inhibited normal cells and actively dividing tumor cells were infected with SVV at one virus particle per cell (ppc). After 72 hours, cells and the medium were subjected to three freeze-thaw cycles and centrifuged to collect the supernatant. Serial log dilutions of supernatants were made and assayed for titer on PER.C6 cells. For each cell line, the efficiency of SVV replication was expressed as pfu/ml (FIG. 49).
Example 10
Toxicity
[0329] The maximum tolerated dose (MTD) is defined as the dosage immediately preceding the dose at which animals (e.g. mice) demonstrate a dose limiting toxicity (DLT) after the treatment with SVV. DLT is defined as the dose at which the animals exhibit a loss in body weight, symptoms, and mortality attributed to SVV administration during the entire duration of the study. Neutralizing antibodies to SVV were assessed at baseline, day 15, and day 21. Neutralization assays were carried as described earlier.
[0330] Escalating doses (1×108-1×1014 vp/kg) of SVV were administered intravenously into both immune deficient nude and caesarean derived-1 (CD-1) out-bred immune competent mice purchased from Harlan Sprague Dawley (Indianapolis, Ind., USA) to determine the MTD with 10 mice per dose level. The virus was well-tolerated at all tested dose levels without exhibiting any clinical symptoms and without loss in body weight (FIG. 50). Mice were bled at day 15 and 21 and the sera was monitored for the presence of SVV-specific neutralizing antibodies in neutralization assays. SVV injected CD1 mice develop neutralizing antibodies and the titers range from 1/1024 to greater than 1/4096.
[0331] Another toxicity study was conducted on the immunocompetent mouse strain (A/J). It has been demonstrated that SVV exhibits cell killing activity and replication in N1E-115 cells (see Table 1). The murine cell line N1E-115 (a neuroblastoma cell line, i.e., neuroendocrine cancer) is derived from the A/J mouse strain. Thus, a syngeneic mouse model was established where N1E-115 cells were implanted subcutaneously in A/J mice to form tumors, and the mice were then treated with SVV to investigate its efficacy and toxicity.
[0332] In the A/J study, mice were i.v. injected with SVV to determine whether A/J mice can tolerate systemic administration of SVV. Blood hematology results were obtained to look for signs of toxicity, and serum chemistry results can also be obtained. The study design is shown in Table 7 below:
TABLE-US-00011 TABLE 7 A/J Study Design Dosage Dosage Group Animals Test Level Volume Dosing Necropsy # (Female) Article (particles/kg) (mL/kg) regimen Day 1 5 Vehicle 0 10 IV on Day 15 Day 1 2 5 SVV .sup. 108 10 IV on Day 15 Day 1 3 5 SVV .sup. 1011 10 IV on Day 15 Day 1 4 5 SVV .sup. 1014 10 IV on Day 15 Day 1
[0333] The A/J mice were 8-10 week old females obtained from The Jackson Laboratory (Bar Harbor, Me.). SVV was prepared by storing isolated virions at -80° C. until use. SVV was prepared fresh by thawing on ice and diluting with HBSS (Hank's balanced salt solution). SVV was diluted to concentrations of 107 particles/mL for group 2, 1010 particles/mL for group 3, and 1013 particle/mL for group 4. HBSS was used as the vehicle control for group 1. All dosing solutions were kept on wet ice until dosing.
[0334] SVV was administered to animals intravenous injection via the tail vein at a dose volume of 10 mL/kg body weight. Animals were weighed on the day of dosing and dose volumes were adjusted based on body weight (i.e., a 0.0200 kg mouse gets 0.200 mL of dosing solution). Mice were monitored twice daily for morbidity and mortality. Mice were weighed twice weekly. Information relating to moribund animals and animals exhibiting any unusual symptoms (physically or behaviorally) are recorded immediately.
[0335] Post-mortem observations and measurements entail the collection of blood from all surviving animals at terminal sacrifice for standard hematology and serum chemistry (AST, ALT, BUN, CK, LDH). The following organs are to be collected at sacrifice: brain, heart, lung, kidney, liver, and gonads. Half of each organ sample is snap frozen on dry ice and the other half will be placed in formalin.
[0336] Initial blood hematology results (CBC, differential) were obtained two weeks after SVV injection and the results are summarized below in Table 8 below. Five mice were tested from each test group (see Table 7):
TABLE-US-00012 TABLE 8 A/J Toxicity Results - Blood Hematology Test Group 1 Test Group 2 Test Group 3 Test Group 4 Body Weight Result ± SD (g): Day 0 21.48 ± 0.88 21.98 ± 1.93 22.58 ± 0.87 21.04 ± 1.67 Day 14 20.26 ± 0.93 20.92 ± 1.71 21.44 ± 0.84 21.26 ± 1.45 CBC Wet (Result ± SD (ref range)): White blood count 3.63 ± 1.57 4.5 ± 1.57 4.26 ± 0.94 4.72 ± 0.62 (THSN/UL) (2.60-10.69) (2.60-10.69) (2.60-10.69) (2.60-10.69) Red blood count 9.87 ± 0.03 9.49 ± 0.07 9.76 ± 0.37 9.71 ± 0.32 (MILL/UL) (6.4-9.4) (6.4-9.4) (6.4-9.4) (6.4-9.4) Hemoglobin 15.37 ± 0.06 14.78 ± 0.29 15.12 ± 0.66 15.02 ± 0.63 (GM/DL) (11.5-16.1) (11.5-16.1) (11.5-16.1) (11.5-16.1) Hematocrit (%) 46.03 ± 0.40 44.52 ± 0.49 45.7 ± 1.82 45.28 ± 1.69 (36.1-49.5) (36.1-49.5) (36.1-49.5) (36.1-49.5) MCV (FL) 46.67 ± 0.58 47.00 ± 0.0 47.0 ± 0.0 46.6 ± 0.55 (45.4-60.3) (45.4-60.3) (45.4-60.3) (45.4-60.3) MHC (PICO GM) 15.57 ± 0.06 15.70 ± 0.17 15.37 ± 0.06 15.43 ± 0.15 (14.1-19.3) (14.1-19.3) (14.1-19.3) (14.1-19.3) MCHC (%) 33.37 ± 0.12 33.14 ± 0.48 33.08 ± 0.22 33.14 ± 0.25 (25.4-34.1) (25.4-34.1) (25.4-34.1) (25.4-34.1) Platelet (THSN/UL) 885.33 ± 28.6 758.2 ± 146.2 874.8 ± 56.7 897.2 ± 105.4 (592-2972) (592-2972) (592-2972) (592-2972) Differential (Result ± SD (ref range)): Bands (THSN/UL) 0.0 0.0 0.0 0.0 (0.0-0.1) (0.0-0.1) (0.0-0.1) (0.0-0.1) Seg. Neutrophils 0.92 ± 0.27 1.16 ± 0.37 1.09 ± 0.38 0.96 ± 0.20 (THSN/UL) (0.13-2.57) (0.13-2.57) (0.13-2.57) (0.13-2.57) Lymphocytes 2.64 ± 1.26 2.98 ± 1.41 3.10 ± 0.56 3.70 ± 0.41 (THSN/UL) (1.43-9.94) (1.43-9.94) (1.43-9.94) (1.43-9.94) Monocytes 0.06 ± 0.04 0.15 ± 0.05 0.06 ± 0.03 0.05 ± 0.02 (THSN/UL) (0.0-0.39) (0.0-0.39) (0.0-0.39) (0.0-0.39) Eosinophils 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.003 ± 0.01 (THSN/UL) (0.0-0.24) (0.0-0.24) (0.0-0.24) (0.0-0.24) Basophils 0.0 0.004 ± 0.005 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Atypical Lympho. 0.0 0.0 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Metamyelocytes 0.0 0.0 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Myelocytes 0.0 0.0 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) NRBC (/100WBC) 0.0 0.0 0.0 0.0 (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Other (Result ± SD (ref range)): AST (SGOT) (U/L) 1762.8 ± 1129.8 899.0 ± 234.6 779.8 ± 312.2 843.2 ± 653.4 (72-288) (72-288) (72-288) (72-288) ALT (SGPT) (U/L) 2171.8 ± 2792.9 535.2 ± 272.8 555 ± 350.8 380.2 ± 385.7 (24-140) (24-140) (24-140) (24-140) BUN (MG/DL) 27.2 ± 0.8 24.8 ± 1.9 24.6 ± 5.5 28.2 ± 12.8 (9-28) (9-28) (9-28) (9-28) Creatine phospho- 28312.8 ± 20534.4 12194.4 ± 4049.2 10157 ± 5420.5 11829 ± 10363.9 kinase (U/L) (0-800) (0-800) (0-800) (0-800) LDH (U/L) 6650.2 ± 4788.6 3661.6 ± 933.6 3450.8 ± 972.6 2808.4 ± 1709.1 (260-680) (260-680) (260-680) (260-680) Hemolytic Index 706.6 ± 423.4 477.6 ± 195.7 589.6 ± 198.6 496.4 ± 321.1 (MG/DL HGB) (0-70) (0-70) (0-70) (0-70)
[0337] These results show that there are no abnormalities in blood hematology profiles obtained from mice treated with low, medium and high doses of SVV compared to blood hematology profiles obtained from untreated mice. From this study, it can be concluded that there are no measureable signs of toxicity following systemic administration of SVV, indicating that SVV is tolerated by A/J mice following i.v. injection.
Example 11
Efficacy
[0338] Athymic female nude mice (nu/nu) aged 6-7 weeks purchased from Harlan Sprague Dawley (Indianapolis, Ind.) were used in efficacy studies. Mice were injected subcutaneously with 5×106 H446 cells into the right flank using manual restraint. Tumor sizes were measured regularly, and the volumes were calculated using the formula π/6×W×L2, where L=length and W=width of the tumor. When the tumors reach approximately 100-150 mm3, mice (n=10) were randomly divided into groups. Mice were injected with escalating doses of SVV by tail vein injections at a dose volume of 10 ml/kg. A control group of mice was injected with an equivalent volume of HBSS. Dose escalation proceeds from 1×107 to 1×1013 particles per kilogram body weight. Antitumoral efficacy was determined by measuring tumor volumes twice weekly following SVV administration. Complete response was defined as complete disappearance of xenograft; partial response as regression of the tumor volume by equal to or more than 50%; and no response as continuous growth of tumor as in the control group.
[0339] Tumors from mice treated with HBSS grew rapidly and the tumor volumes reached more than 2000 mm3 by study day 20 (FIG. 51; see line with open diamond). In contrast, mice given one systemic injection of SVV at all tested doses (with the exception of the lowest dose) became tumor free by study day 20. In the lowest dose group, 8 mice became tumor free, one mouse had a very large tumor and the other had a small palpable tumor (25 mm3) by study day 31. To evaluate the antitumor activity of SVV on large sized tumors, five mice from HBSS group bearing tumors >2000 mm3 were systemically injected with a single dose of 1×1011 vp/kg on study day 20. For the duration of the follow-up period (11 days of after SVV injection), a dramatic regression of the tumor volumes were noted (FIG. 51).
[0340] Additional experiments to test the efficacy of a single intravenous dose of SVV was conducted in murine tumor models that express neuroendocrine markers. The tumor models tested included H446 (human SCLC) (see FIG. 90A), Y79 (human retinoblastoma) (see FIG. 90B), H69AR (human multi-drug resistant SCLC) (see FIG. 90C), H1299 (human NSCLC) (see FIG. 90D), and N1E-115 (murine neuroblastoma) (see FIG. 90E).
[0341] The results show that a single intravenous dose of SVV has efficacy in all of the murine neuroendocrine tumor models. The results also show that SVV is efficacious in the N1E-115 immunocompetent murine neuroblastoma model.
[0342] FIG. 52 shows a picture of mice that were "untreated" with SVV (i.e., treated with HBSS) or "treated" with SVV. As can be seen, the untreated mice had very large tumors and the treated mice showed no visible signs of tumor. Further, for unsacrificed mice treated with SVV, no tumor regrowth was observed for the duration of the study, 200 days.
[0343] In vitro efficacy data for SVV for specific tumor cell lines is shown in Tables 1, 1A, and 5. The data shows that SVV specifically infects particular tumor cell types and does not infect normal adult cells (except for porcine normal cells), a significant advantage over any other known oncolytic virus. SVV has been shown to have 1,000 times better cell killing specificity than chemotherapy treatments (cell killing specificity values for SVV have been shown to be greater than 10,000, whereas cell killing specificity values for chemotherapy are around 10).
[0344] Specific cytotoxic activity of SVV was demonstrated in H446 human SCLC cells. Following a two-day incubation with increasing concentrations of SVV, cell viability was determined. The results are shown in FIG. 53. FIG. 53 shows cell survival following incubation of SVV with either H446 SCLC tumor cells (top graph) or normal human H460 cells (bottom graph). SVV specifically killed the tumor cells with an EC50 of approximately 10-3 particles per cell. In contrast, normal human cells were not killed at any concentration of SVV. Further, as summarized in Tables 1, 1A-3, SVV was also cytotoxic toward a number of other tumor cell lines, including SCLC-multidrug resistant tumor cells, and some fetal cells and cell lines. The EC50 values for SVV cytotoxicity for the other tumor cell lines ranged from 10-3 to greater than 20,000 particles per cell. SVV was non-cytotoxic against a variety other non-neural tumors and normal human tissues. Additionally, SVV was not cytotoxic to primary human hepatocytes, as measured by LDH release at up to 1000 particles per cell (see FIG. 48).
Example 12
Biodistribution and Pharmacokinetic Study in Rodents
[0345] Pharmacokinetic and biodistribution study of SVV is performed in normal mice and immunocompromised athymic nude mice bearing H446 SCLC tumors. This study evaluates the biodistribution, elimination and persistence of SVV following a single intravenous administration to both normal and immunocompromised tumor-bearing mice. Groups of mice each receive a single i.v. dose of control buffer or one of three doses of SVV (108, 1010, or 1012 vp/kg) and are monitored for clinical signs. Blood samples are obtained from groups of 5 mice at 1, 6, 24 and 48 hours post dose, and at 1, 2, 4, and 12 weeks post dose. Dose levels include a known low efficacious dose and two higher dose levels to determine linearity of virus elimination. Groups of mice are sacrificed at 24 hours, and 2, 4 and 12 weeks post dose. Selected tissues, including liver, heart, lung, spleen, kidney, lymph nodes, bone marrow, brain and spinal cord tissues are aseptically collected and tested for the presence of SVV RNA using a validated RT-PCR assay.
[0346] Samples of urine and feces are obtained at sacrifice, at 24 hours, and at 2, 4 and 12 weeks post dose and are examined for the presence of infectious virus. The design of the experiments in this Example are shown in Table 9 below:
TABLE-US-00013 TABLE 9 Biodistribution of SVV in CD-1 Mice and Athymic Nude Mice Bearing SCLC Tumors # of # of Mice/ Dose Mice/Timepoint Timepoint Level for Blood for PCR Tissue Group Treatment (vp/kg) Route Sampling Distribution Normal CD-1 Mice 1 Saline 0 i.v. 5 5 2 SVV 108 i.v. 5 5 3 SVV 1010 i.v. 5 5 4 SVV 1012 i.v. 5 5 Athymic Tumor Bearing Mice 5 Saline 0 i.v. 5 5 6 SVV 108 i.v. 5 5 7 SVV 1010 i.v. 5 5 8 SVV 1012 i.v. 5 5
[0347] Acute i.v. toxicology studies were also performed in both normal and immunocompromised athymic nude mice bearing H446 SCLC tumors. Preliminary i.v. studies in normal and SCLC tumor bearing mice indicate safety of SVV at doses up to 1014 vp/kg. No adverse clinical signs were observed and there was no loss of body weight up to 2 weeks following a single i.v. dose of 1014 vp/kg.
Example 13
Viral Transmission Study in Normal Adult and Pregnant Mice
[0348] The purpose of this Example is to determine if SVV is transmissible following cohabitation of noninfected normal mice with mice injected with a high concentration of SVV. Because SVV does not replicate in normal, non-tumor bearing mice, tumor bearing mice can also be injected with high concentrations of SVV and subsequently exposed to normal, healthy animals to better simulate the clinical scenario. A secondary purpose is to assess the potential transmissibility of SVV from an infected female to an uninfected pregnant DAM, and subsequently to the developing fetus.
[0349] Three groups of five naive male and female CD-1 mice are exposed to a single mouse of the same sex infected with either 108, 1010 or 1012/kg, and are monitored for the presence of SVV by blood sampling.
[0350] Similarly, an SVV exposed female is co-mingled with a number of timed pregnant females, and the ability of the virus to transmit from the infected female to an uninfected pregnant female, and subsequently to the developing fetus is determined
Example 14
Non-Human Primate Studies
[0351] The safety, toxicity and toxicokinetics of SVV are also determined in non-human primates. In a dose range-finding phase, individual monkeys receive a single i.v. dose of SVV at 108 vp/kg and are closely monitored for clinical signs of infection or toxicity. If this dose is well tolerated, additional animals are treated with a higher i.v. dose until a dose of 1012 vp/kg is achieved. Subsequently, the main study consists of groups of three male and female monkeys, and each monkey is dosed once weekly for six weeks with either vehicle alone or one of three doses of SVV and monitored for signs of toxicity. An additional two monkeys per sex are dosed with the vehicle alone and with the high dose level of SVV for six weeks, and are allowed an additional four weeks recovery prior to sacrifice.
[0352] Blood samples are obtained following dosing during week 1 and week 6. Clinical pathological and hematology blood samples are obtained prior to the initial dose and prior to sacrifice. Additional blood samples are obtained following each dose for assessing the presence of neutralizing antibodies to SVV.
[0353] Surviving monkeys are euthanized and subjected to a full gross necropsy and a full tissue list is collected from the main study and recovery monkeys. Tissues from the control and high dose groups are evaluated histopathologically. Urine and fecal samples are collected following dosing on weeks 1 and 6 and are evaluated for presence of infectious SVV. The overall design of this Example is shown in Table 10 below.
TABLE-US-00014 TABLE 10 Multiple Dose Toxicology Study of SVV in Primates Dose Range-finding Phase Group Treatment Dose (vp/kg) Route Males Females 1 SVV 108 IV 1 1 2 SVV 1010* IV 1 1 3 SVV 1012* IV 1 1 Main Phase Dose Main Phase Recovery Group Treatment (vp/kg) Route Male Female Male Female 1 Control -- IV 3 3 2 2 2 SVV 108*.sup. IV 3 3 -- -- 3 SVV 1010* IV 3 3 -- -- 4 SVV 1012* IV 3 3 2 2 *Doses can vary based on results of Dose Range-finding phase
Example 15
Construction of an Infectious Full-Length and Functional Genomic SVV Plasmid
[0354] With SEQ ID NO:1, only about 1.5-2 Kb of the 5' genomic sequence of SVV remains to be sequenced, representing the nucleotide region covering the 5' UTR, 1A (VP4) and part of 1B (VP2). To clone the 5' end missing in SEQ ID NO:1, polymerases that function at high temperatures and reagents that can enable a polymerase to read through secondary structures were used. Additional SVV cDNAs were prepared from isolated SVV of ATCC deposit number PTA-5343. SVV particles were infected into a permissive cell line, such as PER.C6, and viruses are isolated. Viral RNA was then recovered from the virus particles such that cDNA copies are made therefrom. Individual cDNA clones were sequenced, such that selected cDNA clones are combined into one full-length clone in a plasmid having a T7 promoter upstream of the 5' end of the SVV sequence. The full-length genomic sequence of SVV is listed in FIGS. 83A-83H and SEQ ID NO:168. The full-length SVV from this plasmid is reverse-transcribed, by utilizing T7 polymerase and an in vitro transcription system, in order to generate full-length RNA (see FIG. 55). The full-length RNA is then transfected into permissive cell lines to test the infectivity of the full-length clone (see FIG. 55).
[0355] The methodology was as follows.
[0356] RNA Isolation:
[0357] SVV genomic RNA was extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen). Briefly, 250 μl of the purified SVV (˜3×1012 virus particles) was mixed with 3 volumes of Trizol and 240 μl of chloroform. The aqueous phase containing RNA was precipitated with 600 μl isopropanol. The RNA pellet was washed twice with 70% ethanol, dried and dissolved in sterile DEPC-treated water. The quantity of RNA extracted can be estimated by optical density measurements at 260 nm. An aliquot of RNA can be resolved through a 1.25% denaturing agarose gel (Cambrex Bio Sciences Rockland Inc., Rockland, Me. USA) and the band visualized by ethidium bromide staining and photographed.
[0358] cDNA Synthesis:
[0359] cDNA of the SVV genome was synthesized by RT-PCR. Synthesis of cDNA was performed under standard conditions using 1 μg of RNA, AMV reverse transcriptase, and oligo-dT primers. Random 14-mer oligonucleotide can also be used. Fragments of the cDNA were amplified and cloned into the plasmid pGEM-3Z (Promega) and the clones were sequenced. The sequence at the 5' end of the viral genome was cloned by RACE and the sequence determined Sequence data was compiled to generate the complete genome sequence of SVV.
[0360] Cloning of Full Length Genome:
[0361] Three cDNA fragments representing the full-length SVV genome were amplified by three PCR reactions employing six sets of SVV-specific primers. Turbo pfu polymerase (Stratagene) was used in PCR reactions. First, a fragment representing the 5' end of SVV genome was amplified with primers 5'SVV-A (SEQ ID NO:219) and SVV1029RT-R1 (SEQ ID NO:220) and the resulting fragment was cut with ApaI and EcoRI and gel purified. The gel purified fragment was ligated to Nde-ApaT7SVV (SEQ ID NO:221), an annealed oligo duplex containing engineered NdeI site at 5' end, T7 core promoter sequence in the middle and first 17 nucleotides of SVV with ready to use ApaI site at 3' end and cloned into Nde I and Eco RI sites of pGEM-3Z (Promega) by three-way ligation to generate pNTX-03. Second, a fragment representing 3' end of viral genome was amplified by PCR with primers SVV6056 (SEQ ID NO:222) and SVV7309NsiB (SEQ ID NO:223). The antisense primer, SVV7309NsiB was used to introduced poly(A) tail of 30 nucleotides in length and Nsi I recognition sequence at 3' end to clone into PstI site of pGEM-3Z plasmid. The resulting PCR product was digested with BamHI and gel purified. A fragment covering the internal part of the viral genome was amplified with primers SVV911L (SEQ ID NO:224) and SVV6157R (SEQ ID NO:225). The resulting PCR product was cut with EcoRI and BamHI and gel purified. The two gel purified fragments representing the middle and 3' end of SVV genome were cloned into EcoRI and SmaI sites of pGEM-4Z by three-way ligation to generate pNTX-02. To generate full-length SVV cDNA, pNTX-02 was digested with EcoRI and NsiI and the resulting 6.3 kb fragment was gel purified cloned into EcoRI and PstI sites of pNTX-03. The resulting full-length plasmid was called pNTX-04.
[0362] The full-length plasmid pNTX-04 was further modified at both 5' and 3' ends to facilitate in vitro transcription and rescuing of the virus following RNA transfection into PER.C6 cells. First, a SwaI restriction enzyme site was inserted immediately downstream of the poly(A) tail to liberate the 3' end of SVV-cDNA from the plasmid backbone prior to in vitro transcription. A PCR approach was used to insert the site utilizing a primer pair of SVV6056 (SEQ ID NO:222) and SVVSwaRev (SEQ ID NO:226) and pNTX-04 as template. The antisense primer SVV3SwaRev contained 58 nucleotides representing the 3' end of the SVV sequence and recognition sequences for SwaI and SphI restriction enzyme sites. The resulting PCR fragment was digested with BamHI and SphI and used to replace the corresponding fragment from pNTX-04 to generate pNTX-06. Second, an extra four nucleotides present between the T7 promoter transcription start site and 5' end of SVV cDNA in pNTX-06 were removed using annealed oligo duplex approach. The duplex nucleotides were engineered to contain KpnI recognition site, T7 core promoter sequence and the first 17 nucleotides of SVV with a ready to use ApaI site at the 3' end (SEQ ID NO:227). The annealed oligos were used to replace the corresponding portion of pNTX-06 using KpnI and ApaI sites to generate pNTX-07. Finally, a two base pair deletion noticed in the polymerase encoding region of pNTX-07 was restored by replacing BamHI and SphI fragment with a corresponding fragment amplified from SVV cDNA by PCR to generate pNTX-09.
[0363] In Vitro Transcription:
[0364] Infectivity of in vitro transcribed RNA was tested by first digesting pNTX-09 with SwaI to liberate 3' end of SVV sequence from plasmid backbone. The linearized plasmid was subjected to in vitro transcription using T7 polymerase (Promega).
[0365] Transfection of In Vitro Transcribed RNA into PER.C6 Cells:
[0366] One day prior to transfection, PER.C6 cells were plated in 6-well tissue-culture dishes. On the next day, Lipofetamine reagent (Invitrogen) was used to transfect in vitro transcribed RNA (1.5 ng) into the cells following the recommendations of the supplier. Cytopathic effect (CPE) due to virus production was noticed within 36 hour post-transfection. The transfected cells were subjected to three cycles of freeze-thaw and the viruses in lysate were further confirmed by infecting PER.C6 cells. Thus, the full-length SVV cDNA clone proved to be infectious.
[0367] As described above, the plasmid with the full-length genome of SVV can be reverse-transcribed following standard protocols. The viral RNA (100 ng) can be used to transfect any cell line known to be permissive for the native SVV, but the most efficient cell line for viral RNA transfection can be empirically determined among a variety of cell lines.
Example 16
Construction of an RGD-Displaying SVV Library
[0368] To find the optimal insertion position for the construction of SVV capsid mutants generated with random with oligonucleotides encoding random peptide sequences, a simple model system (RGD) is employed. RGD (arginine, glycine, aspartic acid) is a short peptide ligand that binds to integrins. A successful RGD-SVV derivative should contain the following characteristics: (1) the genetic insertion should not alter any of SVV's unique and desirable properties; and (2) a successful RGD derivative virus should have tropism toward αVβ5 integrin containing cells.
[0369] A SVV plasmid containing just the contiguous capsid region will be singly cleaved at random positions and a short model peptide sequence, referred to as RGD, will be inserted at each position. The virus SVV-RGD library will be constructed from this plasmid library utilizing the general technology described in FIGS. 56 and 57.
[0370] Random insertion of the cRGD oligonucleotide into the capsid region is conducted. In brief, a plasmid is constructed that just encodes the contiguous 2.1 Kb capsid region of SVV (see FIG. 56, "pSVVcapsid"). A single random cleavage is made in pSVVcapsid by partially digesting the plasmid utilizing either CviJI or an endonuclease V method as described below (see FIG. 57). After isolating the single cleaved plasmid the RGD oligonucleotide will be inserted to create a pSVVcapsid-RGD library.
[0371] The restriction enzyme CviJI has several advantages over other random cleavage methods such as sonication or shearing. First, as CviJI is a blunt ended cutter no repair is necessary. Second, CviJI has been demonstrated to cleave at random locations such that no hot spots will occur. The procedure is also simple and rapid. Briefly, the concentration of CviJI and/or time of digestion are increasingly lowered until the majority of cleaved DNA is a linearized plasmid, i.e. a single cleavage. This can be observed by standard agarose gel electrophoresis as depicted in FIG. 57. The band is then isolated, purified and ligated with the RGD oligo.
[0372] Another method that may be utilized to randomly cleave DNA is the endonuclease V method (Kiyazaki, K., Nucleic Acids Res., 2002, 30(24): e139). Endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites. This method is also expected to randomly cleave DNA, the frequency is simply determined by adjusting the concentration of dUTP in the polymerase chain reaction. Although the cleavage sites are always two or three bases downstream of a thymidine (substituted by uracil) site, this method is expected to produce much fewer hot and cold spots than other methodologies.
[0373] The randomly linearized plasmids are ligated with the cRGD oligonucleotides. The resultant pSVV capsid library is then amplified, such that a population of polynucleotides encoding the capsid region with randomly inserted cRGD regions can be purified (see FIGS. 57 and 58). The population of capsid polynucleotides is then subcloned into a vector containing the full-length SVV sequence minus the capsid region, such that a library of full-length SVV sequences are generated (where the library manifests sequence diversity in the capsid region as the cRGD sequence is randomly inserted). This library is then reverse transcribed into RNA, and the RNA is transfected into a permissive cell line to generate a population of SVV particles having different capsids (see FIG. 59). Once this RGD-SVV population of virus particles is recovered ("RGD-SVV library"), a number of viruses (i.e., 10 or more) will be randomly picked for sequencing to confirm the insertion of the RGD sequence and diversity of insertion site.
[0374] In Vitro Selection of the RGD-Displaying SVV Library.
[0375] The SVV-RGD library is screened to determine which insertion site enabled an expanded tropism of SVV. The RGD-SVV library is allowed to infect αVβ5 integrin-expressing NSCLC lines (non-small cell lung cancer cell lines, i.e., A549 expressing αVβ5). Only those SVV derivatives that contain a functional and properly displayed RGD motif can infect these cells and replicate.
[0376] In vitro screening is carried out by a high throughput automation system (TECAN) that is capable of liquid handling, concurrent incubation of 20 cell lines and measurement in 384-well plates (see FIG. 62 and FIG. 63). The cells are harvested 30 hr after infection when complete CPE is noticed and then cells are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellets are then resuspended in the cell culture supernatant and subjected to three cycles of freeze and thaw. The resulting suspension is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus is purified by two rounds of CsCl gradients: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A260=9.5×1012 particles (Scraba, D. G. and Palmenberg, A. C., 1999). The process may be repeated multiple times until a sufficient amount of virus is recovered from αVβ5 cells.
[0377] Analysis of Recovered RGD-SVV Derivatives.
[0378] A small pool of individual RGD-displaying SVV derivatives (about 10-50 different derivatives) are analyzed. The viral mixture is diluted and single viral particles are expanded for analysis. Each derivative is tested to determine whether they have gained the ability to infect αVβ5-expressing cells efficiently and specifically. The capsid region of each derivative with this property is then be sequenced to determine the site of RGD insertion. The recovered cRGD-displaying SVV derivatives should possess the following properties: (1) the original properties of the virus are still intact; and (2) the derivatives have gained the ability to infect cells that express high levels of integrins that bind to RGD. This approach aims to identify one or more sites that enable an expanded tropism with RGD insertion, such that random oligonucleotides can be inserted into these sites to generate SVV derivatives with altered tropism.
[0379] The sequenced cRGD-SVV derivatives are numbered and ranked by their binding abilities to integrin. To test the binding activity, recombinant β2 integrin is immobilized on a 96-well microtiter plate in PBS, washed twice with PBS, blocked with 3% BSA in PBS, and then incubated with a unique RGD-displaying virus. The native virus without peptide insertions is used as a negative control. After 1-5 hr of incubation, the wells are washed at least three times with PBS. Then, the viruses that are bound to the plate will be detected by anti-SVV antibodies. RGD peptide or antibodies against integrin should be able to compete with the binding of the RGD-SVV derivatives to the integrin-bound plate.
[0380] The cRGD-SVV derivatives (20) that have the strongest binding to integrin are analyzed to determine the `successful` location(s) of cRGD oligonucleotide insertion. The insertion sites provide insights into the tropism of SVV. Based on the analysis of the insertion sites and other known structures, an ideal location to place a random peptide library can be determined (this method is an alternative method for generating SVV derivatives, where oligonucleotides (known sequence or random sequence) are inserted into random locations in the capsid). SVV derivatives generated with random sequence oligonucleotides are constructed in essentially the same manner as described above for the RGD-SVV library, except for two additional and novel methodologies. To avoid unwanted stop codons and deleterious amino acid insertions (e.g. cysteines or prolines) within a desired coding region, TRIM (trinucleotide-mutagenesis) technology developed by Morphosys (Munich, Germany) can be used to generate random oligonucleotides for capsid insertion. TRIM utilizes tri-nucleotides which only code for amino acids at the desired position (Virnekas, B. et al., Nucleic Acids Res, 1994, 22(25): 5600-5607). The random-peptide displaying SVV with a diversity of 108 is believed to be sufficient and expected to yield peptides that specifically direct the virus to targeted tumor tissues. Random-peptide displaying SVV is tested in vitro as described above, or in vivo using tumor-bearing mice.
Example 17
Serum Studies
[0381] Pigs are a permissive host for the USDA virus isolates identified above. The isolate MN 88-36695 was inoculated into a gnotobiotic pig and antisera generated (GP102). The antisera binds to all of the other USDA isolates listed above and to SVV. The antisera does not react with 24 common porcine virus pathogens indicating its specificity. Porcine sera was also tested for neutralizing antibodies to 1278 (Plum Island virus). Sera were collected in the US and 8/29 sera were positive with titers ranging from 1:57 to 1:36,500.
[0382] To test whether the pig is the natural source for SVV, serum samples from various animals were obtained and tested for their ability to act as neutralizing antibodies against SVV infection of permissive cells. The Serum Neutralization Assay is conducted as follows: (1) Dilute various serums 1:2 and 1:4 and serially in increasing dilutions if necessary; (2) Mix with 100 TCID50 of virus (SVV; but any virus can be tested to determine whether a serum can neutralize its infection); (3) Incubate at 37° C. for 1 hour; (4) Add the mixture to 1×104 PER.C6® cells (or other permissive cell type); (5) Incubate at 37° C. for 3 days; and (6) Measure CPE using a tetrazolium based dye cytotoxicity (such as MTS) assay. The neutralization titer is defined as the highest dilution of sera that neutralizes SVV (or other virus in question) at 100%.
[0383] The serum neutralization results showed that there is a minimal or no presence of neutralizing antibodies in human and primate populations. In one experiment, 0/22 human sera contained neutralizing antibodies to SVV. In another experiment, only 1/28 human sera contained neutralizing antibodies. In a third experiment, 0/50 human sera from Amish farmers were neutralizing. In another experiment, 0/52 primate sera from four species were neutralizing.
[0384] The serum neutralization results showed that there is a prevalence of SVV neutralizing antibodies in farm animal populations. In one experiment, 27/71 porcine sera from farms were neutralizing. In another experiment, 4/30 porcine sera from a disease-free farm were neutralizing. In another experiment, 10/50 bovine sera were neutralizing. In yet another experiment, 5/35 wild mouse sera were neutralizing.
[0385] Antisera to MN 88-36694 were tested in serum neutralization assays on SVV (see Example 2). Anti-MN 88-36695 gnotobiotic pig serum was able to neutralize infection by SVV (neutralization titer on infection was 1:100 for SVV). As stated above, the antisera binds to all of the other USDA isolates and to SVV, indicating that the herein disclosed USDA isolates are SVV-like picornaviruses due to their serological cross-reactivity with the gnotobiotic pig serum as measured in an indirect immunofluorescence assay.
[0386] These data indicate that SVV is genetically and serologically linked to the porcine USDA virus isolates.
Example 18
SVV and SVV-Like Picornaviruses
[0387] The grouping of the following isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649, was deduced in part from indirect immunofluorescence experiments. Antisera GP102 was raised against isolate MN 88-36695 by inoculation of the virus into a gnotobiotic pig. The antisera binds to all twelve isolates demonstrating that they are serologically related to one another.
[0388] The GP102 antisera was tested in a neutralization assay with SVV. In this assay, serial dilutions of antisera are mixed with a known quantity of SVV (100 TCID50). The mixtures are placed at 37° C. for 1 hour. An aliquot of the mixture is then added to 1×104 PER.C6® cells, or another cell line that is also permissive for SVV, and the mixtures are placed at 37° C. for 3 days. The wells are then checked for a cytopathic effect of the virus (CPE). If the serum contains neutralizing antibodies, it would neutralize the virus and inhibit the infection of the PER.C6® cells by the virus. CPE is measured quantitatively by using a tetrazolium based dye reagent that changes absorbance based on the number of live cells present. The results are expressed as the percent of viable cells of an uninfected control vs. the log dilution of serum, and are shown in FIG. 93. This data indicates that SVV is serologically linked to the porcine USDA virus isolates.
[0389] Additionally, the viral lysate of MN 88-36695 was tested in cytotoxicity assays with four different cell lines and the results are shown in Table 4. The permissivity profile is identical to that of SVV in that NCI-H446 and HEK293 are permissive for SVV, and NCI-H460 and S8 are not. Additionally, MN 88-36695, like SVV, was cytotoxic to PER.C6® cells. Further, polyclonal antisera to SVV raised in mice was used in a neutralization assay along with MN 88-36695 virus. The results are shown in FIG. 94. The anti-SVV antisera neutralized MN 88-36695, further linking SVV to the USDA viruses serologically.
TABLE-US-00015 TABLE 11 MN 88-36695 Cytotoxicity Results Cell Line TCID50 (pfu/ml) Result NCI-H446 1.6 × 10-6 Permissive HEK293 1.3 × 10-2 Permissive NCI-H460 0 Nonpermissive S8 0 Nonpermissive
[0390] Partial genomic sequence analysis of several of the USDA isolates revealed that they are all very closely related to SVV (see FIGS. 87-89 for sequence alignments). Table 12 shows the percent sequence identity between SVV and six of the isolates. It was found that about 95-98% identity exists at the nucleotide (nt) level over 460 nt of the 3' end of the genome encoding 3Dpol and the 3'UTR (FIG. 89). Each of the USDA viruses is unique and is about 95-98% identical to SVV at the nucleotide level.
TABLE-US-00016 TABLE 12 Percent Sequence Identity Between SVV and Six USDA Isolates 1 2 3 4 5 6 7 Virus Name 96.5 99.1 97.2 97.0 97.4 97.0 1 NJ 90-10324 97.0 95.7 94.8 95.0 98.3* 2 CA 13195 97.6 97.2 97.6 97.2 3 IA 89-47752 95.4 96.1 96.3 4 IL 92-48963 98.9 95.2 5 MN 88-36695 95.4 6 NC 88-23626 7 SVV-001 (SVV)
[0391] Further sequencing of parts of the P1 (FIG. 87) and 2C (FIG. 88) genes of two of the isolates has confirmed this close relationship with SVV. The USDA isolates are more highly related to SVV than any other known viruses, including members of the genus Cardiovirus. Sequences from several regions of seven of the USDA viruses were compared with SVV and neighbor-joining trees were constructed (FIGS. 95A and 95B). These trees further confirm the high degree of relation between the viruses, and identifying CA 131395 as SVV's current closest relative.
Sequence CWU
1
1
22715752DNASeneca Valley Virusmodified_base(1)..(2)a, t, c or g
1nntctagccc accatggcaa caagaagagc ttacaggagc tgaatgaaga acagtgggtg
60gaaatgtctg acgattaccg gaccgggaaa aacatgcctt ttcagtctct tggcacatac
120tatcggcccc ctaactggac ttggggtccc aatttcatca acccctatca agtaacggtt
180ttcccacacc aaattctgaa cgcgagaacc tctacctcgg tagacataaa cgtcccatac
240atcggggaga cccccacgca atcctcagag acacagaact cctggaccct cctcgttatg
300gtgctcgttc ccctagacta taaggaagga gccacaactg acccagaaat tacattttct
360gtaaggccta caagtcccta cttcaatggg cttcgcaacc gctacacggc cgggacggac
420gaagaacagg ggcccattcc tacggcaccc agagaaaatt cgcttatgtt tctctcaacc
480ctccctgacg acactgtccc tgcttacggg aatgtgcgta cccctcctgt caattacctc
540cctggtgaaa taaccgacct tttgcaactg gcccgcatac ccactctcat ggcatttgag
600cgggtgcctg aacccgtgcc tgcctcagac acatatgtgc cctacgttgc cgttcccacc
660cagttcgatg acaggcctct catctccttc ccgatcaccc tttcagatcc cgtctatcag
720aacaccctgg ttggcgccat cagttcaaat ttcgccaatt accgtgggtg tatccaaatc
780actctgacat tttgtggacc catgatggcg agagggaaat tcctgctctc gtattctccc
840ccaaatggaa cgcaaccaca gactctttcc gaagctatgc agtgcacata ctctatttgg
900gacataggct tgaactctag ttggaccttc gtcgtcccct acatctcgcc cagtgactac
960cgtgaaactc gagccattac caactcggtt tactccgctg atggttggtt tagcctgcac
1020aagttgacca aaattactct accacctgac tgtccgcaaa gtccctgcat tctctttttc
1080gcttctgctg gtgaggatta cactctccgt ctccccgttg attgtaatcc ttcctatgtg
1140ttccactcca ccgacaacgc cgagaccggg gttattgagg cgggtaacac tgacaccgat
1200ttctctggtg aactggcggc tcctggccct aaccacacta atgtcaagtt cctgtttgat
1260cgatctcgat tattgaatgt aatcaaggta ctggagaagg acgccgtttt cccccgccct
1320ttccctacac aagaaggtgc gcagcaggat gatggttact tttgtcttct gaccccccgc
1380ccaacagtcg cttcccgacc cgccactcgt ttcggcctgt acgccaatcc gtccggcagt
1440ggtgttcttg ctaacacttc actggacttc aatttttata gcttggcctg tttcacttac
1500tttagatcgg accttgaggt tacggtggtc tcactagagc cggatctgga atttgctgta
1560gggtggtttc cttctggcag tgaataccag gcttccagct ttgtctacga ccagctgcat
1620gtgcccttcc actttactgg gcgcactccc cgcgctttcg ctagcaaggg tgggaaggta
1680tctttcgtgc tcccttggaa ctctgtctcg tctgtgctcc ccgtgcgctg ggggggggct
1740tccaagctct cttctgctac gcggggtcta ccggcgcatg ctgattgggg gactatttac
1800gcctttgtcc cccgtcctaa tgagaagaaa agcaccgctg taaaacacgt ggccgtgtac
1860attcggtaca agaacgcacg tgcctggtgc cccagcatgc ttccctttcg cagctacaag
1920cagaagatgc tgatgcaatc tggcgatatc gagaccaatc ctggtcctgc ttctgacaac
1980ccaattttgg agtttcttga agcagaaaat gatctagtca ctctggcctc tctctggaag
2040atggtgcact ctgttcaaca gacctggaga aagtatgtga agaacgatga tttttggccc
2100aatttactca gcgagctagt gggggaaggc tctgtcgcct tggccgccac gctatccaac
2160caagcttcag taaaggctct tttgggcctg cactttctct ctcgggggct caattacact
2220gacttttact ctttactgat agagaaatgc tctagtttct ttaccgtaga accacctcct
2280ccaccagctg aaaacctgat gaccaagccc tcagtgaagt cgaaattccg aaaactgttt
2340aagatgcaag gacccatgga caaagtcaaa gactggaacc aaatagctgc cggcttgaag
2400aattttcaat ttgttcgtga cctagtcaaa gaggtggtcg attggctgca ggcctggatc
2460aacaaagaga aagccagccc tgtcctccag taccagttgg agatgaagaa gctcgggcct
2520gtggccttgg ctcatgacgc tttcatggct ggttccgggc cccctcttag cgacgaccag
2580attgaatacc tccagaacct caaatctctt gccctaacac tggggaagac taatttggcc
2640caaagtctca ccactatgat caatgccaaa caaagttcag cccaacgagt tgaacccgtt
2700gtggtggtcc ttagaggcaa gccgggatgc ggcaagggct tggcctctac gttgattgcc
2760caggctgtgt ccaagcgcct ctatggctcc caaagtgtat attctcttcc cccagatcca
2820gatttcttcg atggatacaa aggacagttc gtgaccttga tggatgattt gggacaaaac
2880ccggatggac aagatttccc caccttttgt cagatggtgt cgaccgccca atttctcccc
2940aacatggcgg accttgcaga gaaagggcgt ccctttacct ccaatctcat cattgcaact
3000acaaatctcc cccacttcag tcctgtcacc attgctgatc cttctgcagt ctctcgccgt
3060atcaactacg atctgactct agaagtatct gaggcctaca agaaacacac acggctgaat
3120tttgacttgg ctttcaggcg cacagacgcc ccccccattt atccttttgc tgcccatgtg
3180ccctttgtgg acgtagctgt gcgcttcaaa aatggtcacc agaattttaa tctcctagag
3240ttggtcgatt ccatttgtac agacattcga gccaagcaac aaggtgcccg aaacatgcag
3300actctggttc tacagagccc caacgagaat gatgacaccc ccgtcgacga ggcgttgggt
3360agagttctct cccccgctgc ggtcgatgag gcgcttgtcg acctcactcc agaggccgac
3420ccggttggcc gtttggctat tcttgccaag ctaggtcttg ccctagctgc ggtcacccct
3480ggtctgataa tcttggcagt gggactctac aggtacttct ctggctctga tgcagaccaa
3540gaagaaacag aaagtgaggg atctgtcaag gcacccagga gcgaaaatgc ttatgacggc
3600ccgaagaaaa actctaagcc ccctggagca ctctctctca tggaaatgca acagcccaac
3660gtggacatgg gctttgaggc tgcggtcgct aagaaagtgg tcgtccccat taccttcatg
3720gttcccaaca gaccttctgg gcttacacag tccgctcttc tggtgaccgg ccggaccttc
3780ctaatcaatg aacatacatg gtccaatccc tcctggacca gcttcacaat ccgcggtgag
3840gtacacactc gtgatgagcc cttccaaacg gttcatttca ctcaccacgg tattcccaca
3900gatctgatga tggtacgtct cggaccgggc aattctttcc ctaacaatct agacaagttt
3960ggacttgacc agatgccggc acgcaactcc cgtgtggttg gcgtttcgtc cagttacgga
4020aacttcttct tctctggaaa tttcctcgga tttgttgatt ccgtcacctc tgaacaagga
4080acttacgcaa gactctttag gtacagggtg acgacctaca aaggatggtg cggctcggcc
4140ctggtctgtg aggccggtgg cgtccgacgc atcattggcc tgcattctgc tggcgccgcc
4200ggtatcggcg ccgggaccta tatctcaaaa ttaggactaa tcaaagccct gaaacacctc
4260ggtgaacctt tggccacaat gcaaggactg atgactgaat tagagcctgg aatcaccgta
4320catgtacccc ggaaatccaa attgagaaag acgaccgcac acgcggtgta caaaccggag
4380tttgagcctg ctgtgttgtc aaaatttgat cccagactga acaaggatgt tgacttggat
4440gaagtaattt ggtctaaaca cactgccaat gtcccttacc aacctccttt gttctacaca
4500tacatgtcag agtacgctca tcgagtcttc tccttcttgg ggaaagacaa tgacattctg
4560accgtcaaag aagcaattct gggcatcccc ggactagacc ccatggatcc ccacacagct
4620ccgggtctgc cttacgccat caacggcctt cgacgtactg atctcgtcga ttttgtgaac
4680ggtacagtag atgcggcgct ggctgtacaa atccagaaat tcttagacgg tgactactct
4740gaccatgtct tccaaacttt tctgaaagat gagatcagac cctcagagaa agtccgagcg
4800ggaaaaaccc gcattgttga tgtgccctcc ctggcgcatt gcattgtggg cagaatgttg
4860cttgggcgct ttgctgccaa gtttcaatcc catcctggct ttctcctcgg ctctgctatc
4920gggtctgacc ctgatgtttt ctggaccgtc ataggggctc aactcgaggg gagaaagaac
4980acgtatgacg tggactacag tgcctttgac tcttcacacg gcactggctc cttcgaggct
5040ctcatctctc actttttcac cgtggacaat ggttttagcc ctgcgctggg accgtatctc
5100agatccctgg ctgtctcggt gcacgcttac ggcgagcgtc gcatcaagat taccggtggc
5160ctcccctccg gttgtgccgc gaccagcctg ctgaacacag tgctcaacaa tgtgatcatc
5220aggactgctc tggcattgac ttacaaggaa tttgagtatg acacggttga tatcatcgcc
5280tacggtgacg accttctggt tggcacggat tacgatctgg acttcaatga ggtggcacga
5340cgcgctgcca agttggggta taagatgact cctgccaaca agggttctgt cttccctccg
5400acttcctctc tttccgatgc tgtttttcta aagcgcaaat tcgtccaaaa caacgacggc
5460ttatacaaac cagttatgga tttaaagaat ttggaagcca tgctctccta cttcaaacca
5520ggaacactac tcgagaagct gcaatctgtt tctatgttgg ctcaacattc tggaaaagaa
5580gaatatgata gattgatgca ccccttcgct gactacggtg ccgtaccgag tcacgagtac
5640ctgcaggcaa gatggagggc cttgttcgac tgacccagat agcccaaggc gcttcggtgc
5700tgccggcgat tctgggagaa ctcagtcgga acagaaaaaa aaaaaaaaaa aa
575221890PRTSeneca Valley VirusMOD_RES(1)variable amino acid 2Xaa Leu Ala
His His Gly Asn Lys Lys Ser Leu Gln Glu Leu Asn Glu 1 5
10 15 Glu Gln Trp Val Glu Met Ser Asp
Asp Tyr Arg Thr Gly Lys Asn Met 20 25
30 Pro Phe Gln Ser Leu Gly Thr Tyr Tyr Arg Pro Pro Asn
Trp Thr Trp 35 40 45
Gly Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe Pro His Gln 50
55 60 Ile Leu Asn Ala Arg
Thr Ser Thr Ser Val Asp Ile Asn Val Pro Tyr 65 70
75 80Ile Gly Glu Thr Pro Thr Gln Ser Ser Glu
Thr Gln Asn Ser Trp Thr 85 90
95 Leu Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly Ala
Thr 100 105 110 Thr
Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser Pro Tyr Phe 115
120 125 Asn Gly Leu Arg Asn Arg
Tyr Thr Ala Gly Thr Asp Glu Glu Gln Gly 130 135
140 Pro Ile Pro Thr Ala Pro Arg Glu Asn Ser Leu
Met Phe Leu Ser Thr 145 150 155
160Leu Pro Asp Asp Thr Val Pro Ala Tyr Gly Asn Val Arg Thr Pro Pro
165 170 175 Val Asn
Tyr Leu Pro Gly Glu Ile Thr Asp Leu Leu Gln Leu Ala Arg 180
185 190 Ile Pro Thr Leu Met Ala
Phe Glu Arg Val Pro Glu Pro Val Pro Ala 195 200
205 Ser Asp Thr Tyr Val Pro Tyr Val Ala Val Pro
Thr Gln Phe Asp Asp 210 215 220
Arg Pro Leu Ile Ser Phe Pro Ile Thr Leu Ser Asp Pro Val Tyr Gln
225 230 235 240Asn Thr
Leu Val Gly Ala Ile Ser Ser Asn Phe Ala Asn Tyr Arg Gly
245 250 255 Cys Ile Gln Ile Thr Leu
Thr Phe Cys Gly Pro Met Met Ala Arg Gly 260
265 270 Lys Phe Leu Leu Ser Tyr Ser Pro Pro Asn
Gly Thr Gln Pro Gln Thr 275 280
285 Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile
Gly Leu 290 295 300
Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp Tyr 305
310 315 320Arg Glu Thr Arg Ala
Ile Thr Asn Ser Val Tyr Ser Ala Asp Gly Trp 325
330 335 Phe Ser Leu His Lys Leu Thr Lys Ile Thr
Leu Pro Pro Asp Cys Pro 340 345
350 Gln Ser Pro Cys Ile Leu Phe Phe Ala Ser Ala Gly Glu Asp Tyr
Thr 355 360 365 Leu
Arg Leu Pro Val Asp Cys Asn Pro Ser Tyr Val Phe His Ser Thr 370
375 380 Asp Asn Ala Glu Thr Gly
Val Ile Glu Ala Gly Asn Thr Asp Thr Asp 385 390
395 400Phe Ser Gly Glu Leu Ala Ala Pro Gly Pro Asn
His Thr Asn Val Lys 405 410
415 Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val Leu Glu
420 425 430 Lys Asp
Ala Val Phe Pro Arg Pro Phe Pro Thr Gln Glu Gly Ala Gln 435
440 445 Gln Asp Asp Gly Tyr Phe Cys
Leu Leu Thr Pro Arg Pro Thr Val Ala 450 455
460 Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Ala Asn
Pro Ser Gly Ser 465 470 475
480Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser Leu Ala
485 490 495 Cys Phe Thr
Tyr Phe Arg Ser Asp Leu Glu Val Thr Val Val Ser Leu 500
505 510 Glu Pro Asp Leu Glu Phe Ala Val
Gly Trp Phe Pro Ser Gly Ser Glu 515 520
525 Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val
Pro Phe His 530 535 540
Phe Thr Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly Gly Lys Val 545
550 555 560Ser Phe Val Leu
Pro Trp Asn Ser Val Ser Ser Val Leu Pro Val Arg 565
570 575 Trp Gly Gly Ala Ser Lys Leu Ser Ser
Ala Thr Arg Gly Leu Pro Ala 580 585
590 His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Val Pro Arg Pro
Asn Glu 595 600 605
Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Ile Arg Tyr Lys 610
615 620 Asn Ala Arg Ala Trp
Cys Pro Ser Met Leu Pro Phe Arg Ser Tyr Lys 625 630
635 640Gln Lys Met Leu Met Gln Ser Gly Asp Ile
Glu Thr Asn Pro Gly Pro 645 650
655 Ala Ser Asp Asn Pro Ile Leu Glu Phe Leu Glu Ala Glu Asn Asp
Leu 660 665 670 Val
Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val Gln Gln Thr 675
680 685 Trp Arg Lys Tyr Val Lys
Asn Asp Asp Phe Trp Pro Asn Leu Leu Ser 690 695
700 Glu Leu Val Gly Glu Gly Ser Val Ala Leu Ala
Ala Thr Leu Ser Asn 705 710 715
720Gln Ala Ser Val Lys Ala Leu Leu Gly Leu His Phe Leu Ser Arg Gly
725 730 735 Leu Asn
Tyr Thr Asp Phe Tyr Ser Leu Leu Ile Glu Lys Cys Ser Ser 740
745 750 Phe Phe Thr Val Glu Pro Pro
Pro Pro Pro Ala Glu Asn Leu Met Thr 755 760
765 Lys Pro Ser Val Lys Ser Lys Phe Arg Lys Leu Phe
Lys Met Gln Gly 770 775 780
Pro Met Asp Lys Val Lys Asp Trp Asn Gln Ile Ala Ala Gly Leu Lys
785 790 795 800Asn Phe
Gln Phe Val Arg Asp Leu Val Lys Glu Val Val Asp Trp Leu
805 810 815 Gln Ala Trp Ile Asn Lys
Glu Lys Ala Ser Pro Val Leu Gln Tyr Gln 820
825 830 Leu Glu Met Lys Lys Leu Gly Pro Val Ala
Leu Ala His Asp Ala Phe 835 840
845 Met Ala Gly Ser Gly Pro Pro Leu Ser Asp Asp Gln Ile Glu
Tyr Leu 850 855 860
Gln Asn Leu Lys Ser Leu Ala Leu Thr Leu Gly Lys Thr Asn Leu Ala 865
870 875 880Gln Ser Leu Thr Thr
Met Ile Asn Ala Lys Gln Ser Ser Ala Gln Arg 885
890 895 Val Glu Pro Val Val Val Val Leu Arg Gly
Lys Pro Gly Cys Gly Lys 900 905
910 Gly Leu Ala Ser Thr Leu Ile Ala Gln Ala Val Ser Lys Arg Leu
Tyr 915 920 925 Gly
Ser Gln Ser Val Tyr Ser Leu Pro Pro Asp Pro Asp Phe Phe Asp 930
935 940 Gly Tyr Lys Gly Gln Phe
Val Thr Leu Met Asp Asp Leu Gly Gln Asn 945 950
955 960Pro Asp Gly Gln Asp Phe Ser Thr Phe Cys Gln
Met Val Ser Thr Ala 965 970
975 Gln Phe Leu Pro Asn Met Ala Asp Leu Ala Glu Lys Gly Arg Pro Phe
980 985 990 Thr Ser
Asn Leu Ile Ile Ala Thr Thr Asn Leu Pro His Phe Ser Pro 995
1000 1005 Val Thr Ile Ala Asp Pro
Ser Ala Val Ser Arg Arg Ile Asn Tyr Asp 1010 1015
1020 Leu Thr Leu Glu Val Ser Glu Ala Tyr Lys
Lys His Thr Arg Leu Asn 1025 1030 1035
1040Phe Asp Leu Ala Phe Arg Arg Thr Asp Ala Pro Pro Ile Tyr Pro
Phe 1045 1050 1055 Ala
Ala His Val Pro Phe Val Asp Val Ala Val Arg Phe Lys Asn Gly
1060 1065 1070 His Gln Asn Phe Asn
Leu Leu Glu Leu Val Asp Ser Ile Cys Thr Asp 1075
1080 1085 Ile Arg Ala Lys Gln Gln Gly Ala Arg
Asn Met Gln Thr Leu Val Leu 1090 1095
1100 Gln Ser Pro Asn Glu Asn Asp Asp Thr Pro Val Asp Glu
Ala Leu Gly 1105 1110 1115
1120Arg Val Leu Ser Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu Thr
1125 1130 1135 Pro Glu Ala
Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly 1140
1145 1150 Leu Ala Leu Ala Ala Val Thr
Pro Gly Leu Ile Ile Leu Ala Val Gly 1155 1160
1165 Leu Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp
Gln Glu Glu Thr Glu 1170 1175 1180
Ser Glu Gly Ser Val Lys Ala Pro Arg Ser Glu Asn Ala Tyr Asp
Gly 1185 1190 1195 1200Pro
Lys Lys Asn Ser Lys Pro Pro Gly Ala Leu Ser Leu Met Glu Met
1205 1210 1215 Gln Gln Pro Asn Val
Asp Met Gly Phe Glu Ala Ala Val Ala Lys Lys 1220
1225 1230 Val Val Val Pro Ile Thr Phe Met Val
Pro Asn Arg Pro Ser Gly Leu 1235 1240
1245 Thr Gln Ser Ala Leu Leu Val Thr Gly Arg Thr Phe Leu
Ile Asn Glu 1250 1255 1260
His Thr Trp Ser Asn Pro Ser Trp Thr Ser Phe Thr Ile Arg Gly Glu 1265
1270 1275 1280Val His Thr Arg
Asp Glu Pro Phe Gln Thr Val His Phe Thr His His 1285
1290 1295 Gly Ile Pro Thr Asp Leu Met Met
Val Arg Leu Gly Pro Gly Asn Ser 1300 1305
1310 Phe Pro Asn Asn Leu Asp Lys Phe Gly Leu Asp Gln
Met Pro Ala Arg 1315 1320 1325
Asn Ser Arg Val Val Gly Val Ser Ser Ser Tyr Gly Asn Phe Phe Phe
1330 1335 1340 Ser Gly Asn
Phe Leu Gly Phe Val Asp Ser Val Thr Ser Glu Gln Gly 1345
1350 1355 1360Thr Tyr Ala Arg Leu Phe Arg
Tyr Arg Val Thr Thr Tyr Lys Gly Trp 1365
1370 1375 Cys Gly Ser Ala Leu Val Cys Glu Ala Gly Gly
Val Arg Arg Ile Ile 1380 1385
1390 Gly Leu His Ser Ala Gly Ala Ala Gly Ile Gly Ala Gly Thr Tyr
Ile 1395 1400 1405 Ser
Lys Leu Gly Leu Ile Lys Ala Leu Lys His Leu Gly Glu Pro Leu 1410
1415 1420 Ala Thr Met Gln Gly
Leu Met Thr Glu Leu Glu Pro Gly Ile Thr Val 1425 1430
1435 1440His Val Pro Arg Lys Ser Lys Leu Arg Lys
Thr Thr Ala His Ala Val 1445 1450
1455 Tyr Lys Pro Glu Phe Glu Pro Ala Val Leu Ser Lys Phe Asp
Pro Arg 1460 1465 1470
Leu Asn Lys Asp Val Asp Leu Asp Glu Val Ile Trp Ser Lys His Thr
1475 1480 1485 Ala Asn Val Pro
Tyr Gln Pro Pro Leu Phe Tyr Thr Tyr Met Ser Glu 1490
1495 1500 Tyr Ala His Arg Val Phe Ser Phe
Leu Gly Lys Asp Asn Asp Ile Leu 1505 1510
1515 1520Thr Val Lys Glu Ala Ile Leu Gly Ile Pro Gly Leu
Asp Pro Met Asp 1525 1530
1535 Pro His Thr Ala Pro Gly Leu Pro Tyr Ala Ile Asn Gly Leu Arg Arg
1540 1545 1550 Thr Asp
Leu Val Asp Phe Val Asn Gly Thr Val Asp Ala Ala Leu Ala 1555
1560 1565 Val Gln Ile Gln Lys Phe
Leu Asp Gly Asp Tyr Ser Asp His Val Phe 1570 1575
1580 Gln Thr Phe Leu Lys Asp Glu Ile Arg Pro
Ser Glu Lys Val Arg Ala 1585 1590 1595
1600Gly Lys Thr Arg Ile Val Asp Val Pro Ser Leu Ala His Cys Ile
Val 1605 1610 1615 Gly
Arg Met Leu Leu Gly Arg Phe Ala Ala Lys Phe Gln Ser His Pro
1620 1625 1630 Gly Phe Leu Leu Gly
Ser Ala Ile Gly Ser Asp Pro Asp Val Phe Trp 1635
1640 1645 Thr Val Ile Gly Ala Gln Leu Glu Gly
Arg Lys Asn Thr Tyr Asp Val 1650 1655
1660 Asp Tyr Ser Ala Phe Asp Ser Ser His Gly Thr Gly Ser
Phe Glu Ala 1665 1670 1675
1680Leu Ile Ser His Phe Phe Thr Val Asp Asn Gly Phe Ser Pro Ala Leu
1685 1690 1695 Gly Pro Tyr
Leu Arg Ser Leu Ala Val Ser Val His Ala Tyr Gly Glu 1700
1705 1710 Arg Arg Ile Lys Ile Thr Gly
Gly Leu Pro Ser Gly Cys Ala Ala Thr 1715 1720
1725 Ser Leu Leu Asn Thr Val Leu Asn Asn Val Ile
Ile Arg Thr Ala Leu 1730 1735 1740
Ala Leu Thr Tyr Lys Glu Phe Glu Tyr Asp Thr Val Asp Ile Ile
Ala 1745 1750 1755 1760Tyr
Gly Asp Asp Leu Leu Val Gly Thr Asp Tyr Asp Leu Asp Phe Asn
1765 1770 1775 Glu Val Ala Arg Arg
Ala Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala 1780
1785 1790 Asn Lys Gly Ser Val Phe Pro Pro Thr
Ser Ser Leu Ser Asp Ala Val 1795 1800
1805 Phe Leu Lys Arg Lys Phe Val Gln Asn Asn Asp Gly Leu
Tyr Lys Pro 1810 1815 1820
Val Met Asp Leu Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys Pro 1825
1830 1835 1840Gly Thr Leu Leu
Glu Lys Leu Gln Ser Val Ser Met Leu Ala Gln His 1845
1850 1855 Ser Gly Lys Glu Glu Tyr Asp Arg
Leu Met His Pro Phe Ala Asp Tyr 1860 1865
1870 Gly Ala Val Pro Ser His Glu Tyr Leu Gln Ala Arg
Trp Arg Ala Leu 1875 1880 1885
Phe Asp
18903426DNASeneca Valley Virus 3ctagcccacc atggcaacaa gaagagctta
caggagctga atgaagaaca gtgggtggaa 60atgtctgacg attaccggac cgggaaaaac
atgccttttc agtctcttgg cacatactat 120cggcccccta actggacttg gggtcccaat
ttcatcaacc cctatcaagt aacggttttc 180ccacaccaaa ttctgaacgc gagaacctct
acctcggtag acataaacgt cccatacatc 240ggggagaccc ccacgcaatc ctcagagaca
cagaactcct ggaccctcct cgttatggtg 300ctcgttcccc tagactataa ggaaggagcc
acaactgacc cagaaattac attttctgta 360aggcctacaa gtccctactt caatgggctt
cgcaaccgct acacggccgg gacggacgaa 420gaacag
4264142PRTSeneca Valley Virus 4Leu Ala
His His Gly Asn Lys Lys Ser Leu Gln Glu Leu Asn Glu Glu 1
5 10 15 Gln Trp Val Glu Met Ser Asp
Asp Tyr Arg Thr Gly Lys Asn Met Pro 20 25
30 Phe Gln Ser Leu Gly Thr Tyr Tyr Arg Pro Pro Asn
Trp Thr Trp Gly 35 40 45
Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe Pro His Gln Ile
50 55 60 Leu Asn Ala Arg
Thr Ser Thr Ser Val Asp Ile Asn Val Pro Tyr Ile 65 70
75 80Gly Glu Thr Pro Thr Gln Ser Ser Glu
Thr Gln Asn Ser Trp Thr Leu 85 90
95 Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly Ala
Thr Thr 100 105 110
Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser Pro Tyr Phe Asn
115 120 125 Gly Leu Arg Asn
Arg Tyr Thr Ala Gly Thr Asp Glu Glu Gln 130
135 140 5717DNASeneca Valley Virus 5gggcccattc
ctacggcacc cagagaaaat tcgcttatgt ttctctcaac cctccctgac 60gacactgtcc
ctgcttacgg gaatgtgcgt acccctcctg tcaattacct ccctggtgaa 120ataaccgacc
ttttgcaact ggcccgcata cccactctca tggcatttga gcgggtgcct 180gaacccgtgc
ctgcctcaga cacatatgtg ccctacgttg ccgttcccac ccagttcgat 240gacaggcctc
tcatctcctt cccgatcacc ctttcagatc ccgtctatca gaacaccctg 300gttggcgcca
tcagttcaaa tttcgccaat taccgtgggt gtatccaaat cactctgaca 360ttttgtggac
ccatgatggc gagagggaaa ttcctgctct cgtattctcc cccaaatgga 420acgcaaccac
agactctttc cgaagctatg cagtgcacat actctatttg ggacataggc 480ttgaactcta
gttggacctt cgtcgtcccc tacatctcgc ccagtgacta ccgtgaaact 540cgagccatta
ccaactcggt ttactccgct gatggttggt ttagcctgca caagttgacc 600aaaattactc
taccacctga ctgtccgcaa agtccctgca ttctcttttt cgcttctgct 660ggtgaggatt
acactctccg tctccccgtt gattgtaatc cttcctatgt gttccac
7176239PRTSeneca Valley Virus 6Gly Pro Ile Pro Thr Ala Pro Arg Glu Asn
Ser Leu Met Phe Leu Ser 1 5 10
15 Thr Leu Pro Asp Asp Thr Val Pro Ala Tyr Gly Asn Val Arg Thr
Pro 20 25 30 Pro
Val Asn Tyr Leu Pro Gly Glu Ile Thr Asp Leu Leu Gln Leu Ala 35
40 45 Arg Ile Pro Thr Leu Met
Ala Phe Glu Arg Val Pro Glu Pro Val Pro 50 55
60 Ala Ser Asp Thr Tyr Val Pro Tyr Val Ala Val
Pro Thr Gln Phe Asp 65 70 75
80Asp Arg Pro Leu Ile Ser Phe Pro Ile Thr Leu Ser Asp Pro Val Tyr
85 90 95 Gln Asn Thr
Leu Val Gly Ala Ile Ser Ser Asn Phe Ala Asn Tyr Arg 100
105 110 Gly Cys Ile Gln Ile Thr Leu Thr
Phe Cys Gly Pro Met Met Ala Arg 115 120
125 Gly Lys Phe Leu Leu Ser Tyr Ser Pro Pro Asn Gly Thr
Gln Pro Gln 130 135 140
Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly 145
150 155 160Leu Asn Ser Ser
Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp 165
170 175 Tyr Arg Glu Thr Arg Ala Ile Thr Asn
Ser Val Tyr Ser Ala Asp Gly 180 185
190 Trp Phe Ser Leu His Lys Leu Thr Lys Ile Thr Leu Pro Pro
Asp Cys 195 200 205
Pro Gln Ser Pro Cys Ile Leu Phe Phe Ala Ser Ala Gly Glu Asp Tyr 210
215 220 Thr Leu Arg Leu Pro
Val Asp Cys Asn Pro Ser Tyr Val Phe His 225 230
235 7777DNASeneca Valley Virus 7tccaccgaca
acgccgagac cggggttatt gaggcgggta acactgacac cgatttctct 60ggtgaactgg
cggctcctgg ccctaaccac actaatgtca agttcctgtt tgatcgatct 120cgattattga
atgtaatcaa ggtactggag aaggacgccg ttttcccccg ccctttccct 180acacaagaag
gtgcgcagca ggatgatggt tacttttgtc ttctgacccc ccgcccaaca 240gtcgcttccc
gacccgccac tcgtttcggc ctgtacgcca atccgtccgg cagtggtgtt 300cttgctaaca
cttcactgga cttcaatttt tatagcttgg cctgtttcac ttactttaga 360tcggaccttg
aggttacggt ggtctcacta gagccggatc tggaatttgc tgtagggtgg 420tttccttctg
gcagtgaata ccaggcttcc agctttgtct acgaccagct gcatgtgccc 480ttccacttta
ctgggcgcac tccccgcgct ttcgctagca agggtgggaa ggtatctttc 540gtgctccctt
ggaactctgt ctcgtctgtg ctccccgtgc gctggggggg ggcttccaag 600ctctcttctg
ctacgcgggg tctaccggcg catgctgatt gggggactat ttacgccttt 660gtcccccgtc
ctaatgagaa gaaaagcacc gctgtaaaac acgtggccgt gtacattcgg 720tacaagaacg
cacgtgcctg gtgccccagc atgcttccct ttcgcagcta caagcag
7778259PRTSeneca Valley Virus 8Ser Thr Asp Asn Ala Glu Thr Gly Val Ile
Glu Ala Gly Asn Thr Asp 1 5 10
15 Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Pro Asn His Thr
Asn 20 25 30 Val
Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val 35
40 45 Leu Glu Lys Asp Ala Val
Phe Pro Arg Pro Phe Pro Thr Gln Glu Gly 50 55
60 Ala Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu
Thr Pro Arg Pro Thr 65 70 75
80Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Ala Asn Pro Ser
85 90 95 Gly Ser Gly
Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser 100
105 110 Leu Ala Cys Phe Thr Tyr Phe Arg
Ser Asp Leu Glu Val Thr Val Val 115 120
125 Ser Leu Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe
Pro Ser Gly 130 135 140
Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val Pro 145
150 155 160Phe His Phe Thr
Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly Gly 165
170 175 Lys Val Ser Phe Val Leu Pro Trp Asn
Ser Val Ser Ser Val Leu Pro 180 185
190 Val Arg Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg
Gly Leu 195 200 205
Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Val Pro Arg Pro 210
215 220 Asn Glu Lys Lys Ser
Thr Ala Val Lys His Val Ala Val Tyr Ile Arg 225 230
235 240Tyr Lys Asn Ala Arg Ala Trp Cys Pro Ser
Met Leu Pro Phe Arg Ser 245 250
255 Tyr Lys Gln
942DNASeneca Valley Virus 9aagatgctga tgcaatctgg cgatatcgag
accaatcctg gt 421014PRTSeneca Valley Virus 10Lys
Met Leu Met Gln Ser Gly Asp Ile Glu Thr Asn Pro Gly 1
5 10 11384DNASeneca Valley Virus
11cctgcttctg acaacccaat tttggagttt cttgaagcag aaaatgatct agtcactctg
60gcctctctct ggaagatggt gcactctgtt caacagacct ggagaaagta tgtgaagaac
120gatgattttt ggcccaattt actcagcgag ctagtggggg aaggctctgt cgccttggcc
180gccacgctat ccaaccaagc ttcagtaaag gctcttttgg gcctgcactt tctctctcgg
240gggctcaatt acactgactt ttactcttta ctgatagaga aatgctctag tttctttacc
300gtagaaccac ctcctccacc agctgaaaac ctgatgacca agccctcagt gaagtcgaaa
360ttccgaaaac tgtttaagat gcaa
38412128PRTSeneca Valley Virus 12Pro Ala Ser Asp Asn Pro Ile Leu Glu Phe
Leu Glu Ala Glu Asn Asp 1 5 10
15 Leu Val Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val Gln
Gln 20 25 30 Thr
Trp Arg Lys Tyr Val Lys Asn Asp Asp Phe Trp Pro Asn Leu Leu 35
40 45 Ser Glu Leu Val Gly Glu
Gly Ser Val Ala Leu Ala Ala Thr Leu Ser 50 55
60 Asn Gln Ala Ser Val Lys Ala Leu Leu Gly Leu
His Phe Leu Ser Arg 65 70 75
80Gly Leu Asn Tyr Thr Asp Phe Tyr Ser Leu Leu Ile Glu Lys Cys Ser
85 90 95 Ser Phe Phe
Thr Val Glu Pro Pro Pro Pro Pro Ala Glu Asn Leu Met 100
105 110 Thr Lys Pro Ser Val Lys Ser Lys
Phe Arg Lys Leu Phe Lys Met Gln 115 120
125 13966DNASeneca Valley Virus 13ggacccatgg acaaagtcaa
agactggaac caaatagctg ccggcttgaa gaattttcaa 60tttgttcgtg acctagtcaa
agaggtggtc gattggctgc aggcctggat caacaaagag 120aaagccagcc ctgtcctcca
gtaccagttg gagatgaaga agctcgggcc tgtggccttg 180gctcatgacg ctttcatggc
tggttccggg ccccctctta gcgacgacca gattgaatac 240ctccagaacc tcaaatctct
tgccctaaca ctggggaaga ctaatttggc ccaaagtctc 300accactatga tcaatgccaa
acaaagttca gcccaacgag ttgaacccgt tgtggtggtc 360cttagaggca agccgggatg
cggcaagggc ttggcctcta cgttgattgc ccaggctgtg 420tccaagcgcc tctatggctc
ccaaagtgta tattctcttc ccccagatcc agatttcttc 480gatggataca aaggacagtt
cgtgaccttg atggatgatt tgggacaaaa cccggatgga 540caagatttcc ccaccttttg
tcagatggtg tcgaccgccc aatttctccc caacatggcg 600gaccttgcag agaaagggcg
tccctttacc tccaatctca tcattgcaac tacaaatctc 660ccccacttca gtcctgtcac
cattgctgat ccttctgcag tctctcgccg tatcaactac 720gatctgactc tagaagtatc
tgaggcctac aagaaacaca cacggctgaa ttttgacttg 780gctttcaggc gcacagacgc
cccccccatt tatccttttg ctgcccatgt gccctttgtg 840gacgtagctg tgcgcttcaa
aaatggtcac cagaatttta atctcctaga gttggtcgat 900tccatttgta cagacattcg
agccaagcaa caaggtgccc gaaacatgca gactctggtt 960ctacag
96614322PRTSeneca Valley
Virus 14Gly Pro Met Asp Lys Val Lys Asp Trp Asn Gln Ile Ala Ala Gly Leu
1 5 10 15 Lys Asn Phe
Gln Phe Val Arg Asp Leu Val Lys Glu Val Val Asp Trp 20
25 30 Leu Gln Ala Trp Ile Asn Lys Glu
Lys Ala Ser Pro Val Leu Gln Tyr 35 40
45 Gln Leu Glu Met Lys Lys Leu Gly Pro Val Ala Leu Ala
His Asp Ala 50 55 60
Phe Met Ala Gly Ser Gly Pro Pro Leu Ser Asp Asp Gln Ile Glu Tyr 65
70 75 80Leu Gln Asn Leu Lys
Ser Leu Ala Leu Thr Leu Gly Lys Thr Asn Leu 85
90 95 Ala Gln Ser Leu Thr Thr Met Ile Asn Ala
Lys Gln Ser Ser Ala Gln 100 105
110 Arg Val Glu Pro Val Val Val Val Leu Arg Gly Lys Pro Gly Cys
Gly 115 120 125 Lys
Gly Leu Ala Ser Thr Leu Ile Ala Gln Ala Val Ser Lys Arg Leu 130
135 140 Tyr Gly Ser Gln Ser Val
Tyr Ser Leu Pro Pro Asp Pro Asp Phe Phe 145 150
155 160Asp Gly Tyr Lys Gly Gln Phe Val Thr Leu Met
Asp Asp Leu Gly Gln 165 170
175 Asn Pro Asp Gly Gln Asp Phe Ser Thr Phe Cys Gln Met Val Ser Thr
180 185 190 Ala Gln
Phe Leu Pro Asn Met Ala Asp Leu Ala Glu Lys Gly Arg Pro 195
200 205 Phe Thr Ser Asn Leu Ile Ile
Ala Thr Thr Asn Leu Pro His Phe Ser 210 215
220 Pro Val Thr Ile Ala Asp Pro Ser Ala Val Ser Arg
Arg Ile Asn Tyr 225 230 235
240Asp Leu Thr Leu Glu Val Ser Glu Ala Tyr Lys Lys His Thr Arg Leu
245 250 255 Asn Phe Asp
Leu Ala Phe Arg Arg Thr Asp Ala Pro Pro Ile Tyr Pro 260
265 270 Phe Ala Ala His Val Pro Phe Val
Asp Val Ala Val Arg Phe Lys Asn 275 280
285 Gly His Gln Asn Phe Asn Leu Leu Glu Leu Val Asp Ser
Ile Cys Thr 290 295 300
Asp Ile Arg Ala Lys Gln Gln Gly Ala Arg Asn Met Gln Thr Leu Val 305
310 315 320Leu Gln
15270DNASeneca Valley
Virus 15agccccaacg agaatgatga cacccccgtc gacgaggcgt tgggtagagt tctctccccc
60gctgcggtcg atgaggcgct tgtcgacctc actccagagg ccgacccggt tggccgtttg
120gctattcttg ccaagctagg tcttgcccta gctgcggtca cccctggtct gataatcttg
180gcagtgggac tctacaggta cttctctggc tctgatgcag accaagaaga aacagaaagt
240gagggatctg tcaaggcacc caggagcgaa
2701690PRTSeneca Valley Virus 16Ser Pro Asn Glu Asn Asp Asp Thr Pro Val
Asp Glu Ala Leu Gly Arg 1 5 10
15 Val Leu Ser Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu Thr
Pro 20 25 30 Glu
Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly Leu 35
40 45 Ala Leu Ala Ala Val Thr
Pro Gly Leu Ile Ile Leu Ala Val Gly Leu 50 55
60 Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp Gln
Glu Glu Thr Glu Ser 65 70 75
80Glu Gly Ser Val Lys Ala Pro Arg Ser Glu
85 901766DNASeneca Valley Virus
17aatgcttatg acggcccgaa gaaaaactct aagccccctg gagcactctc tctcatggaa
60atgcaa
661822PRTSeneca Valley Virus 18Asn Ala Tyr Asp Gly Pro Lys Lys Asn Ser
Lys Pro Pro Gly Ala Leu 1 5 10
15 Ser Leu Met Glu Met Gln
20 19633DNASeneca Valley Virus 19cagcccaacg
tggacatggg ctttgaggct gcggtcgcta agaaagtggt cgtccccatt 60accttcatgg
ttcccaacag accttctggg cttacacagt ccgctcttct ggtgaccggc 120cggaccttcc
taatcaatga acatacatgg tccaatccct cctggaccag cttcacaatc 180cgcggtgagg
tacacactcg tgatgagccc ttccaaacgg ttcatttcac tcaccacggt 240attcccacag
atctgatgat ggtacgtctc ggaccgggca attctttccc taacaatcta 300gacaagtttg
gacttgacca gatgccggca cgcaactccc gtgtggttgg cgtttcgtcc 360agttacggaa
acttcttctt ctctggaaat ttcctcggat ttgttgattc cgtcacctct 420gaacaaggaa
cttacgcaag actctttagg tacagggtga cgacctacaa aggatggtgc 480ggctcggccc
tggtctgtga ggccggtggc gtccgacgca tcattggcct gcattctgct 540ggcgccgccg
gtatcggcgc cgggacctat atctcaaaat taggactaat caaagccctg 600aaacacctcg
gtgaaccttt ggccacaatg caa
63320211PRTSeneca Valley Virus 20Gln Pro Asn Val Asp Met Gly Phe Glu Ala
Ala Val Ala Lys Lys Val 1 5 10
15 Val Val Pro Ile Thr Phe Met Val Pro Asn Arg Pro Ser Gly Leu
Thr 20 25 30 Gln
Ser Ala Leu Leu Val Thr Gly Arg Thr Phe Leu Ile Asn Glu His 35
40 45 Thr Trp Ser Asn Pro Ser
Trp Thr Ser Phe Thr Ile Arg Gly Glu Val 50 55
60 His Thr Arg Asp Glu Pro Phe Gln Thr Val His
Phe Thr His His Gly 65 70 75
80Ile Pro Thr Asp Leu Met Met Val Arg Leu Gly Pro Gly Asn Ser Phe
85 90 95 Pro Asn Asn
Leu Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg Asn 100
105 110 Ser Arg Val Val Gly Val Ser Ser
Ser Tyr Gly Asn Phe Phe Phe Ser 115 120
125 Gly Asn Phe Leu Gly Phe Val Asp Ser Val Thr Ser Glu
Gln Gly Thr 130 135 140
Tyr Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys Gly Trp Cys 145
150 155 160Gly Ser Ala Leu
Val Cys Glu Ala Gly Gly Val Arg Arg Ile Ile Gly 165
170 175 Leu His Ser Ala Gly Ala Ala Gly Ile
Gly Ala Gly Thr Tyr Ile Ser 180 185
190 Lys Leu Gly Leu Ile Lys Ala Leu Lys His Leu Gly Glu Pro
Leu Ala 195 200 205
Thr Met Gln 210
211389DNASeneca Valley Virus 21ggactgatga ctgaattaga gcctggaatc
accgtacatg taccccggaa atccaaattg 60agaaagacga ccgcacacgc ggtgtacaaa
ccggagtttg agcctgctgt gttgtcaaaa 120tttgatccca gactgaacaa ggatgttgac
ttggatgaag taatttggtc taaacacact 180gccaatgtcc cttaccaacc tcctttgttc
tacacataca tgtcagagta cgctcatcga 240gtcttctcct tcttggggaa agacaatgac
attctgaccg tcaaagaagc aattctgggc 300atccccggac tagaccccat ggatccccac
acagctccgg gtctgcctta cgccatcaac 360ggccttcgac gtactgatct cgtcgatttt
gtgaacggta cagtagatgc ggcgctggct 420gtacaaatcc agaaattctt agacggtgac
tactctgacc atgtcttcca aacttttctg 480aaagatgaga tcagaccctc agagaaagtc
cgagcgggaa aaacccgcat tgttgatgtg 540ccctccctgg cgcattgcat tgtgggcaga
atgttgcttg ggcgctttgc tgccaagttt 600caatcccatc ctggctttct cctcggctct
gctatcgggt ctgaccctga tgttttctgg 660accgtcatag gggctcaact cgaggggaga
aagaacacgt atgacgtgga ctacagtgcc 720tttgactctt cacacggcac tggctccttc
gaggctctca tctctcactt tttcaccgtg 780gacaatggtt ttagccctgc gctgggaccg
tatctcagat ccctggctgt ctcggtgcac 840gcttacggcg agcgtcgcat caagattacc
ggtggcctcc cctccggttg tgccgcgacc 900agcctgctga acacagtgct caacaatgtg
atcatcagga ctgctctggc attgacttac 960aaggaatttg agtatgacac ggttgatatc
atcgcctacg gtgacgacct tctggttggc 1020acggattacg atctggactt caatgaggtg
gcacgacgcg ctgccaagtt ggggtataag 1080atgactcctg ccaacaaggg ttctgtcttc
cctccgactt cctctctttc cgatgctgtt 1140tttctaaagc gcaaattcgt ccaaaacaac
gacggcttat acaaaccagt tatggattta 1200aagaatttgg aagccatgct ctcctacttc
aaaccaggaa cactactcga gaagctgcaa 1260tctgtttcta tgttggctca acattctgga
aaagaagaat atgatagatt gatgcacccc 1320ttcgctgact acggtgccgt accgagtcac
gagtacctgc aggcaagatg gagggccttg 1380ttcgactga
138922462PRTSeneca Valley Virus 22Gly
Leu Met Thr Glu Leu Glu Pro Gly Ile Thr Val His Val Pro Arg 1
5 10 15 Lys Ser Lys Leu Arg Lys
Thr Thr Ala His Ala Val Tyr Lys Pro Glu 20
25 30 Phe Glu Pro Ala Val Leu Ser Lys Phe Asp Pro
Arg Leu Asn Lys Asp 35 40 45
Val Asp Leu Asp Glu Val Ile Trp Ser Lys His Thr Ala Asn Val Pro
50 55 60 Tyr Gln Pro
Pro Leu Phe Tyr Thr Tyr Met Ser Glu Tyr Ala His Arg 65
70 75 80Val Phe Ser Phe Leu Gly Lys Asp
Asn Asp Ile Leu Thr Val Lys Glu 85 90
95 Ala Ile Leu Gly Ile Pro Gly Leu Asp Pro Met Asp Pro
His Thr Ala 100 105 110
Pro Gly Leu Pro Tyr Ala Ile Asn Gly Leu Arg Arg Thr Asp Leu Val
115 120 125 Asp Phe Val Asn
Gly Thr Val Asp Ala Ala Leu Ala Val Gln Ile Gln 130
135 140 Lys Phe Leu Asp Gly Asp Tyr Ser
Asp His Val Phe Gln Thr Phe Leu 145 150
155 160Lys Asp Glu Ile Arg Pro Ser Glu Lys Val Arg Ala
Gly Lys Thr Arg 165 170
175 Ile Val Asp Val Pro Ser Leu Ala His Cys Ile Val Gly Arg Met Leu
180 185 190 Leu Gly Arg
Phe Ala Ala Lys Phe Gln Ser His Pro Gly Phe Leu Leu 195
200 205 Gly Ser Ala Ile Gly Ser Asp Pro
Asp Val Phe Trp Thr Val Ile Gly 210 215
220 Ala Gln Leu Glu Gly Arg Lys Asn Thr Tyr Asp Val Asp
Tyr Ser Ala 225 230 235
240Phe Asp Ser Ser His Gly Thr Gly Ser Phe Glu Ala Leu Ile Ser His
245 250 255 Phe Phe Thr Val
Asp Asn Gly Phe Ser Pro Ala Leu Gly Pro Tyr Leu 260
265 270 Arg Ser Leu Ala Val Ser Val His Ala
Tyr Gly Glu Arg Arg Ile Lys 275 280
285 Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr Ser Leu
Leu Asn 290 295 300
Thr Val Leu Asn Asn Val Ile Ile Arg Thr Ala Leu Ala Leu Thr Tyr 305
310 315 320Lys Glu Phe Glu Tyr
Asp Thr Val Asp Ile Ile Ala Tyr Gly Asp Asp 325
330 335 Leu Leu Val Gly Thr Asp Tyr Asp Leu Asp
Phe Asn Glu Val Ala Arg 340 345
350 Arg Ala Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala Asn Lys Gly
Ser 355 360 365 Val
Phe Pro Pro Thr Ser Ser Leu Ser Asp Ala Val Phe Leu Lys Arg 370
375 380 Lys Phe Val Gln Asn Asn
Asp Gly Leu Tyr Lys Pro Val Met Asp Leu 385 390
395 400Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys
Pro Gly Thr Leu Leu 405 410
415 Glu Lys Leu Gln Ser Val Ser Met Leu Ala Gln His Ser Gly Lys Glu
420 425 430 Glu Tyr
Asp Arg Leu Met His Pro Phe Ala Asp Tyr Gly Ala Val Pro 435
440 445 Ser His Glu Tyr Leu Gln Ala
Arg Trp Arg Ala Leu Phe Asp 450 455
460 232292PRTEncephalomyocarditis virus 23Met Ala Thr Thr
Met Glu Gln Glu Thr Cys Ala His Ser Leu Thr Phe 1 5
10 15 Glu Glu Cys Pro Lys Cys Ser Ala Leu
Gln Tyr Arg Asn Gly Phe Tyr 20 25
30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Leu Leu
Thr Asp 35 40 45
Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val Val Phe 50
55 60 Glu Leu Gln Gly Asn
Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70
75 80Glu Gly Asn Glu Gly Val Ile Ile Asn Asn
Phe Tyr Ser Asn Gln Tyr 85 90
95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Ala Gly Ser Asp Pro
Pro 100 105 110 Arg
Thr Tyr Gly Gln Phe Ser Asn Leu Phe Ser Gly Ala Val Asn Ala 115
120 125 Phe Ser Asn Met Leu Pro
Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135
140 Glu Asn Leu Ser Asp Arg Val Ser Gln Asp Thr
Ala Gly Asn Thr Val 145 150 155
160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Thr Val
165 170 175 His Asp
Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180
185 190 Ile Leu Ala Val Glu Arg Tyr
Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200
205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro
Leu Pro His Val 210 215 220
Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala Leu Arg Arg His
225 230 235 240Tyr Leu
Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser
245 250 255 Gln Phe His Ala Gly Ser
Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260
265 270 Thr Leu Asp Ala Phe Ala Met Asp Asn Arg
Trp Ser Lys Asp Asn Leu 275 280
285 Pro Asn Gly Thr Arg Thr Gln Thr Asn Lys Lys Gly Pro Phe
Ala Met 290 295 300
Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305
310 315 320Asn Leu Arg Thr Asn
Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325
330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His
Ala Ser Trp Thr Leu Val 340 345
350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr
Ser 355 360 365 Leu
Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370
375 380 Leu Arg His Glu Thr Leu
Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390
395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu
Pro Asp Ser Thr Val 405 410
415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ser Asn Tyr Met Val Gly
420 425 430 Glu Tyr
Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435
440 445 Asn Lys Ile Pro Asn Ala Val
Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455
460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val Thr
Leu Ser Cys Ser 465 470 475
480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln
485 490 495 Tyr Arg Gly
Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500
505 510 Met Lys Gly Lys Phe Leu Ile Ala
Tyr Thr Pro Pro Gly Ala Gly Lys 515 520
525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala
Ile Trp Asp 530 535 540
Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545
550 555 560Thr His Phe Arg
Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Ala 565
570 575 Asp Gly Trp Val Thr Val Trp Gln Leu
Thr Pro Leu Thr Tyr Pro Pro 580 585
590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala
Gly Lys 595 600 605
Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610
615 620 Gly Val Glu Asn Ala
Glu Lys Gly Val Thr Glu Asn Thr Asn Ala Thr 625 630
635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu
Pro Glu Asn Gln Thr Lys 645 650
655 Val Ala Phe Phe Tyr Asn Arg Ser Ser Pro Ile Gly Ala Phe Thr
Val 660 665 670 Lys
Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Gly Thr 675
680 685 Cys Pro Asn Ser Val Ile
Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695
700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu
Ile Trp Gly Asn Gly 705 710 715
720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys Ser Lys Gln Asp Tyr
725 730 735 Ser Phe
Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740
745 750 Val Thr Leu Ser Pro His Thr
Ser Gly Asn His Gly Leu Leu Val Arg 755 760
765 Trp Cys Pro Thr Gly Thr Pro Thr Lys Pro Thr Thr
Gln Val Leu His 770 775 780
Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala
785 790 795 800Gly Pro
Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser
805 810 815 Pro Leu Ser Val Leu Ser
Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820
825 830 Asp Asn Thr Gly Ser Leu Gly Ile Ala Pro
Asn Ser Asp Phe Gly Thr 835 840
845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val
Tyr Leu 850 855 860
Arg Tyr Lys Asn Lys Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865
870 875 880Pro Trp Pro Thr Ser
Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885
890 895 Val Leu Met Leu Glu Ser Pro Asn Ala Leu
Asp Ile Ser Arg Thr Tyr 900 905
910 Pro Thr Leu His Val Leu Ile Gln Phe Asn His Arg Gly Leu Glu
Val 915 920 925 Arg
Leu Phe Arg His Gly His Phe Trp Ala Glu Thr Arg Ala Asp Val 930
935 940 Ile Leu Arg Ser Lys Thr
Lys Gln Val Ser Phe Leu Ser Asn Gly Asn 945 950
955 960Tyr Pro Ser Met Asp Ser Arg Ala Pro Trp Asn
Pro Trp Lys Asn Thr 965 970
975 Tyr Gln Ala Val Leu Arg Ala Glu Pro Cys Arg Val Thr Met Asp Ile
980 985 990 Tyr Tyr
Lys Arg Val Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995
1000 1005 Trp Pro Val Arg Glu Glu Asn
Val Phe Gly Leu Tyr Arg Ile Phe Asn 1010 1015
1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu
Ile His Asp Ile Glu 1025 1030 1035
1040Thr Asn Pro Gly Pro Phe Met Phe Arg Pro Arg Lys Gln Val Phe Gln
1045 1050 1055 Thr Gln
Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn 1060
1065 1070 Asp Leu Ala Ser Lys Ala
Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075 1080
1085 Ala Asn Glu Asp Ala Gln Lys Ala Met Lys
Ile Ile Lys Thr Leu Ser 1090 1095 1100
Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr Leu Asn
Asn Pro 1105 1110 1115
1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly
1125 1130 1135 Met Thr Ile
Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140
1145 1150 Gly Thr Leu Thr Ala Ala Glu
Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160
1165 Glu Ile Ala Ala Lys Phe Lys Thr Ile Phe Ile
Thr Pro Pro Pro Arg 1170 1175 1180
Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln
Val 1185 1190 1195 1200Asn
Asp Ile Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr
1205 1210 1215 Val Glu Lys Val Val
Asp Trp Phe Gly Thr Trp Ile Val Gln Glu Glu 1220
1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu
Gln Arg Phe Pro Glu His Ala 1235 1240
1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ala Ala Tyr
Val Glu Cys 1250 1255 1260
Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265
1270 1275 1280Glu Lys Arg Thr
Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285
1290 1295 His Asp His Ala Thr Ala Arg Cys
Glu Pro Val Val Ile Val Leu Arg 1300 1305
1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln
Val Ile Ala Gln 1315 1320 1325
Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro
1330 1335 1340 Pro Asp Ser
Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345
1350 1355 1360Met Asp Asp Leu Gly Gln Asn
Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365
1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro
Asn Met Ala Ser Leu 1380 1385
1390 Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr
Thr 1395 1400 1405 Asn
Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410
1415 1420 Glu Arg Arg Ile Thr
Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430
1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val Leu
Asp Val Glu Arg Ala Phe 1445 1450
1455 Arg Pro Thr Gly Glu Ala Pro Leu Pro Cys Phe Gln Asn Asn
Cys Leu 1460 1465 1470
Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu
1475 1480 1485 Ile Ile Ser Leu
Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490
1495 1500 Arg Lys Lys Lys Val Leu Thr Thr
Val Gln Thr Leu Val Ala Gln Gly 1505 1510
1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln
Gln Leu Lys Ala 1525 1530
1535 Arg Gln Gln Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe
1540 1545 1550 Ala Lys
Val Gln Glu Arg Asn Ser Val Phe Ser Asp Trp Leu Lys Ile 1555
1560 1565 Ser Ala Met Leu Cys Ala
Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575
1580 Met Ala Lys Ala Val Lys Gln Met Val Lys
Pro Asp Leu Val Arg Val 1585 1590 1595
1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Thr Ala Arg
Val 1605 1610 1615 Lys
Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val
1620 1625 1630 Met Asp Phe Glu Lys
Tyr Val Ala Lys His Val Thr Ala Pro Ile Gly 1635
1640 1645 Phe Val Tyr Pro Thr Gly Val Ser Thr
Gln Thr Cys Leu Leu Val Arg 1650 1655
1660 Gly Arg Thr Leu Val Val Asn Arg His Met Ala Glu Ser
Asp Trp Thr 1665 1670 1675
1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Lys Ile
1685 1690 1695 Leu Ala Ile
Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700
1705 1710 Leu Ser Ser Gly Pro Leu Phe
Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720
1725 Ala Gly Asp Val Leu Pro Thr Gly Ala Ala Pro
Val Thr Gly Ile Met 1730 1735 1740
Asn Thr Asp Ile Pro Met Met Tyr Thr Gly Thr Phe Leu Lys Ala
Gly 1745 1750 1755 1760Val
Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His
1765 1770 1775 Tyr Lys Ala Asn Thr
Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780
1785 1790 Asp Leu Gly Gly Ser Lys Lys Ile Leu
Gly Ile His Ser Ala Gly Ser 1795 1800
1805 Met Gly Ile Ala Ala Ala Ser Ile Val Ser Gln Glu Met
Ile Arg Ala 1810 1815 1820
Val Val Asn Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825
1830 1835 1840Gly Pro Arg Ile
His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845
1850 1855 Ala Arg Gln Val Phe Gln Pro Ala
Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865
1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val
Ala Phe Ser Lys 1875 1880 1885
His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala
1890 1895 1900 Lys Glu Tyr
Ala Asn Arg Val Phe Thr Leu Leu Gly Lys Asp Asn Gly 1905
1910 1915 1920Arg Leu Thr Val Lys Gln Ala
Leu Glu Gly Leu Glu Gly Met Asp Pro 1925
1930 1935 Met Asp Arg Asn Thr Ser Pro Gly Leu Pro Tyr
Thr Ala Leu Gly Met 1940 1945
1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro
Phe 1955 1960 1965 Ala
Ala Glu Arg Leu Arg Lys Met Asn Glu Gly Asp Phe Ser Glu Val 1970
1975 1980 Val Tyr Gln Thr Phe
Leu Lys Asp Glu Leu Arg Pro Ile Glu Lys Val 1985 1990
1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp
Val Pro Pro Phe Glu His Cys 2005 2010
2015 Ile Leu Gly Arg Gln Leu Leu Gly Lys Phe Ala Ser Lys
Phe Gln Thr 2020 2025 2030
Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val
2035 2040 2045 His Trp Thr Ala
Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050
2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp
Ser Thr His Ser Val Ala Met Phe 2065 2070
2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn
Gly Phe Asp Pro 2085 2090
2095 Leu Thr Arg Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe
2100 2105 2110 Glu Glu
Lys Arg Phe Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115
2120 2125 Ala Thr Ser Met Leu Asn
Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135
2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu
Phe Asp Asp Val Lys Val 2145 2150 2155
2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu
Asp 2165 2170 2175 Phe
Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr
2180 2185 2190 Pro Ala Asn Lys Thr
Ser Thr Phe Pro Leu Asn Ser Thr Leu Glu Asp 2195
2200 2205 Val Val Phe Leu Lys Arg Lys Phe Lys
Lys Glu Gly Pro Leu Tyr Arg 2210 2215
2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser
Tyr Tyr Arg 2225 2230 2235
2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val
2245 2250 2255 His Ser Gly
Lys Gln Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260
2265 2270 Val Gly Val Val Val Pro Ser
Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280
2285 Ser Leu Phe Trp
2290 242292PRTEncephalomyocarditis virus
24Met Ala Thr Thr Met Glu Gln Glu Thr Cys Ala His Ser Leu Thr Phe 1
5 10 15 Glu Glu Cys Pro
Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20
25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr
Pro Glu Glu Leu Leu Thr Asp 35 40
45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val
Val Phe 50 55 60
Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65
70 75 80Glu Gly Asn Glu Gly
Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85
90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala
Ala Gly Ser Asp Pro Pro 100 105
110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Phe Ser Gly Ala Val Asn
Ala 115 120 125 Phe
Ser Asn Met Leu Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130
135 140 Glu Asn Leu Ser Asp Arg
Val Ser Gln Asp Thr Ala Gly Asn Thr Val 145 150
155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val
Gly Tyr Gly Thr Val 165 170
175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys
180 185 190 Ile Leu
Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195
200 205 Ser Thr Gln Lys Pro Phe Glu
Tyr Ile Arg Ile Pro Leu Pro His Val 210 215
220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala
Leu Arg Arg His 225 230 235
240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser
245 250 255 Gln Phe His
Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260
265 270 Thr Leu Asp Thr Phe Val Met Asp
Asn Arg Trp Ser Lys Asp Asn Leu 275 280
285 Pro Asn Gly Ala Arg Thr Gln Thr Asn Lys Lys Gly Pro
Phe Ala Met 290 295 300
Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305
310 315 320Asn Leu Arg Thr
Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325
330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln
His Ala Ser Trp Thr Leu Val 340 345
350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser
Thr Ser 355 360 365
Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370
375 380 Leu Arg His Glu Thr
Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390
395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr
Leu Pro Asp Ser Thr Val 405 410
415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ser Asn Tyr Met Val
Gly 420 425 430 Glu
Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435
440 445 Asn Lys Ile Pro Asn Ala
Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455
460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val
Thr Leu Ser Cys Ser 465 470 475
480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln
485 490 495 Tyr Arg
Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500
505 510 Met Lys Gly Lys Phe Leu Ile
Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520
525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr
Ala Ile Trp Asp 530 535 540
Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro
545 550 555 560Thr His
Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Ala
565 570 575 Asp Gly Trp Val Thr Val
Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580
585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr
Met Val Ser Ala Gly Lys 595 600
605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser
Pro Gln 610 615 620
Gly Val Glu Asn Ala Glu Lys Gly Val Thr Glu Asn Ala Asp Ala Thr 625
630 635 640Ala Asp Phe Val Ala
Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645
650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro
Ile Gly Ala Phe Thr Val 660 665
670 Gln Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Lys
Thr 675 680 685 Cys
Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690
695 700 Tyr Asp Gln Leu Arg Pro
Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710
715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys
Ser Lys Gln Asp Tyr 725 730
735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu
740 745 750 Val Thr
Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755
760 765 Trp Cys Pro Thr Gly Thr Pro
Thr Lys Pro Thr Thr Gln Val Leu His 770 775
780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln
Val Tyr Ser Ala 785 790 795
800Gly Pro Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser
805 810 815 Pro Leu Ser
Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820
825 830 Asp Asn Thr Gly Ser Leu Gly Ile
Ala Pro Asn Ser Asp Phe Gly Thr 835 840
845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr
Val Tyr Leu 850 855 860
Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865
870 875 880Pro Trp Pro Thr
Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885
890 895 Val Leu Met Leu Glu Ser Pro Asn Ala
Leu Asp Ile Ser Arg Thr Tyr 900 905
910 Pro Thr Leu His Val Leu Ile Gln Phe Asn His Arg Gly Leu
Glu Val 915 920 925
Arg Leu Phe Arg His Gly Gln Phe Trp Ala Glu Thr Arg Ala Asp Val 930
935 940 Ile Leu Arg Ser Lys
Thr Lys Gln Val Ser Phe Leu Ser Asn Gly Asn 945 950
955 960Tyr Pro Ser Met Asp Ser Arg Ala Pro Trp
Asn Pro Trp Lys Asn Thr 965 970
975 Tyr Gln Ala Val Leu Arg Ala Glu Pro Cys Arg Val Thr Met Asp
Ile 980 985 990 Tyr
Tyr Lys Arg Val Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995
1000 1005 Trp Arg Val Arg Glu Glu
Asn Val Phe Gly Leu Tyr Arg Ile Phe Asn 1010 1015
1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu
Leu Ile His Asp Ile Glu 1025 1030 1035
1040Thr Asn Pro Gly Pro Phe Met Phe Arg Pro Arg Lys Gln Val
Phe Gln 1045 1050 1055
Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn
1060 1065 1070 Asp Leu Ala Ser
Lys Ala Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075
1080 1085 Ala Asn Glu Asp Ala Arg Lys Ala
Met Lys Ile Ile Lys Thr Leu Ser 1090 1095
1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr
Leu Asn Asn Pro 1105 1110 1115
1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly
1125 1130 1135 Met Thr Ile
Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140
1145 1150 Gly Thr Leu Thr Ala Ala Glu
Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160
1165 Glu Ile Ala Ala Lys Phe Lys Thr Ile Phe Ile
Thr Pro Pro Pro Arg 1170 1175 1180
Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln
Val 1185 1190 1195 1200Asn
Asp Phe Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr
1205 1210 1215 Val Glu Lys Val Val
Asp Trp Phe Gly Thr Trp Ile Val Gln Glu Glu 1220
1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu
Gln Arg Phe Pro Glu His Ala 1235 1240
1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ala Ala Tyr
Val Glu Cys 1250 1255 1260
Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265
1270 1275 1280Glu Lys Arg Thr
Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285
1290 1295 His Asp His Ala Thr Ala Arg Cys
Glu Pro Val Val Ile Val Leu Arg 1300 1305
1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln
Val Ile Ala Gln 1315 1320 1325
Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro
1330 1335 1340 Pro Asp Ser
Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345
1350 1355 1360Met Asp Asp Leu Gly Gln Asn
Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365
1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro
Asn Met Ala Ser Leu 1380 1385
1390 Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr
Thr 1395 1400 1405 Asn
Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410
1415 1420 Glu Arg Arg Ile Thr
Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430
1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val
Leu Asp Val Glu Arg Ala Phe 1445 1450
1455 Arg Pro Thr Gly Glu Ala Pro Leu Pro Cys Phe Gln Asn
Asn Cys Leu 1460 1465 1470
Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu
1475 1480 1485 Ile Ile Ser Leu
Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490
1495 1500 Arg Lys Lys Lys Val Leu Thr Thr
Val Gln Thr Leu Val Ala Gln Ala 1505 1510
1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln
Gln Leu Lys Ala 1525 1530
1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe
1540 1545 1550 Ala Lys
Val Gln Glu Arg Asn Ser Val Phe Ser Asp Trp Leu Lys Ile 1555
1560 1565 Ser Ala Met Leu Cys Ala
Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575
1580 Met Ala Lys Ala Val Lys Gln Met Val Lys
Pro Asp Leu Val Arg Val 1585 1590 1595
1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Thr Ala
Arg Ala 1605 1610 1615
Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val
1620 1625 1630 Met Asp Phe Glu
Lys Tyr Val Ala Lys His Val Thr Ala Pro Ile Asp 1635
1640 1645 Phe Val Tyr Pro Thr Gly Val Ser
Thr Gln Thr Cys Leu Leu Val Arg 1650 1655
1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu
Ser Asp Trp Thr 1665 1670 1675
1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Lys Ile
1685 1690 1695 Leu Ala Ile
Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700
1705 1710 Leu Ser Ser Gly Pro Leu Phe
Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720
1725 Ala Gly Asp Val Leu Pro Thr Gly Ala Ala Pro
Val Thr Gly Ile Met 1730 1735 1740
Asn Thr Asp Ile Pro Met Met Tyr Thr Gly Thr Phe Leu Lys Ala
Gly 1745 1750 1755 1760Val
Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His
1765 1770 1775 Tyr Lys Ala Asn Thr
Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780
1785 1790 Asp Leu Gly Gly Ser Lys Lys Ile Leu
Gly Ile His Ser Ala Gly Ser 1795 1800
1805 Met Gly Ile Ala Ala Ala Ser Ile Val Ser Gln Glu Met
Ile Arg Ala 1810 1815 1820
Val Val Asn Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825
1830 1835 1840Gly Pro Arg Ile
His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845
1850 1855 Ala Arg Gln Val Phe Gln Pro Ala
Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865
1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val
Ala Phe Ser Lys 1875 1880 1885
His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala
1890 1895 1900 Lys Glu Tyr
Ala Asn Arg Val Phe Thr Leu Leu Gly Lys Asp Asn Gly 1905
1910 1915 1920Arg Leu Thr Val Lys Gln Ala
Leu Glu Gly Leu Glu Gly Met Asp Pro 1925
1930 1935 Met Asp Arg Asn Thr Ser Pro Gly Leu Pro Tyr
Thr Ala Leu Gly Met 1940 1945
1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro
Phe 1955 1960 1965 Ala
Ala Glu Arg Leu Arg Lys Met Asn Glu Gly Asp Phe Ser Glu Val 1970
1975 1980 Val Tyr Gln Thr Phe
Leu Lys Asp Glu Leu Arg Pro Ile Glu Lys Val 1985 1990
1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp
Val Pro Pro Phe Glu His Cys 2005 2010
2015 Ile Leu Gly Arg Gln Leu Leu Gly Lys Phe Ala Ser Lys
Phe Gln Thr 2020 2025 2030
Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val
2035 2040 2045 His Trp Thr Ala
Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050
2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp
Ser Thr His Ser Val Ala Met Phe 2065 2070
2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn
Gly Phe Asp Pro 2085 2090
2095 Leu Thr Arg Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe
2100 2105 2110 Glu Glu
Lys Arg Phe Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115
2120 2125 Ala Thr Ser Met Leu Asn
Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135
2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu
Phe Asp Asp Val Lys Val 2145 2150 2155
2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln
Leu Asp 2165 2170 2175
Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr
2180 2185 2190 Pro Ala Asn Lys
Thr Ser Thr Phe Pro Leu Asn Ser Thr Leu Glu Asp 2195
2200 2205 Val Val Phe Leu Lys Arg Lys Phe
Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215
2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu
Ser Tyr Tyr Arg 2225 2230 2235
2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val
2245 2250 2255 His Ser Gly
Lys Gln Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260
2265 2270 Val Gly Val Val Val Pro Ser
Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280
2285 Ser Leu Phe Trp
2290 252292PRTEncephalomyocarditis virus
25Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Leu 1
5 10 15 Lys Gly Cys Pro
Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20
25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr
Pro Glu Glu Leu Leu Thr Asp 35 40
45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val
Val Phe 50 55 60
Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65
70 75 80Asp Gly Asn Glu Gly
Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85
90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala
Thr Gly Ser Asp Pro Pro 100 105
110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn
Ala 115 120 125 Phe
Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130
135 140 Glu Asn Leu Ser Asp Arg
Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150
155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val
Gly Tyr Gly Ala Val 165 170
175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys
180 185 190 Ile Leu
Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195
200 205 Ser Thr Gln Lys Pro Phe Glu
Tyr Ile Arg Ile Pro Leu Pro His Val 210 215
220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala
Leu Arg Arg His 225 230 235
240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser
245 250 255 Gln Phe His
Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260
265 270 Thr Leu Asp Ala Phe Ala Met Asp
Asn Arg Trp Ser Lys Asp Asn Leu 275 280
285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro
Phe Ala Met 290 295 300
Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305
310 315 320Asn Leu Arg Thr
Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325
330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln
His Ala Ser Trp Thr Leu Val 340 345
350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser
Thr Ser 355 360 365
Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370
375 380 Leu Arg His Glu Thr
Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390
395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr
Leu Pro Asp Ser Thr Val 405 410
415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val
Gly 420 425 430 Glu
Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435
440 445 Asn Lys Ile Pro Asn Ala
Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455
460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val
Thr Leu Ser Cys Ser 465 470 475
480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln
485 490 495 Tyr Arg
Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500
505 510 Met Lys Gly Lys Phe Leu Ile
Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520
525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr
Ala Ile Trp Asp 530 535 540
Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro
545 550 555 560Thr His
Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val
565 570 575 Asp Gly Trp Val Thr Val
Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580
585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr
Met Val Ser Ala Gly Lys 595 600
605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser
Pro Gln 610 615 620
Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625
630 635 640Ala Asp Phe Val Ala
Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645
650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro
Ile Gly Ala Phe Thr Val 660 665
670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Thr Pro Phe Ser Asn Gln
Thr 675 680 685 Cys
Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690
695 700 Tyr Asp Gln Leu Arg Pro
Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710
715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys
Ser Lys Gln Asp Tyr 725 730
735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu
740 745 750 Val Thr
Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755
760 765 Trp Cys Pro Thr Gly Thr Pro
Thr Lys Pro Thr Thr Gln Val Leu His 770 775
780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln
Val Tyr Ser Ala 785 790 795
800Gly Pro Gly Ile Thr Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser
805 810 815 Pro Leu Ser
Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820
825 830 Asp Asn Thr Gly Ser Leu Gly Ile
Ala Pro Asn Ser Asp Phe Gly Thr 835 840
845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr
Val Tyr Leu 850 855 860
Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865
870 875 880Pro Trp Pro Ser
Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885
890 895 Val Leu Met Leu Glu Ser Pro Asn Ala
Leu Asp Ile Ser Arg Thr Tyr 900 905
910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu
Glu Ile 915 920 925
Arg Leu Phe Arg His Gly Met Phe Trp Ala Glu Ala His Ala Asp Val 930
935 940 Ile Leu Arg Ser Arg
Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950
955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp
Asn Pro Trp Lys Asn Thr 965 970
975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp
Val 980 985 990 Tyr
His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995
1000 1005 Trp Asn Val Arg Glu
Glu Asn Val Phe Gly Leu Tyr Gly Ile Phe Asn 1010 1015
1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp
Leu Leu Ile His Asp Ile Glu 1025 1030 1035
1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln
Val Phe Gln 1045 1050 1055
Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn
1060 1065 1070 Asp Leu Ala Ser
Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075
1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala
Met Arg Ile Ile Lys Thr Leu Ser 1090 1095
1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr
Leu Asn Asn Pro 1105 1110 1115
1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly
1125 1130 1135 Met Thr Ile
Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140
1145 1150 Gly Thr Leu Thr Ala Ala Glu
Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160
1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr
Thr Pro Pro Pro Arg 1170 1175 1180
Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln
Val 1185 1190 1195 1200Asn
Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr
1205 1210 1215 Val Glu Lys Val Val
Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220
1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu
Gln Arg Phe Pro Glu His Ala 1235 1240
1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr
Val Glu Cys 1250 1255 1260
Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265
1270 1275 1280Glu Lys Arg Thr
Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285
1290 1295 His Asp His Ala Thr Ala Arg Cys
Glu Pro Val Val Ile Val Leu Arg 1300 1305
1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln
Val Ile Ala Gln 1315 1320 1325
Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro
1330 1335 1340 Pro Asp Ser
Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345
1350 1355 1360Met Asp Asp Leu Gly Gln Asn
Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365
1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro
Asn Met Ala Ser Leu 1380 1385
1390 Glu Arg Asn Gly Thr Pro Phe Thr Ser Gln Ile Val Val Ala Thr
Thr 1395 1400 1405 Asn
Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410
1415 1420 Glu Arg Arg Ile Thr
Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430
1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val
Leu Asp Val Glu Arg Ala Phe 1445 1450
1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn
Asn Cys Leu 1460 1465 1470
Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu
1475 1480 1485 Ile Leu Ser Leu
Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490
1495 1500 Arg Lys Lys Lys Val Leu Thr Thr
Val Gln Thr Leu Val Ala Gln Ala 1505 1510
1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln
Gln Leu Lys Ala 1525 1530
1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe
1540 1545 1550 Ala Lys
Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555
1560 1565 Ser Ala Met Leu Cys Ala
Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575
1580 Met Ala Lys Thr Val Lys Gln Met Val Arg
Pro Asp Leu Val Arg Val 1585 1590 1595
1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val
Arg Ala 1605 1610 1615
Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val
1620 1625 1630 Met Asp Phe Glu
Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635
1640 1645 Phe Val Tyr Pro Thr Gly Val Ser
Thr Gln Thr Cys Leu Leu Val Lys 1650 1655
1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu
Ser Asp Trp Ser 1665 1670 1675
1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile
1685 1690 1695 Leu Ala Ile
Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700
1705 1710 Leu Ser Ser Gly Pro Leu Phe
Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720
1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro
Val Ile Gly Ile Met 1730 1735 1740
Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala
Gly 1745 1750 1755 1760Val
Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His
1765 1770 1775 Tyr Lys Ala Asn Thr
Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780
1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu
Gly Met His Ser Ala Gly Ser 1795 1800
1805 Met Gly Arg Thr Ala Ala Ser Ile Val Ser Gln Glu Met
Ile Cys Ala 1810 1815 1820
Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825
1830 1835 1840Gly Pro Arg Ile
His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845
1850 1855 Ala Arg Arg Val Phe Gln Pro Ala
Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865
1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val
Ala Phe Ser Lys 1875 1880 1885
His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala
1890 1895 1900 Lys Glu Tyr
Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905
1910 1915 1920Arg Leu Thr Val Lys Gln Ala
Leu Glu Gly Leu Glu Gly Met Asp Pro 1925
1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr
Thr Ala Leu Gly Met 1940 1945
1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro
Tyr 1955 1960 1965 Ala
Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970
1975 1980 Val Tyr Gln Thr Phe
Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990
1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp
Val Pro Pro Phe Glu His Cys 2005 2010
2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys
Phe Gln Thr 2020 2025 2030
Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val
2035 2040 2045 His Trp Thr Ala
Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050
2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp
Ser Thr His Ser Val Ala Met Phe 2065 2070
2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn
Gly Phe Asp Pro 2085 2090
2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe
2100 2105 2110 Glu Glu
Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115
2120 2125 Ala Thr Ser Met Leu Asn
Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135
2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu
Phe Asp Asp Val Lys Val 2145 2150 2155
2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln
Leu Asn 2165 2170 2175
Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr
2180 2185 2190 Pro Ala Asn Lys
Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195
2200 2205 Val Val Phe Leu Lys Arg Lys Phe
Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215
2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu
Ser Tyr Tyr Arg 2225 2230 2235
2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val
2245 2250 2255 His Ser Gly
Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260
2265 2270 Val Gly Val Val Val Pro Ser
Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280
2285 Ser Leu Phe Trp
2290 262292PRTEncephalomyocarditis virus
26Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Phe 1
5 10 15 Lys Gly Cys Pro
Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20
25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr
Pro Glu Glu Leu Leu Thr Asp 35 40
45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val
Val Phe 50 55 60
Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65
70 75 80Asp Gly Asn Glu Gly
Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85
90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala
Thr Gly Ser Asp Pro Pro 100 105
110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn
Ala 115 120 125 Phe
Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130
135 140 Glu Asn Leu Ser Asp Arg
Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150
155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val
Gly Tyr Gly Ala Val 165 170
175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys
180 185 190 Ile Leu
Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195
200 205 Ser Thr Gln Lys Pro Phe Glu
Tyr Ile Arg Ile Pro Leu Pro His Val 210 215
220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala
Leu Arg Arg His 225 230 235
240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser
245 250 255 Gln Phe His
Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260
265 270 Thr Leu Asp Ala Phe Ala Met Asp
Asn Arg Trp Ser Lys Asp Asn Leu 275 280
285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro
Phe Ala Met 290 295 300
Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305
310 315 320Asn Leu Arg Thr
Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325
330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln
His Ala Ser Trp Thr Leu Val 340 345
350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser
Thr Ser 355 360 365
Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370
375 380 Leu Arg His Glu Thr
Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390
395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr
Leu Pro Asp Ser Thr Val 405 410
415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val
Gly 420 425 430 Glu
Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435
440 445 Asn Lys Ile Pro Asn Ala
Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455
460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val
Thr Leu Ser Cys Ser 465 470 475
480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln
485 490 495 Tyr Arg
Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500
505 510 Met Lys Gly Lys Phe Leu Ile
Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520
525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr
Ala Ile Trp Asp 530 535 540
Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro
545 550 555 560Thr His
Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val
565 570 575 Asp Gly Trp Val Thr Val
Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580
585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr
Met Val Ser Ala Gly Lys 595 600
605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser
Pro Gln 610 615 620
Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625
630 635 640Ala Asp Phe Val Ala
Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645
650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro
Ile Gly Ala Phe Ala Val 660 665
670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Glu
Thr 675 680 685 Cys
Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690
695 700 Tyr Asp Gln Leu Arg Pro
Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710
715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys
Ser Lys Gln Asp Tyr 725 730
735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu
740 745 750 Val Thr
Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755
760 765 Trp Cys Pro Thr Gly Thr Pro
Ala Lys Pro Thr Thr Gln Val Leu His 770 775
780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln
Val Tyr Ser Ala 785 790 795
800Gly Pro Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser
805 810 815 Pro Leu Ser
Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820
825 830 Asp Asn Thr Gly Ser Leu Gly Ile
Ala Pro Asn Ser Asp Phe Gly Thr 835 840
845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr
Val Tyr Leu 850 855 860
Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865
870 875 880Pro Trp Pro Ser
Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885
890 895 Val Leu Met Leu Glu Ser Pro Asn Ala
Leu Asp Ile Ser Arg Thr Tyr 900 905
910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu
Glu Ile 915 920 925
Arg Leu Phe Arg His Gly Met Phe Trp Ala Glu Ala His Ala Asp Val 930
935 940 Ile Leu Arg Ser Arg
Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950
955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp
Asn Pro Trp Lys Asn Thr 965 970
975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp
Val 980 985 990 Tyr
His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995
1000 1005 Trp Asn Val Arg Glu
Glu Asn Val Phe Gly Leu Tyr Gly Ile Phe Asn 1010 1015
1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp
Leu Leu Ile His Asp Ile Glu 1025 1030 1035
1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln
Val Phe Gln 1045 1050 1055
Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn
1060 1065 1070 Asp Leu Ala Ser
Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075
1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala
Met Arg Ile Ile Lys Thr Leu Ser 1090 1095
1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr
Leu Asn Asn Pro 1105 1110 1115
1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly
1125 1130 1135 Met Thr Ile
Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140
1145 1150 Gly Thr Leu Thr Ala Ala Glu
Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160
1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr
Thr Pro Pro Pro Arg 1170 1175 1180
Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln
Val 1185 1190 1195 1200Asn
Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr
1205 1210 1215 Val Glu Lys Val Val
Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220
1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu
Gln Arg Phe Pro Glu His Ala 1235 1240
1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr
Val Glu Cys 1250 1255 1260
Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265
1270 1275 1280Glu Lys Arg Thr
Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285
1290 1295 His Asp His Ala Thr Ala Arg Cys
Glu Pro Val Val Ile Val Leu Arg 1300 1305
1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln
Val Ile Ala Gln 1315 1320 1325
Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro
1330 1335 1340 Pro Asp Ser
Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345
1350 1355 1360Met Asp Asp Leu Gly Gln Asn
Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365
1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro
Asn Met Ala Ser Leu 1380 1385
1390 Glu Arg Asn Gly Thr Pro Phe Thr Ser Gln Ile Val Val Ala Thr
Thr 1395 1400 1405 Asn
Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410
1415 1420 Glu Arg Arg Ile Thr
Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430
1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val
Leu Asp Val Glu Arg Ala Phe 1445 1450
1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn
Asn Cys Leu 1460 1465 1470
Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu
1475 1480 1485 Ile Leu Ser Leu
Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490
1495 1500 Arg Lys Lys Lys Val Leu Thr Thr
Val Gln Thr Leu Val Ala Gln Ala 1505 1510
1515 1520Pro Val Ala Glu Val Ser Phe His Ser Val Val Gln
Gln Leu Lys Ala 1525 1530
1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe
1540 1545 1550 Ala Lys
Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555
1560 1565 Ser Ala Met Leu Cys Ala
Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575
1580 Met Ala Lys Thr Val Lys Gln Met Val Arg
Pro Asp Leu Val Arg Val 1585 1590 1595
1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val
Arg Ala 1605 1610 1615
Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val
1620 1625 1630 Met Asp Phe Glu
Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635
1640 1645 Phe Val Tyr Pro Thr Gly Val Ser
Thr Gln Thr Cys Leu Leu Val Lys 1650 1655
1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu
Ser Asp Trp Ser 1665 1670 1675
1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile
1685 1690 1695 Leu Ala Ile
Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700
1705 1710 Leu Ser Ser Gly Pro Leu Phe
Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720
1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro
Val Ile Gly Ile Met 1730 1735 1740
Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala
Gly 1745 1750 1755 1760Val
Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His
1765 1770 1775 Tyr Lys Ala Asn Thr
Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780
1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu
Gly Met His Ser Ala Gly Ser 1795 1800
1805 Met Gly Arg Thr Ala Ala Ser Ile Val Ser Gln Glu Met
Ile Cys Ala 1810 1815 1820
Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825
1830 1835 1840Gly Pro Arg Ile
His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845
1850 1855 Ala Arg Arg Val Phe Gln Pro Ala
Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865
1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val
Ala Phe Ser Lys 1875 1880 1885
His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala
1890 1895 1900 Lys Glu Tyr
Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905
1910 1915 1920Arg Leu Thr Val Lys Gln Ala
Leu Glu Gly Leu Glu Gly Met Asp Pro 1925
1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr
Thr Ala Leu Gly Met 1940 1945
1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro
Tyr 1955 1960 1965 Ala
Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970
1975 1980 Val Tyr Gln Thr Phe
Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990
1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp
Val Pro Pro Phe Glu His Cys 2005 2010
2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys
Phe Gln Thr 2020 2025 2030
Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val
2035 2040 2045 His Trp Thr Ala
Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050
2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp
Ser Thr His Ser Val Ala Met Phe 2065 2070
2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn
Gly Phe Asp Pro 2085 2090
2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe
2100 2105 2110 Glu Glu
Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115
2120 2125 Ala Thr Ser Met Leu Asn
Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135
2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu
Phe Asp Asp Val Lys Val 2145 2150 2155
2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln
Leu Asn 2165 2170 2175
Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr
2180 2185 2190 Pro Ala Asn Lys
Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195
2200 2205 Val Val Phe Leu Lys Arg Lys Phe
Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215
2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu
Ser Tyr Tyr Arg 2225 2230 2235
2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val
2245 2250 2255 His Ser Gly
Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260
2265 2270 Val Gly Val Val Val Pro Ser
Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280
2285 Ser Leu Phe Trp
2290 272292PRTEncephalomyocarditis virus
27Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Phe 1
5 10 15 Lys Gly Cys Pro
Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20
25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr
Pro Glu Glu Leu Leu Thr Asp 35 40
45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val
Val Phe 50 55 60
Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65
70 75 80Asp Gly Asn Glu Gly
Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85
90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala
Thr Gly Ser Asp Pro Pro 100 105
110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn
Ala 115 120 125 Phe
Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130
135 140 Glu Asn Leu Ser Asp Arg
Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150
155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val
Gly Tyr Gly Ala Val 165 170
175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys
180 185 190 Ile Leu
Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195
200 205 Ser Thr Gln Lys Pro Phe Glu
Tyr Ile Arg Ile Pro Leu Pro His Val 210 215
220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala
Leu Arg Arg His 225 230 235
240Tyr Leu Val Lys Thr Gly Trp Pro Val Gln Val Gln Cys Asn Ala Ser
245 250 255 Gln Phe His
Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260
265 270 Thr Leu Asp Ala Phe Ala Met Asp
Asn Arg Trp Ser Lys Asp Asn Leu 275 280
285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro
Phe Ala Met 290 295 300
Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305
310 315 320Asn Leu Arg Thr
Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325
330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln
His Ala Ser Trp Thr Leu Val 340 345
350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser
Thr Ser 355 360 365
Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370
375 380 Leu Arg His Glu Thr
Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390
395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr
Leu Pro Asp Ser Thr Val 405 410
415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val
Gly 420 425 430 Glu
Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435
440 445 Asn Lys Ile Pro Asn Ala
Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455
460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val
Thr Leu Ser Cys Ser 465 470 475
480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln
485 490 495 Tyr Arg
Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500
505 510 Met Lys Gly Lys Phe Leu Ile
Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520
525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr
Ala Ile Trp Asp 530 535 540
Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro
545 550 555 560Thr His
Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val
565 570 575 Asp Gly Trp Val Thr Val
Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580
585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr
Met Val Ser Ala Gly Lys 595 600
605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser
Pro Gln 610 615 620
Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625
630 635 640Ala Asp Phe Val Ala
Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645
650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro
Ile Gly Ala Phe Thr Val 660 665
670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Glu
Thr 675 680 685 Cys
Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690
695 700 Tyr Asp Gln Leu Arg Pro
Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710
715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys
Ser Lys Gln Asp Tyr 725 730
735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu
740 745 750 Val Thr
Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755
760 765 Trp Cys Pro Thr Gly Thr Pro
Ala Lys Pro Thr Thr Gln Val Leu His 770 775
780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln
Val Tyr Ser Ala 785 790 795
800Gly Pro Gly Val Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser
805 810 815 Pro Leu Ser
Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820
825 830 Asp Asn Thr Gly Ser Leu Gly Ile
Ala Pro Asn Ser Asp Phe Gly Thr 835 840
845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr
Val Tyr Leu 850 855 860
Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865
870 875 880Pro Trp Pro Ser
Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885
890 895 Val Leu Met Leu Glu Ser Pro Asn Ala
Leu Asp Ile Ser Arg Thr Tyr 900 905
910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu
Glu Ile 915 920 925
Arg Leu Phe Arg His Val Gln Phe Trp Ala Glu Ala His Ala Asp Val 930
935 940 Ile Leu Arg Ser Arg
Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950
955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp
Asn Pro Trp Lys Asn Thr 965 970
975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp
Val 980 985 990 Tyr
His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995
1000 1005 Trp Asn Val Arg Glu
Glu Asn Val Phe Gly Leu Tyr Gly Ile Phe Asn 1010 1015
1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp
Leu Leu Ile His Asp Ile Glu 1025 1030 1035
1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln
Val Phe Gln 1045 1050 1055
Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn
1060 1065 1070 Asp Leu Ala Ser
Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075
1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala
Met Arg Ile Ile Lys Thr Leu Ser 1090 1095
1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr
Leu Asn Asn Pro 1105 1110 1115
1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly
1125 1130 1135 Met Thr Ile
Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140
1145 1150 Gly Thr Leu Thr Ala Ala Glu
Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160
1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr
Thr Pro Pro Pro Arg 1170 1175 1180
Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln
Val 1185 1190 1195 1200Asn
Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr
1205 1210 1215 Val Glu Lys Val Val
Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220
1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu
Gln Arg Phe Pro Glu His Ala 1235 1240
1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr
Val Glu Cys 1250 1255 1260
Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265
1270 1275 1280Glu Lys Arg Thr
Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285
1290 1295 His Asp His Ala Thr Ala Arg Cys
Glu Pro Val Val Ile Val Leu Arg 1300 1305
1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln
Val Ile Ala Gln 1315 1320 1325
Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro
1330 1335 1340 Pro Asp Ser
Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345
1350 1355 1360Met Asp Asp Leu Gly Gln Asn
Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365
1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro
Asn Met Ala Ser Leu 1380 1385
1390 Glu Arg Asn Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr
Thr 1395 1400 1405 Asn
Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410
1415 1420 Glu Arg Arg Ile Thr
Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430
1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val
Leu Asp Val Glu Arg Ala Phe 1445 1450
1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn
Asn Cys Leu 1460 1465 1470
Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu
1475 1480 1485 Ile Leu Ser Leu
Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490
1495 1500 Arg Lys Lys Lys Val Leu Thr Thr
Val Gln Thr Leu Val Ala Gln Ala 1505 1510
1515 1520Pro Val Ala Glu Val Ser Phe His Ser Val Val Gln
Gln Leu Lys Ala 1525 1530
1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe
1540 1545 1550 Ala Lys
Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555
1560 1565 Ser Ala Met Leu Cys Ala
Ala Thr Leu Ala Leu Thr Gln Val Val Lys 1570 1575
1580 Met Ala Lys Thr Val Lys Gln Met Val Arg
Pro Asp Leu Val Arg Val 1585 1590 1595
1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val
Arg Ala 1605 1610 1615
Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val
1620 1625 1630 Met Asp Phe Glu
Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635
1640 1645 Phe Val Tyr Pro Thr Gly Val Ser
Thr Gln Thr Cys Leu Leu Val Lys 1650 1655
1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu
Ser Asp Trp Ser 1665 1670 1675
1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile
1685 1690 1695 Leu Ala Ile
Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700
1705 1710 Leu Ser Ser Gly Pro Leu Phe
Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720
1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro
Val Ile Gly Ile Met 1730 1735 1740
Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala
Gly 1745 1750 1755 1760Val
Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His
1765 1770 1775 Tyr Lys Ala Asn Thr
Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780
1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu
Gly Met His Ser Ala Gly Ser 1795 1800
1805 Met Gly Val Ala Ala Ala Ser Ile Val Ser Gln Glu Met
Ile Cys Ala 1810 1815 1820
Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825
1830 1835 1840Gly Pro Arg Ile
His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845
1850 1855 Ala Arg Gln Val Phe Gln Pro Ala
Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865
1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val
Ala Phe Ser Lys 1875 1880 1885
His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala
1890 1895 1900 Lys Glu Tyr
Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905
1910 1915 1920Arg Leu Thr Val Lys Gln Ala
Leu Glu Gly Leu Glu Gly Met Asp Pro 1925
1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr
Thr Ala Leu Gly Met 1940 1945
1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro
Tyr 1955 1960 1965 Ala
Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970
1975 1980 Val Tyr Gln Thr Phe
Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990
1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp
Val Pro Pro Phe Glu His Cys 2005 2010
2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys
Phe Gln Thr 2020 2025 2030
Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val
2035 2040 2045 His Trp Thr Ala
Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050
2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp
Ser Thr His Ser Val Ala Met Phe 2065 2070
2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn
Gly Phe Asp Pro 2085 2090
2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe
2100 2105 2110 Glu Glu
Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115
2120 2125 Ala Thr Ser Met Leu Asn
Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135
2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu
Phe Asp Asp Val Lys Val 2145 2150 2155
2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln
Leu Asn 2165 2170 2175
Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr
2180 2185 2190 Pro Ala Asn Lys
Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195
2200 2205 Val Val Phe Leu Lys Arg Lys Phe
Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215
2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu
Ser Tyr Tyr Arg 2225 2230 2235
2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val
2245 2250 2255 His Ser Gly
Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260
2265 2270 Val Gly Val Val Val Pro Ser
Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280
2285 Ser Leu Phe Trp
2290 282292PRTEncephalomyocarditis virus
28Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Phe 1
5 10 15 Lys Gly Cys Pro
Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20
25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr
Pro Glu Glu Leu Leu Thr Asp 35 40
45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val
Val Phe 50 55 60
Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65
70 75 80Asp Gly Asn Glu Gly
Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85
90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala
Thr Gly Ser Asp Pro Pro 100 105
110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn
Ala 115 120 125 Phe
Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130
135 140 Glu Asn Leu Ser Asp Arg
Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150
155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val
Gly Tyr Gly Ala Val 165 170
175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys
180 185 190 Ile Leu
Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195
200 205 Ser Thr Gln Lys Pro Phe Glu
Tyr Ile Arg Ile Pro Leu Pro His Val 210 215
220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala
Leu Arg Arg His 225 230 235
240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser
245 250 255 Gln Phe His
Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260
265 270 Thr Leu Asp Ala Phe Ala Met Asp
Asn Arg Trp Ser Lys Asp Asn Leu 275 280
285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro
Phe Ala Met 290 295 300
Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305
310 315 320Asn Leu Arg Thr
Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325
330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln
His Ala Ser Trp Thr Leu Val 340 345
350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser
Thr Ser 355 360 365
Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370
375 380 Leu Arg His Glu Thr
Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390
395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr
Leu Pro Asp Ser Thr Val 405 410
415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val
Gly 420 425 430 Glu
Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435
440 445 Asn Lys Ile Pro Asn Ala
Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455
460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val
Thr Leu Ser Cys Ser 465 470 475
480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln
485 490 495 Tyr Arg
Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500
505 510 Met Lys Gly Lys Phe Leu Ile
Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520
525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr
Ala Ile Trp Asp 530 535 540
Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro
545 550 555 560Thr His
Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val
565 570 575 Asp Gly Trp Val Thr Val
Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580
585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr
Met Val Ser Ala Gly Lys 595 600
605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser
Pro Gln 610 615 620
Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625
630 635 640Ala Asp Phe Val Ala
Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645
650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro
Ile Gly Ala Phe Thr Val 660 665
670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Lys
Thr 675 680 685 Cys
Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690
695 700 Tyr Asp Gln Leu Arg Pro
Gln Arg Leu Thr Glu Ile Trp Gly Asn Arg 705 710
715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys
Ser Lys Gln Asp Tyr 725 730
735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu
740 745 750 Val Thr
Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755
760 765 Trp Cys Pro Thr Gly Thr Pro
Ala Lys Pro Thr Thr Gln Val Leu His 770 775
780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln
Val Tyr Ser Ala 785 790 795
800Gly Pro Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser
805 810 815 Pro Leu Ser
Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820
825 830 Asp Asn Thr Gly Ser Leu Gly Ile
Ala Pro Asn Ser Asp Phe Gly Thr 835 840
845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr
Val Tyr Leu 850 855 860
Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865
870 875 880Pro Trp Pro Ser
Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885
890 895 Val Leu Met Leu Glu Ser Pro Asn Ala
Leu Asp Ile Ser Arg Thr Tyr 900 905
910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu
Glu Ile 915 920 925
Arg Leu Phe Arg His Gly Gln Phe Trp Ala Glu Ala His Ala Asp Val 930
935 940 Ile Leu Arg Ser Arg
Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950
955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp
Asn Pro Trp Lys Asn Thr 965 970
975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp
Val 980 985 990 Tyr
His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995
1000 1005 Trp Asn Val Arg Glu Glu
Asn Val Phe Gly Leu Tyr Ser Ile Phe Asn 1010 1015
1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu
Leu Ile His Asp Ile Glu 1025 1030 1035
1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln Val
Phe Gln 1045 1050 1055
Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn
1060 1065 1070 Asp Leu Ala Ser
Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075
1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala
Met Arg Ile Ile Lys Thr Leu Ser 1090 1095
1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr
Leu Asn Asn Pro 1105 1110 1115
1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly
1125 1130 1135 Met Thr Ile
Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140
1145 1150 Gly Thr Leu Thr Ala Ala Glu
Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160
1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr
Thr Pro Pro Pro Arg 1170 1175 1180
Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln
Val 1185 1190 1195 1200Asn
Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr
1205 1210 1215 Val Glu Lys Val Val
Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220
1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu
Gln Arg Phe Pro Glu His Ala 1235 1240
1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr
Val Glu Cys 1250 1255 1260
Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265
1270 1275 1280Glu Lys Arg Thr
Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285
1290 1295 His Asp His Ala Thr Ala Arg Cys
Glu Pro Val Val Ile Val Leu Arg 1300 1305
1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln
Val Ile Ala Gln 1315 1320 1325
Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro
1330 1335 1340 Pro Asp Ser
Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345
1350 1355 1360Met Asp Asp Leu Gly Gln Asn
Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365
1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro
Asn Met Ala Ser Leu 1380 1385
1390 Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr
Thr 1395 1400 1405 Asn
Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410
1415 1420 Glu Arg Arg Ile Thr
Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430
1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val
Leu Asp Val Glu Arg Ala Phe 1445 1450
1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn
Asn Cys Leu 1460 1465 1470
Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu
1475 1480 1485 Ile Leu Ser Leu
Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490
1495 1500 Arg Lys Lys Lys Val Leu Thr Thr
Val Gln Thr Leu Val Ala Gln Ala 1505 1510
1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln
Gln Leu Lys Ala 1525 1530
1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe
1540 1545 1550 Ala Lys
Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555
1560 1565 Ser Ala Met Leu Cys Ala
Ala Thr Leu Ala Leu Thr Gln Val Val Lys 1570 1575
1580 Met Ala Lys Thr Val Lys Gln Met Val Arg
Pro Asp Leu Val Arg Val 1585 1590 1595
1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val
Arg Ala 1605 1610 1615
Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val
1620 1625 1630 Met Asp Phe Glu
Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635
1640 1645 Phe Val Tyr Pro Thr Gly Val Ser
Thr Gln Thr Cys Leu Leu Val Lys 1650 1655
1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu
Ser Asp Trp Ser 1665 1670 1675
1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile
1685 1690 1695 Leu Ala Ile
Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700
1705 1710 Leu Ser Ser Gly Pro Leu Phe
Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720
1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro
Val Ile Gly Ile Met 1730 1735 1740
Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala
Gly 1745 1750 1755 1760Val
Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His
1765 1770 1775 Tyr Lys Ala Asn Thr
Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780
1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu
Gly Met His Ser Ala Gly Ser 1795 1800
1805 Met Gly Arg Thr Ala Ala Ser Ile Val Ser Gln Glu Met
Ile Cys Ala 1810 1815 1820
Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825
1830 1835 1840Gly Pro Arg Ile
His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845
1850 1855 Ala Arg Gln Val Phe Gln Pro Ala
Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865
1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val
Ala Phe Ser Lys 1875 1880 1885
His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala
1890 1895 1900 Lys Glu Tyr
Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905
1910 1915 1920Arg Leu Thr Val Lys Gln Ala
Leu Glu Gly Leu Glu Gly Met Asp Pro 1925
1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr
Thr Ala Leu Gly Met 1940 1945
1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro
Tyr 1955 1960 1965 Ala
Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970
1975 1980 Val Tyr Gln Thr Phe
Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990
1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp
Val Pro Pro Phe Glu His Cys 2005 2010
2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys
Phe Gln Thr 2020 2025 2030
Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val
2035 2040 2045 His Trp Thr Ala
Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050
2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp
Ser Thr His Ser Val Ala Met Phe 2065 2070
2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn
Gly Phe Asp Pro 2085 2090
2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe
2100 2105 2110 Glu Glu
Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115
2120 2125 Ala Thr Ser Met Leu Asn
Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135
2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu
Phe Asp Asp Val Lys Val 2145 2150 2155
2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln
Leu Asn 2165 2170 2175
Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr
2180 2185 2190 Pro Ala Asn Lys
Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195
2200 2205 Val Val Phe Leu Lys Arg Lys Phe
Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215
2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu
Ser Tyr Tyr Arg 2225 2230 2235
2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val
2245 2250 2255 His Ser Gly
Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260
2265 2270 Val Gly Val Val Val Pro Ser
Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280
2285 Ser Leu Phe Trp
2290 292293PRTEncephalomyocarditis virus
29Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Met Thr Phe 1
5 10 15 Glu Glu Cys Pro
Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20
25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr
Pro Glu Glu Ser Leu Thr Asp 35 40
45 Gly Glu Asp Asp Val Phe Asp Pro Asp Leu Asp Met Glu Val
Val Phe 50 55 60
Glu Thr Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65
70 75 80Glu Gly Asn Glu Gly
Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85
90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala
Thr Gly Ser Asp Pro Pro 100 105
110 Lys Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn
Ala 115 120 125 Phe
Ser Asn Met Leu Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130
135 140 Glu Asn Leu Ser Asp Arg
Val Ser Gln Asp Thr Ala Gly Asn Thr Val 145 150
155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val
Gly Tyr Gly Thr Val 165 170
175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys
180 185 190 Ile Leu
Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195
200 205 Ser Thr Gln Lys Pro Phe Glu
Tyr Ile Arg Ile Pro Leu Pro His Val 210 215
220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Thr
Leu Arg Arg His 225 230 235
240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser
245 250 255 Gln Phe His
Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260
265 270 Thr Leu Asp Val Phe Ala Met Asp
Asn Arg Trp Ser Lys Asp Asn Leu 275 280
285 Pro Asn Gly Thr Arg Thr Gln Thr Asn Arg Lys Gly Pro
Phe Ala Met 290 295 300
Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305
310 315 320Asn Leu Arg Thr
Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325
330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln
His Ala Ser Trp Thr Leu Val 340 345
350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser
Thr Ser 355 360 365
Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370
375 380 Leu Arg His Glu Val
Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390
395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr
Leu Pro Asp Ser Thr Val 405 410
415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val
Gly 420 425 430 Glu
Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435
440 445 Asn Lys Val Pro Asn Ala
Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455
460 Val Lys Thr Gln Pro Leu Ala Val Tyr Gln Val
Thr Leu Ser Cys Ser 465 470 475
480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln
485 490 495 Tyr Arg
Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500
505 510 Met Lys Gly Lys Phe Leu Ile
Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520
525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr
Ala Ile Trp Asp 530 535 540
Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro
545 550 555 560Thr His
Phe Arg Met Val Gly Thr Asp Gln Ala Asn Ile Thr Asn Val
565 570 575 Asp Gly Trp Val Thr Val
Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580
585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr
Met Val Ser Ala Gly Lys 595 600
605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser
Pro Gln 610 615 620
Gly Val Glu Asn Ala Glu Lys Gly Val Thr Glu Asn Thr Asp Ala Thr 625
630 635 640Ala Asp Phe Val Ala
Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645
650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro
Ile Gly Ala Phe Ala Val 660 665
670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Lys
Ala 675 680 685 Cys
Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690
695 700 Tyr Asp Gln Leu Arg Pro
Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710
715 720Asn Glu Glu Thr Ser Glu Val Phe Pro Leu Lys
Thr Lys Gln Asp Tyr 725 730
735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu
740 745 750 Val Thr
Leu Ser Pro His Thr Ser Gly Ala His Gly Leu Leu Val Arg 755
760 765 Trp Cys Pro Thr Gly Thr Pro
Thr Lys Pro Thr Thr Gln Val Leu His 770 775
780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln
Val Tyr Ser Ala 785 790 795
800Gly Pro Gly Thr Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser
805 810 815 Pro Leu Ser
Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820
825 830 Asp Asn Thr Gly Asp Leu Gly Ile
Ala Pro Asn Ser Asp Phe Gly Thr 835 840
845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr
Val Tyr Leu 850 855 860
Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865
870 875 880Pro Trp Pro Thr
Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885
890 895 Val Leu Met Leu Glu Ser Pro Asn Pro
Leu Asp Val Ser Lys Thr Tyr 900 905
910 Pro Thr Leu His Ile Leu Leu Gln Phe Asn His Arg Gly Leu
Glu Ala 915 920 925
Arg Ile Phe Arg His Gly Gln Leu Trp Ala Glu Thr His Ala Glu Val 930
935 940 Val Leu Arg Ser Lys
Thr Lys Gln Ile Ser Phe Leu Ser Asn Gly Ser 945 950
955 960Tyr Pro Ser Met Asp Ala Thr Thr Pro Leu
Asn Pro Trp Lys Ser Thr 965 970
975 Tyr Gln Ala Val Leu Arg Ala Glu Pro His Arg Val Thr Met Asp
Val 980 985 990 Tyr
His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995
1000 1005 Trp Arg Thr Cys Glu Glu
Asn Val Phe Gly Leu Tyr His Val Phe Glu 1010 1015
1020 Thr His Tyr Ala Gly Tyr Phe Ser Asp Leu Leu
Ile His Asp Val Glu 1025 1030 1035
1040Thr Asn Pro Gly Pro Phe Thr Phe Lys Pro Arg Gln Arg Pro Val Phe
1045 1050 1055 Gln Thr
Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro 1060
1065 1070 Asn Asp Leu Ala Ser Lys
Ala Met Gly Ser Ala Phe Thr Ala Leu Leu 1075 1080
1085 Asp Ala Asn Glu Asp Ala Gln Lys Ala Met
Lys Ile Ile Lys Thr Leu 1090 1095 1100
Ser Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Gly Thr Leu
Asn Asn 1105 1110 1115
1120Pro Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala
1125 1130 1135 Gly Met Thr
Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys 1140
1145 1150 Leu Gly Val Leu Thr Ala Ala
Glu Ile Thr Ser Gln Thr Ser Leu Cys 1155 1160
1165 Glu Glu Ile Ala Ala Lys Phe Lys Thr Ile Phe
Thr Thr Pro Pro Pro 1170 1175 1180
Arg Phe Pro Val Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys
Gln 1185 1190 1195 1200Val
Asn Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys
1205 1210 1215 Thr Val Glu Lys Val
Val Asp Trp Phe Gly Thr Trp Val Ala Gln Glu 1220
1225 1230 Glu Arg Glu Gln Thr Leu Asp Gln Leu
Leu Gln Arg Phe Pro Glu His 1235 1240
1245 Ala Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ala Ala
Tyr Val Glu 1250 1255 1260
Cys Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val 1265
1270 1275 1280Lys Glu Lys Arg
Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln 1285
1290 1295 Lys His Asp His Ala Thr Ala Arg
Cys Glu Pro Val Val Ile Val Leu 1300 1305
1310 Arg Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser
Gln Ile Ile Ala 1315 1320 1325
Gln Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu
1330 1335 1340 Pro Pro Asp
Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala 1345
1350 1355 1360Ile Met Asp Asp Leu Gly Gln
Asn Pro Asp Gly Ser Asp Phe Thr Thr 1365
1370 1375 Phe Cys Gln Met Val Ser Thr Thr Asn Leu Leu
Pro Asn Met Ala Ser 1380 1385
1390 Leu Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala
Thr 1395 1400 1405 Thr
Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala 1410
1415 1420 Val Glu Arg Arg Ile
Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val 1425 1430
1435 1440Cys Ser Lys Thr Glu Ala Gly Cys Lys
Val Leu Asp Val Glu Arg Ala 1445 1450
1455 Phe Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln
Asn Asn Cys 1460 1465 1470
Leu Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Ser Lys
1475 1480 1485 Glu Ile Leu Ser
Leu Val Asp Val Ile Glu Arg Ala Val Thr Arg Ile 1490
1495 1500 Glu Arg Lys Lys Lys Val Leu Thr
Ala Val Gln Thr Leu Val Ala Gln 1505 1510
1515 1520Gly Pro Val Asp Glu Val Ser Phe Tyr Ser Val Val
Gln Gln Leu Lys 1525 1530
1535 Ala Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala
1540 1545 1550 Phe Ala
Arg Val Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys 1555
1560 1565 Ile Ser Ala Met Leu Cys
Ala Ala Thr Leu Ala Leu Thr Gln Val Val 1570 1575
1580 Lys Met Ala Lys Ala Val Lys Gln Met Val
Arg Pro Asp Leu Val Arg 1585 1590 1595
1600Val Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Thr
Thr Arg 1605 1610 1615
Ile Lys Pro Lys Thr Leu Gln Leu Leu Asp Val Gln Gly Pro Asn Pro
1620 1625 1630 Thr Met Asp Phe
Glu Lys Phe Val Ala Lys Phe Val Thr Ala Pro Ile 1635
1640 1645 Gly Phe Val Tyr Pro Thr Gly Val
Ser Thr Gln Thr Cys Leu Leu Val 1650 1655
1660 Lys Gly Arg Thr Leu Ala Val Asn Arg His Met Ala
Glu Ser Asp Trp 1665 1670 1675
1680Thr Ser Ile Val Val Arg Gly Val Ser His Thr Arg Ser Ser Val Lys
1685 1690 1695 Ile Ile Ala
Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile 1700
1705 1710 Arg Leu Ser Ser Gly Pro Leu
Phe Arg Asp Asn Thr Ser Lys Phe Val 1715 1720
1725 Lys Ala Ser Asp Val Leu Pro His Ser Ser Ser
Pro Leu Ile Gly Ile 1730 1735 1740
Met Asn Val Asp Ile Pro Met Met Tyr Thr Gly Thr Phe Leu Lys
Ala 1745 1750 1755 1760Gly
Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile
1765 1770 1775 His Tyr Lys Ala Asn
Thr Arg Lys Gly Trp Cys Gly Ser Ala Ile Leu 1780
1785 1790 Ala Asp Leu Gly Gly Ser Lys Lys Ile
Leu Gly Phe His Ser Ala Gly 1795 1800
1805 Ser Met Gly Val Ala Ala Ala Ser Ile Ile Ser Gln Glu
Met Ile Asp 1810 1815 1820
Ala Val Val Gln Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro 1825
1830 1835 1840Asp Gly Pro Arg
Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr 1845
1850 1855 Val Ala Arg Gln Val Phe Gln Pro
Ala Phe Ala Pro Ala Val Leu Ser 1860 1865
1870 Lys Phe Asp Pro Arg Thr Asp Ala Asp Val Asp Glu
Val Ala Phe Ser 1875 1880 1885
Lys His Thr Ser Asn Gln Glu Thr Leu Pro Pro Val Phe Arg Met Val
1890 1895 1900 Ala Arg Glu
Tyr Ala Asn Arg Val Phe Ala Leu Leu Gly Arg Asp Asn 1905
1910 1915 1920Gly Arg Leu Ser Val Lys Gln
Ala Leu Asp Gly Leu Glu Gly Met Asp 1925
1930 1935 Pro Met Asp Lys Asn Thr Ser Pro Gly Leu Pro
Tyr Thr Thr Leu Gly 1940 1945
1950 Met Arg Arg Thr Asp Val Val Asp Trp Glu Thr Ala Thr Leu Ile
Pro 1955 1960 1965 Phe
Ala Ala Glu Arg Leu Glu Lys Met Asn Asn Lys Asp Phe Ser Asp 1970
1975 1980 Ile Val Tyr Gln Thr
Phe Leu Lys Asp Glu Leu Arg Pro Ile Glu Lys 1985 1990
1995 2000Val Gln Ala Ala Lys Thr Arg Ile Val
Asp Val Pro Pro Phe Glu His 2005 2010
2015 Cys Ile Leu Gly Arg Gln Leu Leu Gly Lys Phe Ala Ser
Lys Phe Gln 2020 2025 2030
Thr Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp
2035 2040 2045 Val His Trp Thr
Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val 2050
2055 2060 Tyr Asp Val Asp Tyr Ser Asn Phe
Asp Ser Thr His Ser Val Ala Ile 2065 2070
2075 2080Phe Arg Leu Leu Ala Glu Glu Phe Phe Ser Glu Glu
Asn Gly Phe Asp 2085 2090
2095 Pro Leu Val Lys Asp Tyr Leu Glu Ser Leu Ala Ile Ser Lys His Ala
2100 2105 2110 Tyr Glu
Glu Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys 2115
2120 2125 Ala Ala Thr Ser Met Leu
Asn Thr Ile Met Asn Asn Ile Ile Ile Arg 2130 2135
2140 Ala Gly Leu Tyr Leu Thr Tyr Lys Asn Phe
Glu Phe Asp Asp Val Lys 2145 2150 2155
2160Val Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr
Gln Leu 2165 2170 2175
Asn Phe Asp Arg Val Arg Thr Ser Leu Ala Lys Thr Gly Tyr Lys Ile
2180 2185 2190 Thr Pro Ala Asn
Lys Thr Ser Thr Phe Pro Leu Glu Ser Thr Leu Glu 2195
2200 2205 Asp Val Val Phe Leu Lys Arg Lys
Phe Lys Lys Glu Gly Pro Leu Tyr 2210 2215
2220 Arg Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met
Leu Ser Tyr Tyr 2225 2230 2235
2240Arg Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala
2245 2250 2255 Val His Ser
Gly Lys Gln Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg 2260
2265 2270 Glu Val Gly Val Ile Val Pro
Thr Phe Glu Ser Val Glu Tyr Arg Trp 2275 2280
2285 Arg Ser Leu Phe Trp
2290 302301PRTTheiler's murine
encephalomyelitis virus 30Met Ala Cys Lys His Gly Tyr Pro Asp Val Cys Pro
Ile Cys Thr Ala 1 5 10
15 Val Asp Val Thr Pro Gly Phe Glu Tyr Leu Leu Leu Ala Asp Gly Glu
20 25 30 Trp Phe Pro
Thr Asp Leu Leu Cys Val Asp Leu Asp Asp Asp Val Phe 35
40 45 Trp Pro Ser Asn Ser Ser Asn Gln
Ser Glu Thr Met Glu Trp Thr Asp 50 55
60 Leu Pro Leu Val Arg Asp Ile Val Met Glu Pro Gln Gly
Asn Ala Ser 65 70 75
80Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser Gly Asn Glu Gly Val Ile
85 90 95 Ile Asn Asn Phe
Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu Ser 100
105 110 Ala Ser Gly Gly Asn Ala Gly Asp Ala
Pro Gln Asn Asn Gly Gln Leu 115 120
125 Ser Asn Ile Leu Gly Gly Ala Ala Asn Ala Phe Ala Thr Met
Ala Pro 130 135 140
Leu Leu Leu Asp Gln Asn Thr Glu Glu Met Glu Asn Leu Ser Asp Arg 145
150 155 160Val Ala Ser Asp Lys
Ala Gly Asn Ser Ala Thr Asn Thr Gln Ser Thr 165
170 175 Val Gly Arg Leu Cys Gly Tyr Gly Glu Ala
His His Gly Glu His Pro 180 185
190 Ala Ser Cys Ala Asp Thr Ala Thr Asp Lys Val Leu Ala Ala Glu
Arg 195 200 205 Tyr
Tyr Thr Ile Asp Leu Ala Ser Trp Thr Thr Thr Gln Glu Ala Phe 210
215 220 Ser His Ile Arg Ile Pro
Leu Pro His Val Leu Ala Gly Glu Asp Gly 225 230
235 240Gly Val Phe Gly Ala Thr Leu Arg Arg His Tyr
Leu Cys Lys Thr Gly 245 250
255 Trp Arg Val Gln Val Gln Cys Asn Ala Ser Gln Phe His Ala Gly Ser
260 265 270 Leu Leu
Val Phe Met Ala Pro Glu Phe Tyr Thr Gly Lys Gly Thr Lys 275
280 285 Thr Gly Asp Met Glu Pro Thr
Asp Pro Phe Thr Met Asp Thr Thr Trp 290 295
300 Arg Ala Pro Gln Gly Ala Pro Thr Gly Tyr Arg Tyr
Asp Ser Arg Thr 305 310 315
320Gly Phe Phe Ala Met Asn His Gln Asn Gln Trp Gln Trp Thr Val Tyr
325 330 335 Pro His Gln
Ile Leu Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu 340
345 350 Val Pro Tyr Val Asn Ile Ala Pro
Thr Ser Ser Trp Thr Gln His Ala 355 360
365 Asn Trp Thr Leu Val Val Ala Val Phe Ser Pro Leu Gln
Tyr Ala Ser 370 375 380
Gly Ser Ser Ser Asp Val Gln Ile Thr Ala Ser Ile Gln Pro Val Asn 385
390 395 400Pro Val Phe Asn
Gly Leu Arg His Glu Thr Val Ile Ala Gln Ser Pro 405
410 415 Ile Ala Val Thr Val Arg Glu His Lys
Gly Cys Phe Tyr Ser Thr Asn 420 425
430 Pro Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ser Thr
Pro Asn 435 440 445
Asp Tyr Met Cys Gly Glu Phe Ser Asp Leu Leu Glu Leu Cys Lys Leu 450
455 460 Pro Thr Phe Leu Gly
Asn Pro Asn Ser Asn Asn Lys Arg Tyr Pro Tyr 465 470
475 480Phe Ser Ala Thr Asn Ser Val Pro Thr Thr
Ser Leu Val Asp Tyr Gln 485 490
495 Val Ala Leu Ser Cys Ser Cys Met Cys Asn Ser Met Leu Ala Ala
Val 500 505 510 Ala
Arg Asn Phe Asn Gln Tyr Arg Gly Ser Leu Asn Phe Leu Phe Val 515
520 525 Phe Thr Gly Ala Ala Met
Val Lys Gly Lys Phe Leu Ile Ala Tyr Thr 530 535
540 Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Asp
Gln Ala Met Gln Ala 545 550 555
560Thr Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser Phe Val Phe Thr
565 570 575 Ala Pro
Phe Ile Ser Pro Thr His Tyr Arg Gln Thr Ser Tyr Thr Ser 580
585 590 Ala Thr Ile Ala Ser Val Asp
Gly Trp Val Thr Val Trp Gln Leu Thr 595 600
605 Pro Leu Thr Tyr Pro Ser Gly Ala Pro Val Asn Ser
Asp Ile Leu Thr 610 615 620
Leu Val Ser Ala Gly Asp Asp Phe Thr Leu Arg Met Pro Ile Ser Pro
625 630 635 640Thr Lys
Trp Ala Pro Gln Gly Ser Asp Asn Ala Glu Lys Gly Lys Val
645 650 655 Ser Asn Asp Asp Ala Ser
Val Asp Phe Val Ala Glu Pro Val Lys Leu 660
665 670 Pro Glu Asn Gln Thr Arg Val Ala Phe Phe
Tyr Asp Arg Ala Val Pro 675 680
685 Ile Gly Met Leu Arg Pro Gly Gln Asn Ile Glu Ser Thr Phe
Val Tyr 690 695 700
Gln Glu Asn Asp Leu Arg Leu Asn Cys Leu Leu Leu Thr Pro Leu Pro 705
710 715 720Ser Phe Cys Pro Asp
Ser Thr Ser Gly Pro Val Lys Thr Lys Ala Pro 725
730 735 Val Gln Trp Arg Trp Val Arg Ser Gly Gly
Thr Thr Asn Phe Pro Leu 740 745
750 Met Thr Lys Gln Asp Tyr Ala Phe Leu Cys Phe Ser Pro Phe Thr
Tyr 755 760 765 Tyr
Lys Cys Asp Leu Glu Val Thr Val Ser Ala Leu Gly Thr Asp Thr 770
775 780 Val Ala Ser Val Leu Arg
Trp Ala Pro Thr Gly Ala Pro Ala Asp Val 785 790
795 800Thr Asp Gln Leu Ile Gly Tyr Thr Pro Ser Leu
Gly Glu Thr Arg Asn 805 810
815 Pro His Met Trp Leu Val Gly Ala Gly Asn Thr Gln Ile Ser Phe Val
820 825 830 Val Pro
Tyr Asn Ser Pro Leu Ser Val Leu Pro Ala Ala Trp Phe Asn 835
840 845 Gly Trp Ser Asp Phe Gly Asn
Thr Lys Asp Phe Gly Val Ala Pro Asn 850 855
860 Ala Asp Phe Gly Arg Leu Trp Ile Gln Gly Asn Thr
Ser Ala Ser Val 865 870 875
880Arg Ile Arg Tyr Lys Lys Met Lys Val Phe Cys Pro Arg Pro Thr Leu
885 890 895 Phe Phe Pro
Trp Pro Val Ser Thr Arg Ser Lys Ile Asn Ala Asp Asn 900
905 910 Pro Val Pro Ile Leu Glu Leu Glu
Asn Pro Ala Ala Phe Tyr Arg Ile 915 920
925 Asp Leu Phe Ile Thr Phe Ile Asp Glu Phe Ile Thr Phe
Asp Tyr Lys 930 935 940
Val His Gly Arg Pro Val Leu Thr Phe Arg Ile Pro Gly Phe Gly Leu 945
950 955 960Thr Pro Ala Gly
Arg Met Leu Val Cys Met Gly Glu Lys Pro Ala His 965
970 975 Gly Pro Phe Thr Ser Ser Arg Ser Leu
Tyr His Val Ile Phe Thr Ala 980 985
990 Thr Cys Ser Ser Phe Ser Phe Ser Ile Tyr Lys Gly Arg Tyr
Arg Ser 995 1000 1005
Trp Lys Lys Pro Ile His Asp Glu Leu Val Asp Arg Gly Tyr Thr Thr 1010
1015 1020 Phe Gly Glu Phe
Phe Arg Ala Val Arg Ala Tyr His Ala Asp Tyr Tyr 1025 1030
1035 1040Lys Gln Arg Leu Ile His Asp Val
Glu Met Asn Pro Gly Pro Val Gln 1045 1050
1055 Ser Val Phe Gln Pro Gln Gly Ala Val Leu Thr Lys
Ser Leu Ala Pro 1060 1065 1070
Gln Ala Gly Ile Gln Asn Leu Leu Leu Arg Leu Leu Gly Ile Asp Gly
1075 1080 1085 Asp Cys Ser
Glu Val Ser Lys Ala Ile Thr Val Val Thr Asp Leu Phe 1090
1095 1100 Ala Ala Trp Glu Arg Ala Lys
Thr Thr Leu Val Ser Pro Glu Phe Trp 1105 1110
1115 1120Ser Lys Leu Ile Leu Lys Thr Thr Lys Phe Ile
Ala Ala Ser Val Leu 1125 1130
1135 Tyr Leu His Asn Pro Asp Phe Thr Thr Thr Val Cys Leu Ser Leu
Met 1140 1145 1150 Thr
Gly Val Asp Leu Leu Thr Asn Asp Ser Val Phe Asp Trp Leu Lys 1155
1160 1165 Asn Lys Leu Ser Ser
Phe Phe Arg Thr Pro Pro Pro Val Cys Pro Asn 1170 1175
1180 Val Leu Gln Pro Gln Gly Pro Leu Arg
Glu Ala Asn Glu Gly Phe Thr 1185 1190 1195
1200Phe Ala Lys Asn Ile Glu Trp Ala Met Lys Thr Ile Gln
Ser Ile Val 1205 1210 1215
Asn Trp Leu Thr Ser Trp Phe Lys Gln Glu Glu Asp His Pro Gln Ser
1220 1225 1230 Lys Leu Asp Lys
Phe Leu Met Glu Phe Pro Asp His Cys Arg Asn Ile 1235
1240 1245 Met Asp Met Arg Asn Gly Arg Lys
Ala Tyr Cys Glu Cys Thr Ala Ser 1250 1255
1260 Phe Lys Tyr Phe Asp Glu Leu Tyr Asn Leu Ala Val
Thr Cys Lys Arg 1265 1270 1275
1280Ile Pro Leu Ala Ser Leu Cys Glu Lys Phe Lys Asn Arg His Asp His
1285 1290 1295 Ser Val Thr
Arg Pro Glu Pro Val Val Val Val Leu Arg Gly Ala Ala 1300
1305 1310 Gly Gln Gly Lys Ser Val Thr
Ser Gln Ile Ile Ala Gln Ser Val Ser 1315 1320
1325 Lys Met Ala Phe Gly Arg Gln Ser Val Tyr Ser
Met Pro Pro Asp Ser 1330 1335 1340
Glu Tyr Phe Asp Gly Tyr Glu Asn Gln Phe Ser Val Ile Met Asp
Asp 1345 1350 1355 1360Leu
Gly Gln Asn Pro Asp Gly Glu Asp Phe Thr Val Phe Cys Gln Met
1365 1370 1375 Val Ser Ser Thr Asn
Phe Leu Pro Asn Met Ala His Leu Glu Arg Lys 1380
1385 1390 Gly Thr Pro Phe Thr Ser Ser Phe Ile
Val Ala Thr Thr Asn Leu Pro 1395 1400
1405 Lys Phe Arg Pro Val Thr Val Ala His Tyr Pro Ala Val
Asp Arg Arg 1410 1415 1420
Ile Thr Phe Asp Phe Thr Val Thr Ala Gly Pro His Cys Thr Thr Ser 1425
1430 1435 1440Asn Gly Met Leu
Asp Ile Glu Lys Ala Phe Asp Glu Ile Pro Gly Ser 1445
1450 1455 Lys Pro Gln Leu Ala Cys Phe Ser
Ala Asp Cys Pro Leu Leu His Lys 1460 1465
1470 Arg Gly Val Met Phe Thr Cys Asn Arg Thr Lys Ala
Val Tyr Asn Leu 1475 1480 1485
Gln Gln Val Val Lys Met Val Asn Asp Thr Ile Thr Arg Lys Thr Glu
1490 1495 1500 Asn Val Lys
Lys Met Asn Ser Leu Val Ala Gln Ser Pro Pro Asp Trp 1505
1510 1515 1520Glu His Phe Glu Asn Ile Leu
Thr Cys Leu Arg Gln Asn Asn Ala Ala 1525
1530 1535 Leu Gln Asp Gln Leu Asp Glu Leu Gln Glu Ala
Phe Ala Gln Ala Arg 1540 1545
1550 Glu Arg Ser Asp Phe Leu Ser Asp Trp Leu Lys Val Ser Ala Ile
Ile 1555 1560 1565 Phe
Ala Gly Ile Ala Ser Leu Ser Ala Val Ile Lys Leu Ala Ser Lys 1570
1575 1580 Phe Lys Glu Ser Ile
Trp Pro Ser Pro Val Arg Val Glu Leu Ser Glu 1585 1590
1595 1600Gly Glu Gln Ala Ala Tyr Ala Gly Arg
Ala Arg Ala Gln Lys Gln Ala 1605 1610
1615 Leu Gln Val Leu Asp Ile Gln Gly Gly Gly Lys Val Leu
Ala Gln Ala 1620 1625 1630
Gly Asn Pro Val Met Asp Phe Glu Leu Phe Cys Ala Lys Asn Met Val
1635 1640 1645 Ala Pro Ile Thr
Phe Tyr Tyr Pro Asp Lys Ala Glu Val Thr Gln Ser 1650
1655 1660 Cys Leu Leu Leu Arg Ala His Leu
Phe Val Val Asn Arg His Val Ala 1665 1670
1675 1680Glu Thr Glu Trp Thr Ala Phe Lys Leu Lys Asp Val
Arg His Glu Arg 1685 1690
1695 Asp Thr Val Val Thr Arg Ser Val Asn Arg Ser Gly Ala Glu Thr Asp
1700 1705 1710 Leu Thr
Phe Ile Lys Val Thr Lys Gly Pro Leu Phe Lys Asp Asn Val 1715
1720 1725 Asn Lys Phe Cys Ser Asn
Lys Asp Asp Phe Pro Ala Arg Asn Asp Ala 1730 1735
1740 Val Thr Gly Ile Met Asn Thr Gly Leu Ala
Phe Val Tyr Ser Gly Asn 1745 1750 1755
1760Phe Leu Ile Gly Asn Gln Pro Val Asn Thr Thr Thr Gly Ala
Cys Phe 1765 1770 1775
Asn His Cys Leu His Tyr Arg Ala Gln Thr Arg Arg Gly Trp Cys Gly
1780 1785 1790 Ser Ala Val Ile
Cys Asn Val Asn Gly Lys Lys Ala Val Tyr Gly Met 1795
1800 1805 His Ser Ala Gly Gly Gly Gly Leu
Ala Ala Ala Thr Ile Ile Thr Arg 1810 1815
1820 Glu Leu Ile Glu Ala Ala Glu Lys Ser Met Leu Ala
Leu Glu Pro Gln 1825 1830 1835
1840Gly Ala Ile Val Asp Ile Ser Thr Gly Ser Val Val His Val Pro Arg
1845 1850 1855 Lys Thr Lys
Leu Arg Arg Thr Val Ala His Asp Val Phe Gln Pro Lys 1860
1865 1870 Phe Glu Pro Ala Val Leu Ser
Arg Tyr Asp Pro Arg Thr Asp Lys Asp 1875 1880
1885 Val Asp Val Val Ala Phe Ser Lys His Thr Thr
Asn Met Glu Ser Leu 1890 1895 1900
Pro Pro Val Phe Asp Ile Val Cys Asp Glu Tyr Ala Asn Arg Val
Phe 1905 1910 1915 1920Thr
Ile Leu Gly Lys Asp Asn Gly Leu Leu Thr Val Glu Gln Ala Val
1925 1930 1935 Leu Gly Leu Pro Gly
Met Asp Pro Met Glu Lys Asp Thr Ser Pro Gly 1940
1945 1950 Leu Pro Tyr Thr Gln Gln Gly Leu Arg
Arg Thr Asp Leu Leu Asn Phe 1955 1960
1965 Asn Thr Ala Lys Met Thr Pro Gln Leu Asp Tyr Ala His
Ser Lys Leu 1970 1975 1980
Val Leu Gly Val Tyr Asp Asp Val Val Tyr Gln Ser Phe Leu Lys Asp 1985
1990 1995 2000Glu Ile Arg Pro
Leu Glu Lys Ile His Glu Ala Lys Thr Arg Ile Val 2005
2010 2015 Asp Val Pro Pro Phe Ala His Cys
Ile Trp Gly Arg Gln Leu Leu Gly 2020 2025
2030 Arg Phe Ala Ser Lys Phe Gln Thr Lys Pro Gly Leu
Glu Leu Gly Ser 2035 2040 2045
Ala Ile Gly Thr Asp Pro Asp Val Asp Trp Thr Pro Tyr Ala Ala Glu
2050 2055 2060 Leu Ser Gly
Phe Asn Tyr Val Tyr Asp Val Asp Tyr Ser Asn Phe Asp 2065
2070 2075 2080Ala Ser His Ser Thr Ala Met
Phe Glu Cys Leu Ile Lys Asn Phe Phe 2085
2090 2095 Thr Glu Gln Asn Gly Phe Asp Arg Arg Ile Ala
Glu Tyr Leu Arg Ser 2100 2105
2110 Leu Ala Val Ser Arg His Ala Tyr Glu Asp Arg Arg Val Leu Ile
Arg 2115 2120 2125 Gly
Gly Leu Leu Ser Gly Cys Ala Ala Thr Ser Met Leu Asn Thr Ile 2130
2135 2140 Met Asn Asn Val Ile
Ile Arg Ala Ala Leu Tyr Leu Thr Tyr Ser Asn 2145 2150
2155 2160Phe Glu Phe Asp Asp Ile Lys Val Leu
Ser Tyr Gly Asp Asp Leu Leu 2165 2170
2175 Ile Gly Thr Asn Tyr Gln Ile Asp Phe Asn Leu Val Lys
Glu Arg Leu 2180 2185 2190
Ala Pro Phe Gly Tyr Lys Ile Thr Pro Ala Asn Lys Thr Thr Thr Phe
2195 2200 2205 Pro Leu Thr Ser
His Leu Gln Asp Val Thr Phe Leu Lys Arg Arg Phe 2210
2215 2220 Val Arg Phe Asn Ser Tyr Leu Phe
Arg Pro Gln Met Asp Ala Val Asn 2225 2230
2235 2240Leu Lys Ala Met Val Ser Tyr Cys Lys Pro Gly Thr
Leu Lys Glu Lys 2245 2250
2255 Leu Met Ser Ile Ala Leu Leu Ala Val His Ser Gly Pro Asp Ile Tyr
2260 2265 2270 Asp Glu
Ile Phe Leu Pro Phe Arg Asn Val Gly Ile Val Val Pro Thr 2275
2280 2285 Tyr Ser Ser Met Leu Tyr
Arg Trp Leu Ser Leu Phe Arg 2290 2295
2300 312303PRTTheiler's murine encephalomyelitis virus
31Met Ala Cys Lys His Gly Tyr Pro Asp Val Cys Pro Ile Cys Thr Ala 1
5 10 15 Val Asp Ala Thr
Pro Asp Phe Glu Tyr Leu Leu Met Ala Asp Gly Glu 20
25 30 Trp Phe Pro Thr Asp Leu Leu Cys Val
Asp Leu Asp Asp Asp Val Phe 35 40
45 Trp Pro Ser Asp Thr Ser Thr Gln Pro Gln Thr Met Glu Trp
Thr Asp 50 55 60
Val Pro Leu Val Cys Asp Thr Val Met Glu Pro Gln Gly Asn Ala Ser 65
70 75 80Ser Ser Asp Lys Ser
Asn Ser Gln Ser Ser Gly Asn Glu Gly Val Ile 85
90 95 Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln
Asn Ser Ile Asp Leu Ser 100 105
110 Ala Ser Gly Gly Asn Ala Gly Asp Ala Pro Gln Asn Asn Gly Gln
Leu 115 120 125 Ser
Ser Ile Leu Gly Gly Ala Ala Asn Ala Phe Ala Thr Met Ala Pro 130
135 140 Leu Leu Met Asp Gln Asn
Thr Glu Glu Met Glu Asn Leu Ser Asp Arg 145 150
155 160Val Ala Ser Asp Lys Ala Gly Asn Ser Ala Thr
Asn Thr Gln Ser Thr 165 170
175 Val Gly Arg Leu Cys Gly Tyr Gly Lys Ser His His Gly Glu His Pro
180 185 190 Thr Ser
Cys Ala Asp Ala Ala Thr Asp Lys Val Leu Ala Ala Glu Arg 195
200 205 Tyr Tyr Thr Ile Asp Leu Ala
Ser Trp Thr Thr Ser Gln Glu Ala Phe 210 215
220 Ser His Ile Arg Ile Pro Leu Pro His Val Leu Ala
Gly Glu Asp Gly 225 230 235
240Gly Val Phe Gly Ala Thr Leu Arg Arg His Tyr Leu Cys Lys Thr Gly
245 250 255 Trp Arg Val
Gln Val Gln Cys Asn Ala Ser Gln Phe His Ala Gly Ser 260
265 270 Leu Leu Val Phe Met Ala Pro Glu
Phe Tyr Thr Gly Lys Gly Thr Lys 275 280
285 Ser Gly Thr Met Glu Pro Ser Asp Pro Phe Thr Met Asp
Thr Thr Trp 290 295 300
Arg Ser Pro Gln Ser Ala Pro Thr Gly Tyr Arg Tyr Asp Arg Gln Ala 305
310 315 320Gly Phe Phe Ala
Met Asn His Gln Asn Gln Trp Gln Trp Thr Val Tyr 325
330 335 Pro His Gln Ile Leu Asn Leu Arg Thr
Asn Thr Thr Val Asp Leu Glu 340 345
350 Val Pro Tyr Val Asn Val Ala Pro Ser Ser Ser Trp Thr Gln
His Ala 355 360 365
Asn Trp Thr Leu Val Val Ala Val Leu Ser Pro Leu Gln Tyr Ala Thr 370
375 380 Gly Ser Ser Pro Asp
Val Gln Ile Thr Ala Ser Leu Gln Pro Val Asn 385 390
395 400Pro Val Phe Asn Gly Leu Arg His Glu Thr
Val Leu Ala Gln Ser Pro 405 410
415 Ile Pro Val Thr Val Arg Glu His Gln Gly Cys Phe Tyr Ser Thr
Asn 420 425 430 Pro
Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ser Thr Pro Ser 435
440 445 Asp Tyr Met Cys Gly Glu
Phe Ser Asp Leu Leu Glu Leu Cys Lys Leu 450 455
460 Pro Thr Phe Leu Gly Asn Pro Ser Thr Asp Asn
Lys Arg Tyr Pro Tyr 465 470 475
480Phe Ser Ala Thr Asn Ser Val Pro Ala Thr Ser Leu Val Asp Tyr Gln
485 490 495 Val Ala
Leu Ser Cys Ser Cys Thr Ala Asn Ser Met Leu Ala Ala Val 500
505 510 Ala Arg Asn Phe Asn Gln Tyr
Arg Gly Ser Leu Asn Phe Leu Phe Val 515 520
525 Phe Thr Gly Ala Ala Met Val Lys Gly Lys Phe Arg
Ile Ala Tyr Thr 530 535 540
Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Asp Gln Ala Met Gln Ala
545 550 555 560Thr Tyr
Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser Phe Asn Phe Thr
565 570 575 Ala Pro Phe Ile Ser Pro
Thr His Tyr Arg Gln Thr Ser Tyr Thr Ser 580
585 590 Pro Thr Ile Thr Ser Val Asp Gly Trp Val
Thr Val Trp Gln Leu Thr 595 600
605 Pro Leu Thr Tyr Pro Ser Gly Thr Pro Thr His Ser Asp Ile
Leu Thr 610 615 620
Leu Val Ser Ala Gly Asp Asp Phe Thr Leu Arg Met Pro Ile Ser Pro 625
630 635 640Thr Lys Trp Val Pro
Gln Gly Ile Asp Asn Ala Glu Lys Gly Lys Val 645
650 655 Ser Asn Asp Asp Ala Ser Val Asp Phe Val
Ala Glu Pro Val Lys Leu 660 665
670 Pro Glu Asn Gln Thr Arg Val Ala Phe Phe Tyr Asp Arg Ala Val
Pro 675 680 685 Ile
Gly Met Leu Arg Pro Gly Gln Asn Met Glu Thr Thr Phe Ser Tyr 690
695 700 Gln Glu Asn Asp Phe Arg
Leu Asn Cys Leu Leu Leu Thr Pro Leu Pro 705 710
715 720Ser Tyr Cys Pro Asp Ser Ser Ser Gly Pro Val
Arg Thr Lys Ala Pro 725 730
735 Val Gln Trp Arg Trp Val Arg Ser Gly Gly Ala Asn Gly Ala Asn Phe
740 745 750 Pro Leu
Met Thr Lys Gln Asp Tyr Ala Phe Leu Cys Phe Ser Pro Phe 755
760 765 Thr Tyr Tyr Lys Cys Asp Leu
Glu Val Thr Val Ser Ala Met Gly Ala 770 775
780 Gly Thr Val Ser Ser Val Leu Arg Trp Ala Pro Thr
Gly Ala Pro Ala 785 790 795
800Asp Val Thr Asp Gln Leu Ile Gly Tyr Thr Pro Ser Leu Gly Glu Thr
805 810 815 Arg Asn Pro
His Met Trp Ile Val Gly Ser Gly Asn Ser Gln Ile Ser 820
825 830 Phe Val Val Pro Tyr Asn Ser Pro
Leu Ser Val Leu Pro Ala Ala Trp 835 840
845 Phe Asn Gly Trp Ser Asp Phe Gly Asn Thr Lys Asp Phe
Gly Val Ala 850 855 860
Pro Thr Ser Asp Phe Gly Arg Ile Trp Ile Gln Gly Asn Ser Ser Ala 865
870 875 880Ser Val Arg Ile
Arg Tyr Lys Lys Met Lys Val Phe Cys Pro Arg Pro 885
890 895 Thr Leu Phe Phe Pro Trp Pro Thr Pro
Thr Thr Thr Lys Ile Asn Ala 900 905
910 Asp Asn Pro Val Pro Ile Leu Glu Leu Glu Asn Pro Ala Ser
Leu Tyr 915 920 925
Arg Ile Asp Leu Phe Ile Thr Phe Thr Asp Glu Leu Ile Thr Phe Asp 930
935 940 Tyr Lys Val His Gly
Arg Pro Val Leu Thr Phe Arg Ile Pro Gly Phe 945 950
955 960Gly Leu Thr Pro Ala Gly Arg Met Leu Val
Cys Met Gly Ala Lys Pro 965 970
975 Ala His Ser Pro Phe Thr Ser Ser Lys Ser Leu Tyr His Val Ile
Phe 980 985 990 Thr
Ser Thr Cys Asn Ser Phe Ser Phe Thr Ile Tyr Lys Gly Arg Tyr 995
1000 1005 Arg Ser Trp Lys Lys Pro
Ile His Asp Glu Leu Val Asp Arg Gly Tyr 1010 1015
1020 Thr Thr Phe Arg Glu Phe Phe Lys Ala Val
Arg Gly Tyr His Ala Asp 1025 1030 1035
1040Tyr Tyr Lys Gln Arg Leu Ile His Asp Val Glu Met Asn Pro
Gly Pro 1045 1050 1055
Val Gln Ser Val Phe Gln Pro Gln Gly Ala Val Leu Thr Lys Ser Leu
1060 1065 1070 Ala Pro Gln Ala
Gly Ile Gln Asn Ile Leu Leu Arg Leu Leu Gly Ile 1075
1080 1085 Glu Gly Asp Cys Ser Glu Val Ser
Lys Ala Ile Thr Val Val Thr Asp 1090 1095
1100 Leu Val Ala Ala Trp Glu Lys Ala Lys Thr Thr Leu
Val Ser Pro Glu 1105 1110 1115
1120Phe Trp Ser Glu Leu Ile Leu Lys Thr Thr Lys Phe Ile Ala Ala Ser
1125 1130 1135 Val Leu Tyr
Leu His Asn Pro Asp Phe Thr Thr Thr Val Cys Leu Ser 1140
1145 1150 Leu Met Thr Gly Val Asp Leu
Leu Thr Asn Asp Ser Val Phe Asp Trp 1155 1160
1165 Leu Lys Ser Lys Leu Ser Ser Phe Phe Arg Thr
Pro Pro Pro Ala Cys 1170 1175 1180
Pro Asn Val Met Gln Pro Gln Gly Pro Leu Arg Glu Ala Asn Glu
Gly 1185 1190 1195 1200Phe
Thr Phe Ala Lys Asn Ile Glu Trp Ala Thr Lys Thr Ile Gln Ser
1205 1210 1215 Ile Val Asn Trp Leu
Thr Ser Trp Phe Lys Gln Glu Glu Asp His Pro 1220
1225 1230 Gln Ser Lys Leu Asp Lys Leu Leu Met
Glu Phe Pro Asp His Cys Arg 1235 1240
1245 Asn Ile Met Asp Met Arg Asn Gly Arg Lys Ala Tyr Cys
Glu Cys Thr 1250 1255 1260
Ala Ser Phe Lys Tyr Phe Asp Asp Leu Tyr Asn Leu Ala Val Thr Cys 1265
1270 1275 1280Lys Arg Ile Pro
Leu Ala Ser Leu Cys Glu Lys Phe Lys Asn Arg His 1285
1290 1295 Asp His Ser Val Thr Arg Pro Glu
Pro Val Val Ala Val Leu Arg Gly 1300 1305
1310 Ala Ala Gly Gln Gly Lys Ser Val Thr Ser Gln Ile
Ile Ala Gln Ser 1315 1320 1325
Val Ser Lys Met Ala Phe Gly Arg Gln Ser Val Tyr Ser Met Pro Pro
1330 1335 1340 Asp Ser Glu
Tyr Phe Asp Gly Tyr Glu Asn Gln Phe Ser Val Ile Met 1345
1350 1355 1360Asp Asp Leu Gly Gln Asn Pro
Asp Gly Glu Asp Phe Thr Val Phe Cys 1365
1370 1375 Gln Met Val Ser Ser Thr Asn Phe Leu Pro Asn
Met Ala His Leu Glu 1380 1385
1390 Arg Lys Gly Thr Pro Phe Thr Ser Ser Phe Ile Val Ala Thr Thr
Asn 1395 1400 1405 Leu
Pro Lys Phe Arg Pro Val Thr Val Ala His Tyr Pro Ala Val Asp 1410
1415 1420 Arg Arg Ile Thr Phe
Asp Phe Thr Val Thr Ala Gly Pro His Cys Lys 1425 1430
1435 1440Thr Pro Ala Gly Met Leu Asp Ile Glu
Lys Ala Phe Asp Glu Ile Pro 1445 1450
1455 Gly Ser Lys Pro Gln Leu Ala Cys Phe Ser Ala Asp Cys
Pro Leu Leu 1460 1465 1470
His Lys Arg Gly Val Met Phe Thr Cys Asn Arg Thr Lys Thr Val Tyr
1475 1480 1485 Asn Leu Gln Gln
Val Val Lys Met Val Asn Asp Thr Ile Thr Arg Lys 1490
1495 1500 Thr Glu Asn Val Lys Lys Met Asn
Ser Leu Val Ala Gln Ser Pro Pro 1505 1510
1515 1520Asp Trp Gln His Phe Glu Asn Ile Leu Thr Cys Leu
Arg Gln Asn Asn 1525 1530
1535 Ala Ala Leu Gln Asp Gln Val Asp Glu Leu Gln Glu Ala Phe Thr Gln
1540 1545 1550 Ala Arg
Glu Arg Ser Asp Phe Leu Ser Asp Trp Leu Lys Val Ser Ala 1555
1560 1565 Ile Ile Phe Ala Gly Ile
Val Ser Leu Ser Ala Val Ile Lys Leu Ala 1570 1575
1580 Ser Lys Phe Lys Glu Ser Ile Trp Pro Thr
Pro Val Arg Val Glu Leu 1585 1590 1595
1600Ser Glu Gly Glu Gln Ala Ala Tyr Ala Gly Arg Ala Arg Ala
Gln Lys 1605 1610 1615
Gln Ala Leu Gln Val Leu Asp Ile Gln Gly Gly Gly Lys Val Leu Ala
1620 1625 1630 Gln Ala Gly Asn
Pro Val Met Asp Phe Glu Leu Phe Cys Ala Lys Asn 1635
1640 1645 Met Val Ser Pro Ile Thr Phe Tyr
Tyr Pro Asp Lys Ala Glu Val Thr 1650 1655
1660 Gln Ser Cys Leu Leu Leu Arg Ala His Leu Phe Val
Val Asn Arg His 1665 1670 1675
1680Val Ala Glu Thr Glu Trp Thr Ala Phe Lys Leu Arg Asp Val Arg His
1685 1690 1695 Glu Arg Asp
Thr Val Val Met Arg Ser Val Asn Arg Ser Gly Ala Glu 1700
1705 1710 Thr Asp Leu Thr Phe Val Lys
Val Thr Lys Gly Pro Leu Phe Lys Asp 1715 1720
1725 Asn Val Asn Lys Phe Cys Ser Asn Lys Asp Asp
Phe Pro Ala Arg Asn 1730 1735 1740
Asp Thr Val Thr Gly Ile Met Asn Thr Gly Leu Ala Phe Val Tyr
Ser 1745 1750 1755 1760Gly
Asn Phe Leu Ile Gly Asn Gln Pro Val Asn Thr Thr Thr Gly Ala
1765 1770 1775 Cys Phe Asn His Cys
Leu His Tyr Arg Ala Gln Thr Arg Arg Gly Trp 1780
1785 1790 Cys Gly Ser Ala Ile Ile Cys Asn Val
Asn Gly Lys Lys Ala Val Tyr 1795 1800
1805 Gly Met His Ser Ala Gly Gly Gly Gly Leu Ala Ala Ala
Thr Ile Ile 1810 1815 1820
Thr Arg Glu Leu Ile Glu Ala Ala Glu Lys Ser Met Leu Ala Leu Glu 1825
1830 1835 1840Pro Gln Gly Ala
Ile Val Asp Ile Ser Thr Gly Ser Val Val His Val 1845
1850 1855 Pro Arg Lys Thr Lys Leu Arg Arg
Thr Val Ala His Asp Val Phe Gln 1860 1865
1870 Pro Lys Phe Glu Pro Ala Val Leu Ser Arg Tyr Asp
Pro Arg Thr Asp 1875 1880 1885
Lys Asp Val Asp Val Val Ala Phe Ser Lys His Thr Thr Asn Met Glu
1890 1895 1900 Ser Leu Pro
Pro Ile Phe Asp Ile Val Cys Gly Glu Tyr Ala Asn Arg 1905
1910 1915 1920Val Phe Thr Ile Leu Gly Lys
Asp Asn Gly Leu Leu Thr Val Glu Gln 1925
1930 1935 Ala Val Leu Gly Leu Ser Gly Met Asp Pro Met
Glu Lys Asp Thr Ser 1940 1945
1950 Pro Gly Leu Pro Tyr Thr Gln Gln Gly Leu Arg Arg Thr Asp Leu
Leu 1955 1960 1965 Asp
Phe Asn Thr Ala Lys Met Thr Pro Gln Leu Asp Tyr Ala His Ser 1970
1975 1980 Lys Leu Val Leu Gly
Val Tyr Asp Asp Val Val Tyr Gln Ser Phe Leu 1985 1990
1995 2000Lys Asp Glu Ile Arg Pro Leu Glu Lys
Ile His Glu Ala Lys Thr Arg 2005 2010
2015 Ile Val Asp Val Pro Pro Phe Ala His Cys Ile Trp Gly
Arg Gln Leu 2020 2025 2030
Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr Lys Pro Gly Phe Glu Leu
2035 2040 2045 Gly Ser Ala Ile
Gly Thr Asp Pro Asp Val Asp Trp Thr Arg Tyr Ala 2050
2055 2060 Ala Glu Leu Ser Gly Phe Asn Tyr
Val Tyr Asp Val Asp Tyr Ser Asn 2065 2070
2075 2080Phe Asp Ala Ser His Ser Thr Ala Met Phe Glu Cys
Leu Ile Asn Asn 2085 2090
2095 Phe Phe Thr Glu Gln Asn Gly Phe Asp Arg Arg Ile Ala Glu Tyr Leu
2100 2105 2110 Arg Ser
Leu Ala Val Ser Arg His Ala Tyr Glu Asp Arg Arg Val Leu 2115
2120 2125 Ile Arg Gly Gly Leu Pro
Ser Gly Cys Ala Ala Thr Ser Met Leu Asn 2130 2135
2140 Thr Ile Met Asn Asn Val Ile Ile Arg Ala
Ala Leu Tyr Leu Thr Tyr 2145 2150 2155
2160Ser Asn Phe Glu Phe Asp Asp Ile Lys Val Leu Ser Tyr Gly
Asp Asp 2165 2170 2175
Leu Leu Ile Gly Thr Asn Tyr Gln Ile Asp Phe Asn Leu Val Lys Glu
2180 2185 2190 Arg Leu Ala Pro
Phe Gly Tyr Lys Ile Thr Pro Ala Asn Lys Thr Thr 2195
2200 2205 Thr Phe Pro Leu Thr Ser His Leu
Gln Asp Val Thr Phe Leu Lys Arg 2210 2215
2220 Arg Phe Val Arg Phe Asn Ser Tyr Leu Phe Arg Pro
Gln Met Asp Ala 2225 2230 2235
2240Val Asn Leu Lys Ala Met Val Ser Tyr Cys Lys Pro Gly Thr Leu Lys
2245 2250 2255 Glu Lys Leu
Met Ser Ile Ala Leu Leu Ala Val His Ser Gly Pro Asp 2260
2265 2270 Ile Tyr Asp Glu Ile Phe Leu
Pro Phe Arg Asn Val Gly Ile Val Val 2275 2280
2285 Pro Thr Tyr Asp Ser Met Leu Tyr Arg Trp Leu
Ser Leu Phe Arg 2290 2295 2300
322303PRTTheiler's murine encephalomyelitis virus 32Met Ala Cys
Lys His Gly Tyr Pro Asp Val Cys Pro Ile Cys Thr Ala 1 5
10 15 Val Asp Ala Thr Pro Gly Phe Glu
Tyr Leu Leu Met Ala Asp Gly Glu 20 25
30 Trp Tyr Pro Thr Asp Leu Leu Cys Val Asp Leu Asp Asp
Asp Val Phe 35 40 45
Trp Pro Ser Asp Thr Ser Asn Gln Ser Gln Thr Met Asp Trp Thr Asp 50
55 60 Val Pro Leu Ile Arg
Asp Ile Val Met Glu Pro Gln Gly Asn Ser Ser 65 70
75 80Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser
Gly Asn Glu Gly Val Ile 85 90
95 Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu
Ser 100 105 110 Ala
Ser Gly Gly Asn Ala Gly Asp Ala Pro Gln Thr Asn Gly Gln Leu 115
120 125 Ser Asn Ile Leu Gly Gly
Ala Ala Asn Ala Phe Ala Thr Met Ala Pro 130 135
140 Leu Leu Leu Asp Gln Asn Thr Glu Glu Met Glu
Asn Leu Ser Asp Arg 145 150 155
160Val Ala Ser Asp Lys Ala Gly Asn Ser Ala Thr Asn Thr Gln Ser Thr
165 170 175 Val Gly
Arg Leu Cys Gly Tyr Gly Lys Ser His His Gly Glu His Pro 180
185 190 Ala Ser Cys Ala Asp Thr Ala
Thr Asp Lys Val Leu Ala Ala Glu Arg 195 200
205 Tyr Tyr Thr Ile Asp Leu Ala Ser Trp Thr Thr Ser
Gln Glu Ala Phe 210 215 220
Ser His Ile Arg Ile Pro Leu Pro His Val Leu Ala Gly Glu Asp Gly
225 230 235 240Gly Val
Phe Gly Ala Thr Leu Arg Arg His Tyr Leu Cys Lys Thr Gly
245 250 255 Trp Arg Val Gln Val Gln
Cys Asn Ala Ser Gln Phe His Ala Gly Ser 260
265 270 Leu Leu Val Phe Met Ala Pro Glu Phe Tyr
Thr Gly Lys Gly Thr Lys 275 280
285 Thr Gly Thr Met Glu Pro Ser Asp Pro Phe Thr Met Asp Thr
Glu Trp 290 295 300
Arg Ser Pro Gln Gly Ala Pro Thr Gly Tyr Arg Tyr Asp Ser Arg Thr 305
310 315 320Gly Phe Phe Ala Thr
Asn His Gln Asn Gln Trp Gln Trp Thr Val Tyr 325
330 335 Pro His Gln Ile Leu Asn Leu Arg Thr Asn
Thr Thr Val Asp Leu Glu 340 345
350 Val Pro Tyr Val Asn Val Ala Pro Ser Ser Ser Trp Thr Gln His
Ala 355 360 365 Asn
Trp Thr Leu Val Val Ala Val Leu Ser Pro Leu Gln Tyr Ala Thr 370
375 380 Gly Ser Ser Pro Asp Val
Gln Ile Thr Ala Ser Leu Gln Pro Val Asn 385 390
395 400Pro Val Phe Asn Gly Leu Arg His Glu Thr Val
Ile Ala Gln Ser Pro 405 410
415 Ile Pro Val Thr Val Arg Glu His Lys Gly Cys Phe Tyr Ser Thr Asn
420 425 430 Pro Asp
Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ser Thr Pro Ser 435
440 445 Asp Tyr Met Cys Gly Glu Phe
Ser Asp Leu Leu Glu Leu Cys Lys Leu 450 455
460 Pro Thr Phe Leu Gly Asn Pro Asn Thr Asn Asn Lys
Arg Tyr Pro Tyr 465 470 475
480Phe Ser Ala Thr Asn Ser Val Pro Ala Thr Ser Met Val Asp Tyr Gln
485 490 495 Val Ala Leu
Ser Cys Ser Cys Met Ala Asn Ser Met Leu Ala Ala Val 500
505 510 Ala Arg Asn Phe Asn Gln Tyr Arg
Gly Ser Leu Asn Phe Leu Phe Val 515 520
525 Phe Thr Gly Ala Ala Met Val Lys Gly Lys Phe Leu Ile
Ala Tyr Thr 530 535 540
Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Asp Gln Ala Met Gln Ser 545
550 555 560Thr Tyr Ala Ile
Trp Asp Leu Gly Leu Asn Ser Ser Phe Asn Phe Thr 565
570 575 Ala Pro Phe Ile Ser Pro Thr His Tyr
Arg Gln Thr Ser Tyr Thr Ser 580 585
590 Pro Thr Ile Thr Ser Val Asp Gly Trp Val Thr Val Trp Lys
Leu Thr 595 600 605
Pro Leu Thr Tyr Pro Ser Gly Thr Pro Thr Asn Ser Asp Ile Leu Thr 610
615 620 Leu Val Ser Ala Gly
Asp Asp Phe Thr Leu Arg Met Pro Ile Ser Pro 625 630
635 640Thr Lys Trp Val Pro Gln Gly Val Asp Asn
Ala Glu Lys Gly Lys Val 645 650
655 Ser Asn Asp Asp Ala Ser Val Asp Phe Val Ala Glu Pro Val Lys
Leu 660 665 670 Pro
Glu Asn Gln Thr Arg Val Ala Phe Phe Tyr Asp Arg Ala Val Pro 675
680 685 Ile Gly Met Leu Arg Pro
Gly Gln Asn Met Glu Thr Thr Phe Asn Tyr 690 695
700 Gln Glu Asn Asp Tyr Arg Leu Asn Cys Leu Leu
Leu Thr Pro Leu Pro 705 710 715
720Ser Phe Cys Pro Asp Ser Ser Ser Gly Pro Gln Lys Thr Lys Ala Pro
725 730 735 Val Gln
Trp Arg Trp Val Arg Ser Gly Gly Val Asn Gly Ala Asn Phe 740
745 750 Pro Leu Met Thr Lys Gln Asp
Tyr Ala Phe Leu Cys Phe Ser Pro Phe 755 760
765 Thr Phe Tyr Lys Cys Asp Leu Glu Val Thr Val Ser
Ala Leu Gly Met 770 775 780
Thr Arg Val Ala Ser Val Leu Arg Trp Ala Pro Thr Gly Ala Pro Ala
785 790 795 800Asp Val
Thr Asp Gln Leu Ile Gly Tyr Thr Pro Ser Leu Gly Glu Thr
805 810 815 Arg Asn Pro His Met Trp
Leu Val Gly Ala Gly Asn Ser Gln Val Ser 820
825 830 Phe Val Val Pro Tyr Asn Ser Pro Leu Ser
Val Leu Pro Ala Ala Trp 835 840
845 Phe Asn Gly Trp Ser Asp Phe Gly Asn Thr Lys Asp Phe Gly
Val Ala 850 855 860
Pro Asn Ala Asp Phe Gly Arg Leu Trp Ile Gln Gly Asn Thr Ser Ala 865
870 875 880Ser Val Arg Ile Arg
Tyr Lys Lys Met Lys Val Phe Cys Pro Arg Pro 885
890 895 Thr Leu Phe Phe Pro Trp Pro Thr Pro Thr
Thr Thr Lys Ile Asn Ala 900 905
910 Asp Asn Pro Val Pro Ile Leu Glu Leu Glu Asn Pro Ala Ala Leu
Tyr 915 920 925 Arg
Ile Asp Leu Phe Ile Thr Phe Thr Asp Glu Phe Ile Thr Phe Asp 930
935 940 Tyr Lys Val His Gly Arg
Pro Val Leu Thr Phe Arg Ile Pro Gly Phe 945 950
955 960Gly Leu Thr Pro Ala Gly Arg Met Leu Val Cys
Met Gly Glu Gln Pro 965 970
975 Ala His Gly Pro Phe Thr Ser Ser Arg Ser Leu Tyr His Val Ile Phe
980 985 990 Thr Ala
Thr Cys Ser Ser Phe Ser Phe Ser Ile Tyr Lys Gly Arg Tyr 995
1000 1005 Arg Ser Trp Lys Lys Pro Ile
His Asp Glu Leu Val Asp Arg Gly Tyr 1010 1015
1020 Thr Thr Phe Gly Glu Phe Phe Lys Ala Val Arg
Gly Tyr His Ala Asp 1025 1030 1035
1040Tyr Tyr Arg Gln Arg Leu Ile His Asp Val Glu Thr Asn Pro Gly
Pro 1045 1050 1055 Val
Gln Ser Val Phe Gln Pro Gln Gly Ala Val Leu Thr Lys Ser Leu
1060 1065 1070 Ala Pro Gln Ala Gly
Ile Gln Asn Leu Leu Leu Arg Leu Leu Gly Ile 1075
1080 1085 Asp Gly Asp Cys Ser Glu Val Ser Lys
Ala Ile Thr Val Val Thr Asp 1090 1095
1100 Leu Val Ala Ala Trp Glu Lys Ala Lys Thr Thr Leu Val
Ser Pro Glu 1105 1110 1115
1120Phe Trp Ser Lys Leu Ile Leu Lys Thr Thr Lys Phe Ile Ala Ala Ser
1125 1130 1135 Val Leu Tyr
Leu His Asn Pro Asp Phe Thr Thr Thr Val Cys Leu Ser 1140
1145 1150 Leu Met Thr Gly Val Asp Leu
Leu Thr Asn Asp Ser Val Phe Asp Trp 1155 1160
1165 Leu Lys Gln Lys Leu Ser Ser Phe Phe Arg Thr
Pro Pro Pro Ala Cys 1170 1175 1180
Pro Asn Val Met Gln Pro Gln Gly Pro Leu Arg Glu Ala Asn Glu
Gly 1185 1190 1195 1200Phe
Thr Phe Ala Lys Asn Ile Glu Trp Ala Met Lys Thr Ile Gln Ser
1205 1210 1215 Val Val Asn Trp Leu
Thr Ser Trp Phe Lys Gln Glu Glu Asp His Pro 1220
1225 1230 Gln Ser Lys Leu Asp Lys Leu Leu Met
Glu Phe Pro Asp His Cys Arg 1235 1240
1245 Asn Ile Met Asp Met Arg Asn Gly Arg Lys Ala Tyr Cys
Glu Cys Thr 1250 1255 1260
Ala Ser Phe Lys Tyr Phe Asp Glu Leu Tyr Asn Leu Ala Val Thr Cys 1265
1270 1275 1280Lys Arg Ile Pro
Leu Ala Ser Leu Cys Glu Lys Phe Lys Asn Arg His 1285
1290 1295 Asp His Ser Val Thr Arg Pro Glu
Pro Val Val Val Val Leu Arg Gly 1300 1305
1310 Ala Ala Gly Gln Gly Lys Ser Val Thr Ser Gln Ile
Ile Ala Gln Ser 1315 1320 1325
Val Ser Lys Met Ala Phe Gly Arg Gln Ser Val Tyr Ser Met Pro Pro
1330 1335 1340 Asp Ser Glu
Tyr Phe Asp Gly Tyr Glu Asn Gln Phe Ser Val Ile Met 1345
1350 1355 1360Asp Asp Leu Gly Gln Asn Pro
Asp Gly Glu Asp Phe Thr Val Phe Cys 1365
1370 1375 Gln Met Val Ser Ser Thr Asn Phe Leu Pro Asn
Met Ala His Leu Glu 1380 1385
1390 Arg Lys Gly Thr Pro Phe Thr Ser Ser Phe Ile Val Ala Thr Thr
Asn 1395 1400 1405 Leu
Pro Lys Phe Arg Pro Val Thr Val Ala His Tyr Pro Ala Val Asp 1410
1415 1420 Arg Arg Ile Thr Phe
Asp Phe Thr Val Thr Ala Gly Pro His Cys Lys 1425 1430
1435 1440Thr Pro Ala Gly Met Leu Asp Val Glu
Lys Ala Phe Asp Glu Ile Pro 1445 1450
1455 Gly Ser Lys Pro Gln Leu Ala Cys Phe Ser Ala Asp Cys
Pro Leu Leu 1460 1465 1470
His Lys Arg Gly Val Met Phe Thr Cys Asn Arg Thr Gln Thr Val Tyr
1475 1480 1485 Asn Leu Gln Gln
Val Val Lys Met Val Asn Asp Thr Ile Thr Arg Lys 1490
1495 1500 Thr Glu Asn Val Lys Lys Met Asn
Ser Leu Val Ala Gln Ser Pro Pro 1505 1510
1515 1520Asp Trp Glu His Phe Glu Asn Ile Leu Thr Cys Leu
Arg Gln Asn Asn 1525 1530
1535 Ala Ala Leu Gln Asp Gln Leu Asp Glu Leu Gln Glu Ala Phe Ala Gln
1540 1545 1550 Ala Arg
Glu Arg Ser Asp Phe Leu Ser Asp Trp Leu Lys Val Ser Ala 1555
1560 1565 Ile Ile Phe Ala Gly Ile
Ala Ser Leu Ser Ala Val Ile Lys Leu Ala 1570 1575
1580 Ser Lys Phe Lys Glu Ser Ile Trp Pro Thr
Pro Val Arg Val Glu Leu 1585 1590 1595
1600Ser Glu Gly Glu Gln Ala Ala Tyr Ala Gly Arg Ala Arg Ala
Gln Lys 1605 1610 1615
Gln Ala Leu Gln Val Leu Asp Ile Gln Gly Gly Gly Lys Val Leu Ala
1620 1625 1630 Gln Ala Gly Asn
Pro Val Met Asp Phe Glu Leu Phe Cys Ala Lys Asn 1635
1640 1645 Ile Val Ala Pro Ile Thr Phe Tyr
Tyr Pro Asp Lys Ala Glu Val Thr 1650 1655
1660 Gln Ser Cys Leu Leu Leu Arg Ala His Leu Phe Val
Val Asn Arg His 1665 1670 1675
1680Val Ala Glu Thr Asp Trp Thr Ala Phe Lys Leu Lys Asp Val Arg His
1685 1690 1695 Glu Arg His
Thr Val Ala Leu Arg Ser Val Asn Arg Ser Gly Ala Lys 1700
1705 1710 Thr Asp Leu Thr Phe Ile Lys
Val Thr Lys Gly Pro Leu Phe Lys Asp 1715 1720
1725 Asn Val Asn Lys Phe Cys Ser Asn Lys Asp Asp
Phe Pro Ala Arg Asn 1730 1735 1740
Asp Thr Val Thr Gly Ile Met Asn Thr Gly Leu Ala Phe Val Tyr
Ser 1745 1750 1755 1760Gly
Asn Phe Leu Ile Gly Asn Gln Pro Val Asn Thr Thr Thr Gly Ala
1765 1770 1775 Cys Phe Asn His Cys
Leu His Tyr Arg Ala Gln Thr Arg Arg Gly Trp 1780
1785 1790 Cys Gly Ser Ala Ile Ile Cys Asn Val
Asn Gly Lys Lys Ala Val Tyr 1795 1800
1805 Gly Met His Ser Ala Gly Gly Gly Gly Leu Ala Ala Ala
Thr Ile Ile 1810 1815 1820
Thr Lys Glu Leu Ile Glu Ala Ala Glu Lys Ser Met Leu Ala Leu Glu 1825
1830 1835 1840Pro Gln Gly Ala
Ile Val Asp Ile Ala Thr Gly Ser Val Val His Val 1845
1850 1855 Pro Arg Lys Thr Lys Leu Arg Arg
Thr Val Ala His Asp Val Phe Gln 1860 1865
1870 Pro Lys Phe Glu Pro Ala Val Leu Ser Arg Tyr Asp
Pro Arg Thr Asp 1875 1880 1885
Lys Asp Val Asp Val Val Ala Phe Ser Lys His Thr Thr Asn Met Glu
1890 1895 1900 Ser Leu Pro
Pro Ile Phe Asp Val Val Cys Gly Glu Tyr Ala Asn Arg 1905
1910 1915 1920Val Phe Thr Ile Leu Gly Lys
Glu Asn Gly Leu Leu Thr Val Glu Gln 1925
1930 1935 Ala Val Leu Gly Leu Pro Gly Met Asp Pro Met
Glu Lys Asp Thr Ser 1940 1945
1950 Pro Gly Leu Pro Tyr Thr Gln Gln Gly Leu Arg Arg Thr Asp Leu
Leu 1955 1960 1965 Asn
Phe Ile Thr Ala Lys Met Thr Pro Gln Leu Asp Tyr Ala His Ser 1970
1975 1980 Lys Leu Val Ile Gly
Val Tyr Asp Asp Val Val Tyr Gln Ser Phe Leu 1985 1990
1995 2000Lys Asp Glu Ile Arg Pro Ile Glu Lys
Ile His Glu Ala Lys Thr Arg 2005 2010
2015 Ile Val Asp Val Pro Pro Phe Ala His Cys Ile Trp Gly
Arg Gln Leu 2020 2025 2030
Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr Lys Pro Gly Leu Glu Leu
2035 2040 2045 Gly Ser Ala Ile
Gly Thr Asp Pro Asp Val Asp Trp Thr Arg Tyr Ala 2050
2055 2060 Val Glu Leu Ser Gly Phe Asn Tyr
Val Tyr Asp Val Asp Tyr Ser Asn 2065 2070
2075 2080Phe Asp Ala Ser His Ser Thr Ala Met Phe Glu Cys
Leu Ile Asn Asn 2085 2090
2095 Phe Phe Thr Glu Gln Asn Gly Phe Asp Arg Arg Ile Ala Glu Tyr Leu
2100 2105 2110 Arg Ser
Leu Ala Val Ser Arg His Ala Tyr Glu Asp Arg Arg Val Leu 2115
2120 2125 Ile Arg Gly Gly Leu Pro
Ser Gly Cys Ala Ala Thr Ser Met Leu Asn 2130 2135
2140 Thr Ile Met Asn Asn Val Ile Ile Arg Ala
Ala Leu Tyr Leu Thr Tyr 2145 2150 2155
2160Ser Asn Phe Asp Phe Asp Asp Ile Lys Val Leu Ser Tyr Gly
Asp Asp 2165 2170 2175
Leu Leu Ile Gly Thr Asn Tyr Gln Ile Asp Phe Asn Leu Val Lys Glu
2180 2185 2190 Arg Leu Ala Pro
Phe Gly Tyr Lys Ile Thr Pro Ala Asn Lys Thr Thr 2195
2200 2205 Thr Phe Pro Leu Thr Ser His Leu
Gln Asp Val Thr Phe Leu Lys Arg 2210 2215
2220 Arg Phe Val Arg Phe Asn Ser Tyr Leu Phe Arg Pro
Gln Met Asp Ala 2225 2230 2235
2240Val Asn Leu Lys Ala Met Val Ser Tyr Cys Lys Pro Gly Thr Leu Lys
2245 2250 2255 Glu Lys Leu
Met Ser Ile Ala Leu Leu Ala Val His Ser Gly Pro Asp 2260
2265 2270 Ile Tyr Asp Glu Ile Phe Leu
Pro Phe Arg Asn Val Gly Ile Val Val 2275 2280
2285 Pro Thr Tyr Ser Ser Met Leu Tyr Arg Trp Leu
Ser Leu Phe Arg 2290 2295 2300
332307PRTTheiler's murine encephalomyelitis virus 33Met Met Ala
Cys Ile His Gly Tyr Pro Ser Val Cys Pro Ile Cys Thr 1 5
10 15 Ala Ile Asp Lys Ser Ser Asp Gly
Met Tyr Leu Leu Leu Ala Asp Asn 20 25
30 Glu Trp Phe Pro Ala Asp Leu Leu Thr Met Asp Leu Asp
Asp Asp Val 35 40 45
Phe Trp Pro Asn Asp Glu Ser Asp Val Ser Glu Thr Met Asp Trp Thr 50
55 60 Asp Leu Pro Phe Ile
Leu Asp Thr Ile Met Glu Pro Gln Gly Asn Ser 65 70
75 80Thr Ser Ser Asp Lys Ser Asn Ser Gln Ser
Ser Gly Asn Glu Gly Val 85 90
95 Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp
Leu 100 105 110 Ser
Ala Asn Gly Gly Asn Ala Gly Gly Ala Pro Lys Thr Glu Gly Gln 115
120 125 Leu Gly Asn Ile Leu Gly
Asn Ala Ala Asn Ala Phe Ser Thr Met Ala 130 135
140 Pro Leu Leu Leu Asp Gln Asn Thr Glu Glu Met
Glu Asn Leu Ser Asp 145 150 155
160Arg Val Asp Ser Asp Lys Ala Gly Asn Ser Ala Val Asn Thr Gln Ser
165 170 175 Ser Val
Gly Arg Leu Cys Gly Tyr Gly Met His His Lys Gly Lys His 180
185 190 Pro Ala Ser Cys Ala Asp Thr
Ala Thr Asp Lys Val Leu Ser Ala Glu 195 200
205 Arg Tyr Tyr Thr Ile Asp Leu Ala Thr Trp Thr Thr
Thr Leu Gly Thr 210 215 220
Phe Ser His Ile Arg Ile Pro Leu Pro His Val Leu Ala Gly Glu Asp
225 230 235 240Gly Gly
Val Phe Gly Ser Thr Leu Arg Arg His Tyr Leu Cys Lys Cys
245 250 255 Gly Trp Arg Ile Gln Val
Gln Cys Asn Ala Ser Gln Phe His Ala Gly 260
265 270 Ser Leu Leu Val Phe Met Ala Pro Glu Phe
Tyr Thr Gly His Thr Pro 275 280
285 Val Thr Gly Thr Thr Glu Pro Ala Thr Pro Phe Thr Met Asp
Ser Ser 290 295 300
Trp Gln Thr Pro Gln Gln Asn Pro Val Gly Phe Arg Tyr Asp Gly Arg 305
310 315 320Thr Gly Tyr Phe Ala
Leu Asn His Gln Asn Tyr Trp Gln Trp Met Val 325
330 335 Tyr Pro His Gln Ile Leu Asn Leu Arg Thr
Asn Thr Ser Val Asp Leu 340 345
350 Glu Val Pro Phe Thr Asn Ile Ala Pro Thr Ser Ser Trp Thr Gln
His 355 360 365 Ala
Asn Trp Thr Leu Val Val Ala Val Leu Thr Pro Leu Gln Tyr Ala 370
375 380 Ala Gly Ser Ala Thr Asp
Val Gln Ile Thr Ala Ser Ile Gln Pro Val 385 390
395 400Lys Pro Val Phe Asn Gly Leu Arg His Glu Ala
Val Val Pro Gln Ser 405 410
415 Pro Ile Pro Val Thr Val Arg Glu His Gln Gly Thr Phe Tyr Ser Thr
420 425 430 Asn Pro
Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ala Thr Pro 435
440 445 Ser Asp Tyr Met Cys Gly Glu
Phe Ser Asp Leu Val Glu Leu Cys Lys 450 455
460 Leu Pro Thr Phe Leu Gly Asn Pro Ala Asn Thr Ser
Pro Ala Gly Gly 465 470 475
480Arg Tyr Pro Tyr Phe Ser Ala Thr Asn Ser Val Pro Ala Thr Ala Leu
485 490 495 Ala Ser Tyr
Gln Val Ala Leu Ser Cys Ser Cys Met Ser Asn Ser Met 500
505 510 Leu Ala Ala Val Ala Arg Asn Phe
Asn Gln Tyr Arg Gly Ser Leu Asn 515 520
525 Phe Leu Phe Val Phe Thr Gly Thr Ala Met Thr Lys Gly
Lys Phe Leu 530 535 540
Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Glu Gln 545
550 555 560Ala Met Gln Ala
Thr Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser 565
570 575 Tyr Asn Phe Thr Val Pro Phe Ile Ser
Pro Thr His Tyr Arg Gln Thr 580 585
590 Ser Tyr Thr Ser Thr Ser Ile Thr Ser Val Asp Gly Trp Leu
Thr Val 595 600 605
Trp Gln Leu Thr Pro Leu Thr Tyr Pro Ala Asn Thr Pro Pro Asn Ala 610
615 620 Asp Ile Leu Thr Leu
Val Ser Ala Gly Asp Asp Phe Thr Leu Arg Met 625 630
635 640Pro Ile Ser Pro Thr Lys Trp Ile Pro Gln
Gly Val Asp Asn Ala Glu 645 650
655 Lys Gly Lys Val Ser Asn Asp Asp Ala Thr Val Asp Phe Val Ala
Glu 660 665 670 Pro
Val Lys Phe Pro Asp Asn Gln Thr Lys Val Ser Phe Phe Tyr Asp 675
680 685 Arg Ser Val Pro Leu Gly
Leu Leu Arg Pro Ala Gln Gly Met Glu Gln 690 695
700 Asp Phe Ala Tyr Ala Ala Asn Asp Ser Arg Ala
Asn Ser Ile Leu Leu 705 710 715
720Thr Pro Leu Pro Ser Tyr Ala Pro Asp Ser Thr Thr Gly Pro Thr Glu
725 730 735 Thr Gln
Ala Pro Ile Gln Trp Arg Trp Leu Arg Gly Thr Ser Asp Gly 740
745 750 Ser Thr Thr Phe Pro Leu Met
Thr Lys Gln Asp Tyr Ala Phe Leu Leu 755 760
765 Phe Ser Pro Phe Thr Tyr Tyr Lys Ala Asp Leu Glu
Val Thr Leu Ser 770 775 780
Ala Ile Ser Asn Ser Asn Asn Val Thr Val Val Arg Trp Ala Pro Thr
785 790 795 800Gly Ala
Pro Ala Asp Ile Ser Arg Gln Leu Ser Gly Tyr Thr Pro Ser
805 810 815 Ile Gly Asp Thr Arg Asp
Pro His Leu Trp Phe Val Gly Ala Gly Asn 820
825 830 Ser Gln Thr Ser Phe Val Val Pro Tyr Asn
Ser Pro Leu Ser Val Leu 835 840
845 Pro Ala Ala Trp Phe Asn Gly Trp Ser Asp Phe Gly Asn Thr
Lys Asp 850 855 860
Phe Gly Val Ala Pro Asn Ala Asp Phe Gly Arg Leu Trp Ile Gln Gly 865
870 875 880Asn Thr Ser Val Ala
Val Arg Val Arg Tyr Lys Lys Met Lys Val Phe 885
890 895 Cys Pro Arg Pro Thr Leu Phe Leu Pro Trp
Pro Ser Thr Thr Thr Thr 900 905
910 Arg Ile His Ala Asp Asn Pro Val Ser Val Met Glu Leu Gln Asn
Pro 915 920 925 Phe
Ser Phe Tyr Arg Val Asp Leu Phe Ile Thr Phe Thr Asp Glu Leu 930
935 940 Ile Thr Phe Asp Tyr Lys
Val His Gly Arg Pro Val Leu Gln Tyr Gln 945 950
955 960Val Pro Gly Leu Gly Leu Thr Cys Ala Gly Arg
Met Leu Val Cys Met 965 970
975 Gly Gln Met Pro Asn His Ala Pro Phe Ser Thr Val Arg His Leu Tyr
980 985 990 His Val
Val Phe Thr Gly Ser Arg Asn Ser Phe Gly Val Val Ile Tyr 995
1000 1005 Tyr Lys Arg His Arg Pro
Trp Lys Lys Pro Leu His Glu Glu Leu His 1010 1015
1020 Asp Tyr Gly Phe Glu Cys Phe Ser Asp Phe
Phe Lys His Val Arg Glu 1025 1030 1035
1040Tyr His Ala Ala Tyr Tyr Lys Gln Arg Leu Met His Asp Val
Glu Thr 1045 1050 1055
Asn Pro Gly Pro Pro Val Gln Ser Val Phe Arg Pro Gln Gly Gly Val
1060 1065 1070 Leu Thr Lys Ser
Gln Ala Pro Met Ser Gly Ile Gln Asn Leu Phe Leu 1075
1080 1085 Arg Ala Leu Gly Ile Asp Ala Asp
His Gly Glu Phe Thr Arg Ala Val 1090 1095
1100 Thr Met Ile Thr Asp Leu Cys Asn Thr Trp Glu Lys
Ala Lys Asn Thr 1105 1110 1115
1120Leu Val Ser Pro Glu Phe Trp Thr Val Leu Ile Met Lys Thr Val Lys
1125 1130 1135 Phe Ile Ala
Ala Ser Val Leu Tyr Leu His Asn Pro Asp Leu Thr Ala 1140
1145 1150 Thr Ile Cys Leu Ser Leu Met
Thr Gly Val Asp Val Leu Thr Asn Glu 1155 1160
1165 Ser Ile Phe Asn Trp Leu Ser Asn Lys Leu Ser
Lys Leu Phe His Thr 1170 1175 1180
Pro Pro Pro Pro Thr Ser Pro Leu Leu Gln Ala Gln Ser Pro Leu
Arg 1185 1190 1195 1200Glu
Ala Asn Asp Gly Phe Asn Leu Ala Lys Asn Ile Glu Trp Ala Ile
1205 1210 1215 Lys Thr Val Gln Lys
Ile Val Asp Trp Leu Met Ser Trp Phe Lys Gln 1220
1225 1230 Glu Glu Ala His Pro Gln Ala Lys Leu
Asp Lys Met Leu Ala Asp Phe 1235 1240
1245 Pro Glu His Cys Ala Ser Ile Leu Ala Met Arg Asn Gly
Arg Lys Ala 1250 1255 1260
Tyr Thr Asp Cys Ala Gly Ala Phe Lys Tyr Phe Glu Asp Leu Tyr Asn 1265
1270 1275 1280Leu Ala Val Gln
Cys Lys Arg Ile Pro Leu Ala Thr Leu Cys Glu Lys 1285
1290 1295 Phe Lys Asn Lys His Asp His Ala
Val Ala Arg Pro Glu Pro Val Val 1300 1305
1310 Val Val Leu Arg Gly Asn Ala Gly Gln Gly Lys Ser
Val Thr Ser Gln 1315 1320 1325
Ile Ile Ala Gln Ala Val Ser Lys Leu Ala Phe Gly Arg Gln Ser Val
1330 1335 1340 Tyr Ser Ile
Pro Pro Asp Ser Asp Tyr Leu Asp Gly Tyr Glu Asn Gln 1345
1350 1355 1360Phe Ser Val Ile Met Asp Asp
Leu Gly Gln Asn Pro Asp Gly Glu Asp 1365
1370 1375 Phe Lys Val Phe Cys Gln Met Val Ser Ser Thr
Asn Phe Leu Pro Asn 1380 1385
1390 Met Ala His Leu Glu Lys Lys Gly Thr Pro Phe Thr Ser Asn Phe
Ile 1395 1400 1405 Val
Ala Thr Thr Asn Leu Pro Lys Phe Arg Pro Val Thr Val Ala His 1410
1415 1420 Tyr Pro Ala Val Asp
Arg Arg Ile Thr Phe Asp Leu Thr Val Glu Ala 1425 1430
1435 1440Gly Pro Ala Cys Lys Thr Pro Thr Gly
Met Leu Asp Val Glu Lys Ala 1445 1450
1455 Phe Gln Glu Ile Pro Gly Glu Pro Gln Leu Asp Cys Phe
Ser Ser Asp 1460 1465 1470
Cys Ala Leu Leu His Lys Arg Gly Val Gln Phe Ile Cys Asn Arg Thr
1475 1480 1485 Lys Lys Ile Tyr
Asn Leu Gln Gln Ile Val Lys Met Val Lys Asp Thr 1490
1495 1500 Ile Asp Asn Lys Val Ala Asn Leu
Lys Lys Met Asn Thr Leu Val Ala 1505 1510
1515 1520Gln Ser Pro Asn Asn Gly Asn Asp Met Glu His Ile
Ile Thr Cys Leu 1525 1530
1535 Arg Gln Asn Asn Ala Ala Leu Gln Asp Gln Ile Asp Glu Leu Gln Glu
1540 1545 1550 Ala Phe
Ala Gln Ala Gln Glu Arg Gln Asn Phe Leu Ser Asp Trp Met 1555
1560 1565 Lys Val Ser Ala Ile Ile
Phe Ala Gly Ile Ala Ser Leu Ser Ala Val 1570 1575
1580 Cys Lys Leu Val Gly Arg Leu Lys Asn Leu
Ile Trp Pro Ser Pro Val 1585 1590 1595
1600His Val Glu Leu Ser Glu Gly Glu Gln Ala Ala Tyr Ala Gly
Ala Lys 1605 1610 1615
Arg Gly Ala Lys Gln Ala Leu Gln Val Leu Asp Leu Gln Gly Gly Gly
1620 1625 1630 Arg Ile Ile Ala
Gln Ala Gly Asn Pro Val Met Asp Tyr Glu Val Cys 1635
1640 1645 Val Ala Lys Asn Met Val Ala Pro
Ile Thr Phe Tyr Tyr Ala Asp Lys 1650 1655
1660 Ala Gln Val Thr Gln Ser Cys Leu Leu Val Lys Gly
Arg Leu Phe Val 1665 1670 1675
1680Val Asn Arg His Val Ala Glu Thr Asp Trp Val Ser Phe Glu Leu Arg
1685 1690 1695 Asp Val Arg
His Glu Arg Asp Thr Val Thr Met Arg Ser Val Asn Arg 1700
1705 1710 Ser Gly Met Glu Val Asp Leu
Thr Phe Ile Lys Val Thr Lys Gly Pro 1715 1720
1725 Leu Phe Lys Asp Asn Thr Lys Lys Phe Cys Ser
Asn Lys Asp Asp Phe 1730 1735 1740
Pro Gln Lys Asn Glu Thr Val Thr Gly Ile Met Asn Thr Gly Leu
Pro 1745 1750 1755 1760Phe
Val Phe Asn Gly Lys Phe Ile Ile Gly Asn His Pro Val Asn Thr
1765 1770 1775 Thr Thr Gly Ala Thr
Phe Asn His Cys Leu His Tyr Arg Ala Asn Thr 1780
1785 1790 Arg Arg Gly Trp Cys Gly Ser Ala Val
Ile Cys Gln Val Asn Gly Lys 1795 1800
1805 Lys Ala Val Tyr Gly Met His Ser Ala Gly Gly Gly Gly
Leu Ala Ala 1810 1815 1820
Ala Thr Ile Ile Thr Gln Glu Leu Val Glu Ala Ala Glu Gln Asn Met 1825
1830 1835 1840Asp Arg Leu Val
Pro Gln Gly Ala Ile Met Glu Ile Gly Thr Gly Ser 1845
1850 1855 Val Val His Val Pro Arg Lys Thr
Lys Leu Arg Arg Thr Val Ala His 1860 1865
1870 Glu Ile Phe Leu Pro Lys Phe Glu Pro Ala Val Leu
Ser Arg Tyr Asp 1875 1880 1885
Pro Arg Thr Glu Lys Asp Val Asp Gln Val Ala Phe Ser Lys His Thr
1890 1895 1900 Thr Asn Met
Glu Glu Leu Pro Ala Val Phe Ser Met Val Ala Lys Glu 1905
1910 1915 1920Tyr Ala Asn Arg Val Phe Thr
Lys Leu Gly Lys Glu Asn Gln Leu Leu 1925
1930 1935 Thr Thr Gln Gln Ala Ile Leu Gly Leu Pro Gly
Met Asp Pro Met Glu 1940 1945
1950 Lys Asp Thr Ser Pro Gly Leu Pro Tyr Thr Gln Gln Gly Leu Arg
Arg 1955 1960 1965 Thr
Asp Leu Val Asn Phe Glu Thr Gly Lys Met Asp His Asn Leu Asp 1970
1975 1980 Tyr Ala His Ser Lys
Leu Met Leu Gly His Tyr Glu Asp Val Val Tyr 1985 1990
1995 2000Gln Ser Phe Leu Lys Asp Glu Ile Arg
Pro Ile Glu Lys Ile His Glu 2005 2010
2015 Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe His His
Cys Ile Trp 2020 2025 2030
Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Arg Phe Gln Thr Asn Pro
2035 2040 2045 Gly Leu Asp Leu
Gly Ser Ala Ile Gly Thr Asp Pro Asp Val Asp Trp 2050
2055 2060 Thr Val Phe Ala His Gln Leu Ala
Glu Phe Lys Tyr Ile Tyr Asp Val 2065 2070
2075 2080Asp Tyr Ser Asn Phe Asp Ala Ser His Ser Thr Ala
Ile Phe Glu Ile 2085 2090
2095 Leu Ile Gln Glu Phe Phe Thr Pro Gln Asn Gly Phe Asp Pro Arg Ile
2100 2105 2110 Gly Glu
Tyr Leu Arg Ser Leu Ala Val Ser Arg His Ala Tyr Glu Asp 2115
2120 2125 Arg Arg Val Leu Ile Arg
Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr 2130 2135
2140 Ser Met Ile Asn Thr Ile Ile Asn Asn Ile
Val Ile Arg Ala Ala Leu 2145 2150 2155
2160Tyr Met Thr Tyr Ala Asn Phe Glu Phe Asp Asp Ile Lys Val
Leu Ser 2165 2170 2175
Tyr Gly Asp Asp Leu Leu Ile Ala Thr Asn Tyr Glu Ile Asn Phe Asn
2180 2185 2190 Leu Val Lys Glu
Arg Leu Ala Pro Phe Asn Tyr Lys Ile Thr Pro Ala 2195
2200 2205 Asn Lys Thr Ser Thr Phe Pro Gln
Thr Ser His Leu Gln Asp Val Val 2210 2215
2220 Phe Leu Lys Arg Arg Phe Val Gln Phe Asn Ser Phe
Leu Phe Arg Pro 2225 2230 2235
2240Gln Met Glu Thr Glu Asn Leu Lys Ala Met Val Ser Tyr Cys Arg Pro
2245 2250 2255 Gly Val Leu
Lys Glu Lys Leu Met Ser Ile Ala Leu Leu Ala Val His 2260
2265 2270 Ser Gly Pro Asp Val Tyr Asp
Glu Ile Phe Met Pro Phe Arg Arg Ile 2275 2280
2285 Gly Val Val Val Pro Glu Tyr Ser Thr Met Leu
Tyr Arg Trp Leu Asn 2290 2295 2300
Leu Phe Arg
2305 34930PRTTheiler's murine encephalomyelitis
virusMOD_RES(557)variable amino acid 34Met Ala Cys Lys His Gly Tyr Pro
Asp Val Cys Pro Ile Cys Thr Ala 1 5 10
15 Ile Asp Val Thr Pro Gly Phe Glu Tyr Leu Leu Leu Ala
Asp Gly Glu 20 25 30
Trp Phe Pro Thr Asp Leu Leu Cys Val Asp Leu Asp Asp Asp Val Phe
35 40 45 Trp Pro Ser Asp Ser
Ser Asn Gln Ser Gln Thr Met Glu Trp Thr Asp 50 55
60 Ile Pro Leu Ile Cys Asp Thr Val Met Glu
Pro Gln Gly Asn Ser Thr 65 70 75
80Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser Gly Asn Glu Gly Val
Ile 85 90 95 Ile
Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu Ser
100 105 110 Ala Asn Gly Gly Asn
Ala Gly Asp Gly Pro Lys Thr Glu Gly Gln Leu 115
120 125 Ser Asn Ile Leu Gly Gly Ala Ala Asn
Ala Phe Ala Thr Met Ala Pro 130 135
140 Leu Leu Leu Asp Glu Asn Thr Glu Glu Met Glu Asn Leu
Ser Asp Arg 145 150 155
160Val Asp Ser Asp Lys Ala Gly Asn Ser Ala Thr Asn Thr Gln Ser Ser
165 170 175 Val Gly Arg Leu
His Gly Tyr Gly Ala Thr His Arg Gly Asp His Pro 180
185 190 Ala Ser Cys Ala Asp Thr Ala Thr Asp
Lys Val Leu Ala Ala Glu Arg 195 200
205 Tyr Tyr Thr Ile Asp Leu Ala Thr Trp Thr Thr Ala Gln Thr
Thr Phe 210 215 220
Ser His Ile Arg Val Pro Leu Pro His Ala Leu Ala Gly Glu His Gly 225
230 235 240Gly Val Phe Gly Ala
Thr Leu Arg Arg His Tyr Leu Ala Lys Cys Gly 245
250 255 Trp Arg Val Gln Val Gln Cys Asn Ala Ser
Gln Phe His Ala Gly Ser 260 265
270 Leu Leu Val Phe Leu Ala Pro Glu Phe Tyr Thr Gly Thr Gly Val
Ala 275 280 285 Thr
Ser Gly Gln Glu Pro Asn Lys Val Phe Leu Met Asp Thr Thr Trp 290
295 300 Gln Glu Pro Gln Ala Ala
Pro Thr Gly Phe Arg Tyr Asp Gly Lys Asn 305 310
315 320Gly Phe Phe Thr Leu Asn His Gln Asn Tyr Trp
Gln Trp Thr Val Tyr 325 330
335 Pro His Gln Ile Leu Asn Leu Arg Thr Asn Thr Ser Val Asp Leu Glu
340 345 350 Val Pro
Tyr Val Asn Val Ala Pro Thr Ser Ser Trp Thr Gln His Ala 355
360 365 Asn Trp Ala Leu Val Val Ala
Val Leu Thr Pro Leu Gln Tyr Ser Thr 370 375
380 Gly Ala Ala Thr Asp Val Ala Ile Thr Val Ser Leu
Gln Pro Val Asn 385 390 395
400Pro Val Phe Asn Gly Leu Arg His Glu Ala Gln Val Pro Gln Ser Pro
405 410 415 Val Ala Val
Thr Val Arg Glu His Gln Gly Ser Phe Tyr Ser Thr Asn 420
425 430 Pro Asp Thr Thr Val Pro Ile Tyr
Gly Lys Thr Ile Val Thr Pro Ser 435 440
445 Asp Tyr Met Cys Gly Glu Phe Thr Asp Leu Leu Glu Leu
Cys Lys Leu 450 455 460
Pro Thr Phe Leu Gly Asn Leu Ser Asn Asp Thr Arg Val Pro Phe Phe 465
470 475 480Thr Ala Thr Asn
Ser Val Pro Thr Glu Ser Leu Val Glu Tyr Gln Val 485
490 495 Thr Leu Ser Cys Ser Cys Met Ser Asn
Ser Met Leu Ala Ser Val Ala 500 505
510 Arg Asn Phe Asn Gln Tyr Arg Gly Ser Leu Asn Phe Leu Phe
Val Phe 515 520 525
Thr Gly Ser Ala Met Thr Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro 530
535 540 Pro Gly Ala Gly Lys
Pro Thr Thr Arg Asp Gln Ala Xaa Gln Ser Thr 545 550
555 560Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser
Ser Tyr Asn Phe Thr Val 565 570
575 Pro Phe Ile Ser Pro Ser His Tyr Arg Gln Thr Ser Tyr Thr Ser
Pro 580 585 590 Ser
Ile Ala Ala Val Asp Gly Trp Leu Thr Val Trp Gln Leu Thr Pro 595
600 605 Leu Thr Phe Pro Ala Asn
Val Pro Pro Ser Ser Asp Ile Leu Thr Leu 610 615
620 Val Ser Ala Gly Asn Asp Phe Thr Leu Arg Met
Pro Ile Ser Pro Thr 625 630 635
640Lys Trp Ile Pro Gln Gly Val Asp Asn Ala Glu Lys Gly Lys Val Ser
645 650 655 Asp Asp
Asn Ala Ser Val Asp Phe Val Ala Glu Pro Ile Lys Leu Pro 660
665 670 Glu Asn Gln Thr Arg Val Asn
Phe Phe Tyr Asp Arg Ser Ser Pro Ile 675 680
685 Gly Leu Leu Arg Pro Asn Gln Ala Ile Glu Ser Asn
Phe Ser Tyr Ser 690 695 700
Ala Asp Ser Asn Gly Ala Thr Asn Cys Ala Leu Leu Thr Pro Leu Pro
705 710 715 720Ser Tyr
Ser Pro Asp Arg Pro Gly Gln Ser Pro Asp Thr Ser Lys Ala
725 730 735 Pro Ile Gln Trp Arg Trp
Ile Ser Ala Val Thr Glu Ser Gly Thr Val 740
745 750 Ser Asn Thr Phe Pro Thr Arg Thr Arg Gln
Asp Tyr Ala Phe Leu Leu 755 760
765 Phe Ser Pro Phe Thr Tyr Tyr Lys Cys Asp Leu Glu Val Thr
Leu Ser 770 775 780
Ser Val Gly Asn Gly Val Val Ala Ser Leu Val Arg Trp Ala Pro Thr 785
790 795 800Gly Ala Pro Ala Asp
Ile Thr Thr Gln Leu Thr Thr Ser Thr Pro Ser 805
810 815 Ile Gly Asp Thr Arg Asp Pro His Met Trp
Leu Val Gly Ala Gly Asn 820 825
830 Ser Gln Thr Ser Phe Val Ile Pro Tyr Asn Ser Pro Leu Ser Val
Leu 835 840 845 Pro
Ala Ala Trp Phe Asn Gly Trp Ser Asn Phe Ser Asn Thr Tyr Asp 850
855 860 Phe Gly Ile Ala Pro Cys
Ser Asp Phe Gly Arg Leu Trp Ile Gln Gly 865 870
875 880Asn Ala Pro Leu Ala Ile Arg Val Arg Tyr Lys
Lys Met Arg Val Phe 885 890
895 Cys Pro Arg Pro Thr Leu Phe Phe Pro Trp Pro Thr Pro Thr Thr Thr
900 905 910 Lys Val
Asn Ala Asp Asn Pro Val Pro Ile Leu Asp Leu Glu Asn Pro 915
920 925 Ala Ala
93035142PRTSeneca Valley Virus
35Leu Ala His His Gly Asn Lys Lys Ser Leu Gln Glu Leu Asn Glu Glu 1
5 10 15 Gln Trp Val Glu
Met Ser Asp Asp Tyr Arg Thr Gly Lys Asn Met Pro 20
25 30 Phe Gln Ser Leu Gly Thr Tyr Tyr Arg
Pro Pro Asn Trp Thr Trp Gly 35 40
45 Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe Pro His
Gln Ile 50 55 60
Leu Asn Ala Arg Thr Ser Thr Ser Val Asp Ile Asn Val Pro Tyr Ile 65
70 75 80Gly Glu Thr Pro Thr
Gln Ser Ser Glu Thr Gln Asn Ser Trp Thr Leu 85
90 95 Leu Val Met Val Leu Val Pro Leu Asp Tyr
Lys Glu Gly Ala Thr Thr 100 105
110 Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser Pro Tyr Phe
Asn 115 120 125 Gly
Leu Arg Asn Arg Tyr Thr Ala Gly Thr Asp Glu Glu Gln 130
135 140 36239PRTSeneca Valley Virus
36Gly Pro Ile Pro Thr Ala Pro Arg Glu Asn Ser Leu Met Phe Leu Ser 1
5 10 15 Thr Leu Pro Asp
Asp Thr Val Pro Ala Tyr Gly Asn Val Arg Thr Pro 20
25 30 Pro Val Asn Tyr Leu Pro Gly Glu Ile
Thr Asp Leu Leu Gln Leu Ala 35 40
45 Arg Ile Pro Thr Leu Met Ala Phe Glu Arg Val Pro Glu Pro
Val Pro 50 55 60
Ala Ser Asp Thr Tyr Val Pro Tyr Val Ala Val Pro Thr Gln Phe Asp 65
70 75 80Asp Arg Pro Leu Ile
Ser Phe Pro Ile Thr Leu Ser Asp Pro Val Tyr 85
90 95 Gln Asn Thr Leu Val Gly Ala Ile Ser Ser
Asn Phe Ala Asn Tyr Arg 100 105
110 Gly Cys Ile Gln Ile Thr Leu Thr Phe Cys Gly Pro Met Met Ala
Arg 115 120 125 Gly
Lys Phe Leu Leu Ser Tyr Ser Pro Pro Asn Gly Thr Gln Pro Gln 130
135 140 Thr Leu Ser Glu Ala Met
Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly 145 150
155 160Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr
Ile Ser Pro Ser Asp 165 170
175 Tyr Arg Glu Thr Arg Ala Ile Thr Asn Ser Val Tyr Ser Ala Asp Gly
180 185 190 Trp Phe
Ser Leu His Lys Leu Thr Lys Ile Thr Leu Pro Pro Asp Cys 195
200 205 Pro Gln Ser Pro Cys Ile Leu
Phe Phe Ala Ser Ala Gly Glu Asp Tyr 210 215
220 Thr Leu Arg Leu Pro Val Asp Cys Asn Pro Ser Tyr
Val Phe His 225 230 235
37259PRTSeneca Valley Virus 37Ser Thr Asp Asn Ala Glu Thr Gly Val Ile
Glu Ala Gly Asn Thr Asp 1 5 10
15 Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Pro Asn His Thr
Asn 20 25 30 Val
Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val 35
40 45 Leu Glu Lys Asp Ala Val
Phe Pro Arg Pro Phe Pro Thr Gln Glu Gly 50 55
60 Ala Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu
Thr Pro Arg Pro Thr 65 70 75
80Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Ala Asn Pro Ser
85 90 95 Gly Ser Gly
Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser 100
105 110 Leu Ala Cys Phe Thr Tyr Phe Arg
Ser Asp Leu Glu Val Thr Val Val 115 120
125 Ser Leu Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe
Pro Ser Gly 130 135 140
Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val Pro 145
150 155 160Phe His Phe Thr
Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly Gly 165
170 175 Lys Val Ser Phe Val Leu Pro Trp Asn
Ser Val Ser Ser Val Leu Pro 180 185
190 Val Arg Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg
Gly Leu 195 200 205
Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Val Pro Arg Pro 210
215 220 Asn Glu Lys Lys Ser
Thr Ala Val Lys His Val Ala Val Tyr Ile Arg 225 230
235 240Tyr Lys Asn Ala Arg Ala Trp Cys Pro Ser
Met Leu Pro Phe Arg Ser 245 250
255 Tyr Lys Gln
3814PRTSeneca Valley Virus 38Lys Met Leu Met Gln Ser Gly Asp Ile Glu
Thr Asn Pro Gly 1 5 10
39128PRTSeneca Valley Virus 39Pro Ala Ser Asp Asn Pro Ile Leu Glu Phe
Leu Glu Ala Glu Asn Asp 1 5 10
15 Leu Val Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val Gln
Gln 20 25 30 Thr
Trp Arg Lys Tyr Val Lys Asn Asp Asp Phe Trp Pro Asn Leu Leu 35
40 45 Ser Glu Leu Val Gly Glu
Gly Ser Val Ala Leu Ala Ala Thr Leu Ser 50 55
60 Asn Gln Ala Ser Val Lys Ala Leu Leu Gly Leu
His Phe Leu Ser Arg 65 70 75
80Gly Leu Asn Tyr Thr Asp Phe Tyr Ser Leu Leu Ile Glu Lys Cys Ser
85 90 95 Ser Phe Phe
Thr Val Glu Pro Pro Pro Pro Pro Ala Glu Asn Leu Met 100
105 110 Thr Lys Pro Ser Val Lys Ser Lys
Phe Arg Lys Leu Phe Lys Met Gln 115 120
125 40322PRTSeneca Valley Virus 40Gly Pro Met Asp Lys
Val Lys Asp Trp Asn Gln Ile Ala Ala Gly Leu 1 5
10 15 Lys Asn Phe Gln Phe Val Arg Asp Leu Val
Lys Glu Val Val Asp Trp 20 25
30 Leu Gln Ala Trp Ile Asn Lys Glu Lys Ala Ser Pro Val Leu Gln
Tyr 35 40 45 Gln
Leu Glu Met Lys Lys Leu Gly Pro Val Ala Leu Ala His Asp Ala 50
55 60 Phe Met Ala Gly Ser Gly
Pro Pro Leu Ser Asp Asp Gln Ile Glu Tyr 65 70
75 80Leu Gln Asn Leu Lys Ser Leu Ala Leu Thr Leu
Gly Lys Thr Asn Leu 85 90
95 Ala Gln Ser Leu Thr Thr Met Ile Asn Ala Lys Gln Ser Ser Ala Gln
100 105 110 Arg Val
Glu Pro Val Val Val Val Leu Arg Gly Lys Pro Gly Cys Gly 115
120 125 Lys Gly Leu Ala Ser Thr Leu
Ile Ala Gln Ala Val Ser Lys Arg Leu 130 135
140 Tyr Gly Ser Gln Ser Val Tyr Ser Leu Pro Pro Asp
Pro Asp Phe Phe 145 150 155
160Asp Gly Tyr Lys Gly Gln Phe Val Thr Leu Met Asp Asp Leu Gly Gln
165 170 175 Asn Pro Asp
Gly Gln Asp Phe Ser Thr Phe Cys Gln Met Val Ser Thr 180
185 190 Ala Gln Phe Leu Pro Asn Met Ala
Asp Leu Ala Glu Lys Gly Arg Pro 195 200
205 Phe Thr Ser Asn Leu Ile Ile Ala Thr Thr Asn Leu Pro
His Phe Ser 210 215 220
Pro Val Thr Ile Ala Asp Pro Ser Ala Val Ser Arg Arg Ile Asn Tyr 225
230 235 240Asp Leu Thr Leu
Glu Val Ser Glu Ala Tyr Lys Lys His Thr Arg Leu 245
250 255 Asn Phe Asp Leu Ala Phe Arg Arg Thr
Asp Ala Pro Pro Ile Tyr Pro 260 265
270 Phe Ala Ala His Val Pro Phe Val Asp Val Ala Val Arg Phe
Lys Asn 275 280 285
Gly His Gln Asn Phe Asn Leu Leu Glu Leu Val Asp Ser Ile Cys Thr 290
295 300 Asp Ile Arg Ala Lys
Gln Gln Gly Ala Arg Asn Met Gln Thr Leu Val 305 310
315 320Leu Gln
4190PRTSeneca Valley Virus 41Ser Pro Asn Glu Asn
Asp Asp Thr Pro Val Asp Glu Ala Leu Gly Arg 1 5
10 15 Val Leu Ser Pro Ala Ala Val Asp Glu Ala
Leu Val Asp Leu Thr Pro 20 25
30 Glu Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly
Leu 35 40 45 Ala
Leu Ala Ala Val Thr Pro Gly Leu Ile Ile Leu Ala Val Gly Leu 50
55 60 Tyr Arg Tyr Phe Ser Gly
Ser Asp Ala Asp Gln Glu Glu Thr Glu Ser 65 70
75 80Glu Gly Ser Val Lys Ala Pro Arg Ser Glu
85 904222PRTSeneca
Valley Virus 42Asn Ala Tyr Asp Gly Pro Lys Lys Asn Ser Lys Pro Pro Gly
Ala Leu 1 5 10 15
Ser Leu Met Glu Met Gln
20 43211PRTSeneca Valley Virus 43Gln Pro Asn Val Asp Met Gly
Phe Glu Ala Ala Val Ala Lys Lys Val 1 5
10 15 Val Val Pro Ile Thr Phe Met Val Pro Asn Arg Pro
Ser Gly Leu Thr 20 25 30
Gln Ser Ala Leu Leu Val Thr Gly Arg Thr Phe Leu Ile Asn Glu His
35 40 45 Thr Trp Ser Asn
Pro Ser Trp Thr Ser Phe Thr Ile Arg Gly Glu Val 50
55 60 His Thr Arg Asp Glu Pro Phe Gln Thr
Val His Phe Thr His His Gly 65 70 75
80Ile Pro Thr Asp Leu Met Met Val Arg Leu Gly Pro Gly Asn
Ser Phe 85 90 95
Pro Asn Asn Leu Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg Asn
100 105 110 Ser Arg Val Val Gly
Val Ser Ser Ser Tyr Gly Asn Phe Phe Phe Ser 115
120 125 Gly Asn Phe Leu Gly Phe Val Asp Ser
Val Thr Ser Glu Gln Gly Thr 130 135
140 Tyr Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys
Gly Trp Cys 145 150 155
160Gly Ser Ala Leu Val Cys Glu Ala Gly Gly Val Arg Arg Ile Ile Gly
165 170 175 Leu His Ser Ala
Gly Ala Ala Gly Ile Gly Ala Gly Thr Tyr Ile Ser 180
185 190 Lys Leu Gly Leu Ile Lys Ala Leu Lys
His Leu Gly Glu Pro Leu Ala 195 200
205 Thr Met Gln
210 44462PRTSeneca Valley Virus 44Gly Leu Met Thr Glu Leu
Glu Pro Gly Ile Thr Val His Val Pro Arg 1 5
10 15 Lys Ser Lys Leu Arg Lys Thr Thr Ala His Ala
Val Tyr Lys Pro Glu 20 25
30 Phe Glu Pro Ala Val Leu Ser Lys Phe Asp Pro Arg Leu Asn Lys Asp
35 40 45 Val Asp
Leu Asp Glu Val Ile Trp Ser Lys His Thr Ala Asn Val Pro 50
55 60 Tyr Gln Pro Pro Leu Phe Tyr
Thr Tyr Met Ser Glu Tyr Ala His Arg 65 70
75 80Val Phe Ser Phe Leu Gly Lys Asp Asn Asp Ile Leu
Thr Val Lys Glu 85 90
95 Ala Ile Leu Gly Ile Pro Gly Leu Asp Pro Met Asp Pro His Thr Ala
100 105 110 Pro Gly Leu
Pro Tyr Ala Ile Asn Gly Leu Arg Arg Thr Asp Leu Val 115
120 125 Asp Phe Val Asn Gly Thr Val Asp
Ala Ala Leu Ala Val Gln Ile Gln 130 135
140 Lys Phe Leu Asp Gly Asp Tyr Ser Asp His Val Phe Gln
Thr Phe Leu 145 150 155
160Lys Asp Glu Ile Arg Pro Ser Glu Lys Val Arg Ala Gly Lys Thr Arg
165 170 175 Ile Val Asp Val
Pro Ser Leu Ala His Cys Ile Val Gly Arg Met Leu 180
185 190 Leu Gly Arg Phe Ala Ala Lys Phe Gln
Ser His Pro Gly Phe Leu Leu 195 200
205 Gly Ser Ala Ile Gly Ser Asp Pro Asp Val Phe Trp Thr Val
Ile Gly 210 215 220
Ala Gln Leu Glu Gly Arg Lys Asn Thr Tyr Asp Val Asp Tyr Ser Ala 225
230 235 240Phe Asp Ser Ser His
Gly Thr Gly Ser Phe Glu Ala Leu Ile Ser His 245
250 255 Phe Phe Thr Val Asp Asn Gly Phe Ser Pro
Ala Leu Gly Pro Tyr Leu 260 265
270 Arg Ser Leu Ala Val Ser Val His Ala Tyr Gly Glu Arg Arg Ile
Lys 275 280 285 Ile
Thr Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr Ser Leu Leu Asn 290
295 300 Thr Val Leu Asn Asn Val
Ile Ile Arg Thr Ala Leu Ala Leu Thr Tyr 305 310
315 320Lys Glu Phe Glu Tyr Asp Thr Val Asp Ile Ile
Ala Tyr Gly Asp Asp 325 330
335 Leu Leu Val Gly Thr Asp Tyr Asp Leu Asp Phe Asn Glu Val Ala Arg
340 345 350 Arg Ala
Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala Asn Lys Gly Ser 355
360 365 Val Phe Pro Pro Thr Ser Ser
Leu Ser Asp Ala Val Phe Leu Lys Arg 370 375
380 Lys Phe Val Gln Asn Asn Asp Gly Leu Tyr Lys Pro
Val Met Asp Leu 385 390 395
400Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys Pro Gly Thr Leu Leu
405 410 415 Glu Lys Leu
Gln Ser Val Ser Met Leu Ala Gln His Ser Gly Lys Glu 420
425 430 Glu Tyr Asp Arg Leu Met His Pro
Phe Ala Asp Tyr Gly Ala Val Pro 435 440
445 Ser His Glu Tyr Leu Gln Ala Arg Trp Arg Ala Leu Phe
Asp 450 455 460
456PRTSeneca Valley Virus 45Ala Gly Phe Cys Thr Glu
1 5 466PRTSeneca Valley Virus 46Ala
Gly Asp Cys Ala Glu 1
5 476PRTSeneca Valley Virus 47Thr Gly Phe Cys Ala Glu
1 5 486PRTSeneca Valley Virus
48Ala Gly Phe Cys Ala Glu 1
5 4922PRTPorcine teschovirus 49Gly Pro Gly Ala Thr Asn Phe
Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5
10 15 Glu Glu Asn Pro Gly Pro
20 5022PRTPorcine teschovirus 50Gly
Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1
5 10 15 Glu Glu Asn Pro Gly Pro
20
5122PRTPorcine teschovirus 51Gly Pro Gly Ala Ser Ser Phe Ser Leu Leu Lys
Gln Ala Gly Asp Val 1 5 10
15 Glu Glu Asn Pro Gly Pro
20 5222PRTPorcine teschovirus 52Gly Pro Gly Ala Ser
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5
10 15 Glu Glu Asn Pro Gly Pro
20 5322PRTPorcine teschovirus
53Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1
5 10 15 Glu Glu Asn Pro
Gly Pro 20
5422PRTPorcine teschovirus 54Gly Pro Gly Ala Ala Asn Phe Ser Leu Leu Arg
Gln Ala Gly Asp Val 1 5 10
15 Glu Glu Asn Pro Gly Pro
20 5522PRTPorcine teschovirus 55Gly Pro Gly Ala Thr
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5
10 15 Glu Glu Asn Pro Gly Pro
20 5622PRTPorcine teschovirus
56Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1
5 10 15 Glu Glu Asn Pro
Gly Pro 20
5722PRTPorcine teschovirus 57Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
Gln Ala Gly Asp Ile 1 5 10
15 Glu Glu Asn Pro Gly Pro
20 5822PRTPorcine teschovirus 58Gly Pro Gly Ala Thr
Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5
10 15 Glu Glu Asn Pro Gly Pro
20 5922PRTPorcine teschovirus
59Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1
5 10 15 Glu Glu Asn Pro
Gly Pro 20
6022PRTPorcine teschovirus 60Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
Arg Ala Gly Asp Val 1 5 10
15 Glu Glu Asn Pro Gly Pro
20 6119PRTFoot and mouth disease virus 61Thr Leu Asn
Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5
10 15 Pro Gly Pro
6219PRTFoot and mouth disease virus 62Leu
Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1
5 10 15 Pro Gly Pro
6319PRTFoot and mouth disease
virus 63Leu Leu Asn Phe Asp Leu Leu Gln Leu Ala Gly Asp Val Glu Ser Asn
1 5 10 15 Pro Gly Pro
6419PRTFoot and
mouth disease virus 64Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val
Glu Pro Asn 1 5 10 15
Pro Gly Pro
6519PRTFoot and mouth disease virus 65Leu Leu Asn Phe Asp Leu Leu Lys Leu
Ala Gly Asp Val Glu Ser Asn 1 5 10
15 Pro Gly Pro
6619PRTFoot and mouth disease virus 66Leu Ser Asn Phe Asp Leu Leu
Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5
10 15 Pro Gly Pro
6719PRTFoot and mouth disease virus 67Leu Leu Asn Phe
Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5
10 15 Pro Gly Pro
6819PRTFoot and mouth disease virus 68Thr Leu
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1
5 10 15 Pro Gly Pro
6919PRTFoot and mouth disease virus
69Ala Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1
5 10 15 Pro Gly Pro
7019PRTFoot and mouth
disease virus 70Val Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu
Ser Asn 1 5 10 15
Pro Gly Pro
7119PRTFoot and mouth disease virus 71Met Cys Ser Phe Asp Leu Leu Lys Leu
Ala Gly Asp Val Glu Ser Asn 1 5 10
15 Pro Gly Pro
7219PRTFoot and mouth disease virus 72Leu Cys Asn Phe Asp Leu Leu
Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5
10 15 Pro Gly Pro
7319PRTFoot and mouth disease virus 73Leu Cys Asn Phe
Asp Leu Leu Met Leu Ala Gly Asp Val Glu Ser Asn 1 5
10 15 Pro Gly Pro
7419PRTFoot and mouth disease virus 74Met Ala
Asn Phe Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1
5 10 15 Pro Gly Pro
7519PRTFoot and mouth disease virus
75Leu Ser Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1
5 10 15 Pro Gly Pro
7619PRTFoot and mouth
disease virus 76Leu Phe Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu
Ser Asn 1 5 10 15
Pro Gly Pro
7719PRTFoot and mouth disease virus 77Leu Cys Asn Cys Asp Leu Leu Lys Leu
Ala Gly Asp Val Glu Ser Asn 1 5 10
15 Pro Gly Pro
7819PRTFoot and mouth disease virus 78Leu Leu Asn Phe Asp Leu Leu
Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5
10 15 Pro Gly Pro
7919PRTFoot and mouth disease virus 79Leu Cys Asn Phe
Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5
10 15 Pro Gly Pro
8019PRTFoot and mouth disease virus 80Met Cys
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1
5 10 15 Pro Gly Pro
8122PRTEquine rhinitis virus 81Asn
Lys Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val 1
5 10 15 Glu Ser Asn Pro Gly Pro
20
8222PRTEquine rhinitis virus 82Ser Glu Gly Ala Thr Asn Phe Ser Leu Leu
Lys Leu Ala Gly Asp Val 1 5 10
15 Glu Leu Asn Pro Gly Pro
20 8322PRTEquine rhinitis virus 83Ser Gln Gly Ala
Thr Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5
10 15 Glu Ser Asn Pro Gly Pro
20
8422PRTEncephalomyocarditis virus 84Asn Ala His Tyr Ala Gly Tyr Phe Ala
Asp Leu Leu Ile His Asp Ile 1 5 10
15 Glu Thr Asn Pro Gly Pro
20 8522PRTEncephalomyocarditis virus 85Asn Ala
His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile 1
5 10 15 Glu Thr Asn Pro Gly Pro
20 8622PRTMengo
virus 86Glu Thr His Tyr Ala Gly Tyr Phe Ser Asp Leu Leu Ile His Asp Val
1 5 10 15 Glu Thr Asn
Pro Gly Pro 20
8722PRTSeneca Valley Virus 87Pro Phe Arg Ser Tyr Lys Gln Lys Met Leu Met
Gln Ser Gly Asp Ile 1 5 10
15 Glu Thr Asn Pro Gly Pro
20 8822PRTTheiler's murine encephalomyelitis virus
88Arg Gly Tyr His Ala Asp Tyr Tyr Arg Gln Arg Leu Ile His Asp Val 1
5 10 15 Glu Thr Asn Pro
Gly Pro 20
8922PRTTheiler's murine encephalomyelitis virus 89Arg Gly Tyr His Ala Asp
Tyr Tyr Lys Gln Arg Leu Ile His Asp Val 1 5
10 15 Glu Met Asn Pro Gly Pro
20 9022PRTTheiler's murine
encephalomyelitis virus 90Arg Ala Tyr His Ala Asp Tyr Tyr Lys Gln Arg Leu
Ile His Asp Val 1 5 10
15 Glu Met Asn Pro Gly Pro
20 9122PRTRat theiler's like virus 91Arg Glu Tyr His Ala
Ala Tyr Tyr Lys Gln Arg Leu Met His Asp Val 1 5
10 15 Glu Thr Asn Pro Gly Pro
20 9222PRTLjungan virus 92Glu
Met Asp Phe Ala Gly Gly Lys Phe Leu Asn Gln Cys Gly Asp Val 1
5 10 15 Glu Thr Asn Pro Gly Pro
20
9322PRTLjungan virus 93Glu Met Asp Phe Ala Gly Gly Lys Leu Phe Asn Gln
Cys Gly Asp Val 1 5 10
15 Glu Thr Asn Pro Gly Pro
20 9422PRTLjungan virus 94Glu Met Asp Tyr Ser Gly Gly Lys
Phe Leu Asn Gln Cys Gly Asp Val 1 5 10
15 Glu Ser Asn Pro Gly Pro
20 9522PRTLjungan virus 95Asp Met Asp Tyr
Ala Gly Gly Lys Leu Phe Asn Gln Cys Gly Asp Val 1 5
10 15 Glu Thr Asn Pro Gly Pro
20 9622PRTTrypanosoma
brucei 96Ala Ile Ser Ser Ile Ile Arg Thr Lys Met Leu Leu Ser Gly Asp Val
1 5 10 15 Glu Glu Asn
Pro Gly Pro 20
9722PRTTrypanosoma cruzi 97Ala Val Cys Asp Ala Gln Arg Gln Lys Leu Leu
Leu Ser Gly Asp Ile 1 5 10
15 Glu Gln Asn Pro Gly Pro
20 9822PRTBovine rotavirus 98Ala Asn Ser Lys Phe Gln
Ile Asp Arg Ile Leu Ile Ser Gly Asp Ile 1 5
10 15 Glu Leu Asn Pro Gly Pro
20 9922PRTHuman rotavirus 99Ala
Asn Ser Lys Phe Gln Ile Asp Lys Ile Leu Ile Ser Gly Asp Ile 1
5 10 15 Glu Leu Asn Pro Gly Pro
20
10023PRTPorcine rotavirus 100Ala Asn Ala Lys Phe Gly Gln Ile Asp Lys Ile
Leu Ile Ser Gly Asp 1 5 10
15 Val Glu Leu Asn Pro Gly Pro
20 10122PRTDrosophila C virus 101Lys Gln Glu Ala
Ala Arg Gln Met Leu Leu Leu Leu Ser Gly Asp Val 1 5
10 15 Glu Thr Asn Pro Gly Pro
20 10222PRTCricket
paralysis virus 102Arg Ala Phe Leu Arg Lys Arg Thr Gln Leu Leu Met Ser
Gly Asp Val 1 5 10 15
Glu Ser Asn Pro Gly Pro
20 10322PRTAcute bee paralysis virus 103His Cys Gly Ser Trp
Thr Asp Ile Leu Leu Leu Leu Ser Gly Asp Val 1 5
10 15 Glu Thr Asn Pro Gly Pro
20 10422PRTCricket paralysis
virus 104Thr Leu Thr Arg Ala Glu Ile Glu Asp Glu Leu Ile Arg Ala Gly Ile
1 5 10 15 Glu Ser Asn
Pro Gly Pro 20
10522PRTPerina nuda picorna like virus 105Val Thr Ala Gln Gly Trp Val
Pro Asp Leu Thr Val Asp Gly Asp Val 1 5
10 15 Glu Ser Asn Pro Gly Pro
20 10622PRTEctropis obliqua picorna
like virus 106Val Thr Ala Gln Gly Trp Ala Pro Asp Leu Thr Gln Asp Gly Asp
Val 1 5 10 15 Glu
Ser Asn Pro Gly Pro
20 10722PRTPerina nuda picorna like virus 107Asn Ile Ile Gly Gly
Gly Gln Lys Asp Leu Thr Gln Asp Gly Asp Ile 1 5
10 15 Glu Ser Asn Pro Gly Pro
20 10822PRTEctropis obliqua
picorna like virus 108Asn Ile Ile Gly Gly Gly Gln Arg Asp Leu Thr Gln Asp
Gly Asp Ile 1 5 10 15
Glu Ser Asn Pro Gly Pro
20 10922PRTDeformed wing virus 109Asn Leu Leu Gln Leu Ser Asn
Pro Val Gln Ala Lys Pro Glu Met Asp 1 5
10 15 Asn Pro Asn Pro Gly Pro
20 11022PRTKakugo virus 110Asn Leu
Leu Gln Leu Ser Asn Pro Val Gln Ala Lys Pro Glu Met Asp 1
5 10 15 Asn Pro Asn Pro Gly Pro
20 1114PRTSeneca
Valley Virus 111Asn Pro Gly Pro
1 1129PRTArtificial SequenceDescription of Artificial
Sequence Illustrative conserved motif 112Gly Xaa Xaa Gly Xaa
Gly Lys Ser Thr 1 5
1136PRTSeneca Valley Virus 113Lys Asp Glu Leu Ile Arg
1 5 1144PRTSeneca Valley Virus
114Tyr Gly Asp Asp 1
1154PRTSeneca Valley Virus 115Phe Leu Lys Arg
1 1166PRTArtificial
SequenceDescription of Artificial Sequence synthetic 6-His tag
peptide 116His His His His His His
1 5 1174PRTSeneca Valley Virus 117Glu Gln Gly Pro
1 1184PRTSeneca
Valley Virus 118Phe His Ser Thr
1 1194PRTSeneca Valley Virus 119Lys Gln Lys Met
1 1204PRTSeneca
Valley Virus 120Met Gln Gly Pro
1 1214PRTSeneca Valley Virus 121Leu Gln Ser Pro
1 1224PRTSeneca
Valley Virus 122Ser Glu Asn Ala
1 1234PRTSeneca Valley Virus 123Met Gln Gln Pro
1 1244PRTSeneca
Valley Virus 124Met Gln Gly Leu
1 1254PRTEncephalomyocarditis virus 125Leu Gln Gly Asn
1
1264PRTEncephalomyocarditis virus 126Leu Ala Asp Gln
1 1274PRTEncephalomyocarditis
virus 127Arg Gln Ser Pro
1 1284PRTEncephalomyocarditis virus 128Pro Gln Gly Val
1
1294PRTEncephalomyocarditis virus 129Leu Glu Ser Pro
1 1304PRTEncephalomyocarditis
virus 130Asn Pro Gly Pro
1 1314PRTEncephalomyocarditis virus 131Gln Gln Ser Pro
1
1324PRTEncephalomyocarditis virus 132Ala Gln Gly Pro
1 1334PRTEncephalomyocarditis
virus 133Ala Gln Ala Pro
1 1344PRTEncephalomyocarditis virus 134Glu Gln Gly Pro
1
1354PRTEncephalomyocarditis virus 135Ile Gln Gly Pro
1 1364PRTEncephalomyocarditis
virus 136Val Gln Gly Pro
1 1374PRTEncephalomyocarditis virus 137Pro Gln Gly Ala
1 1384PRTTheiler's
murine encephalomyelitis virus 138Pro Gln Gly Asn
1 1394PRTTheiler's murine
encephalomyelitis virus 139Leu Leu Asp Gln
1 1404PRTTheiler's murine encephalomyelitis
virus 140Leu Met Asp Gln
1 1414PRTTheiler's murine encephalomyelitis virus 141Ala Gln
Ser Pro 1
1424PRTTheiler's murine encephalomyelitis virus 142Pro Gln Gly Val
1 1434PRTTheiler's
murine encephalomyelitis virus 143Pro Gln Gly Ile
1 1444PRTTheiler's murine
encephalomyelitis virus 144Pro Gln Gly Ser
1 1454PRTTheiler's murine encephalomyelitis
virus 145Leu Glu Asn Pro
1 1464PRTTheiler's murine encephalomyelitis virus 146Asn Pro
Gly Pro 1
1474PRTTheiler's murine encephalomyelitis virus 147Pro Gln Gly Pro
1 1484PRTTheiler's
murine encephalomyelitis virus 148Ala Gln Ser Pro
1 1494PRTTheiler's murine
encephalomyelitis virus 149Glu Gln Ala Ala
1 1504PRTTheiler's murine encephalomyelitis
virus 150Ile Gln Gly Gly
1 1514PRTTheiler's murine encephalomyelitis virus 151Pro Gln
Gly Ala 1
1524PRTRat Theilovirus 152Pro Gln Gly Asn
1 1534PRTRat Theilovirus 153Leu Leu Asp Gln
1 1544PRTRat
Theilovirus 154Pro Gln Ser Pro
1 1554PRTRat Theilovirus 155Pro Gln Gly Val
1 1564PRTRat Theilovirus
156Leu Gln Asn Pro 1
1574PRTRat Theilovirus 157Asn Pro Gly Pro
1 1584PRTRat Theilovirus 158Ala Gln
Ser Pro 1
1594PRTRat Theilovirus 159Ala Gln Ser Pro
1 1604PRTRat Theilovirus 160Glu Gln Ala Ala
1 1614PRTRat
Theilovirus 161Ile Gln Gly Gly
1 1624PRTRat Theilovirus 162Pro Gln Gly Ala
1 1634PRTVilyuisk human
encephalomyelitis virus 163Pro Gln Gly Asn
1 1644PRTVilyuisk human encephalomyelitis
virus 164Leu Leu Asp Glu
1 1654PRTVilyuisk human encephalomyelitis virus 165Pro Gln Ser
Pro 1
1664PRTVilyuisk human encephalomyelitis virus 166Pro Gln Gly Val
1 1674PRTVilyuisk
human encephalomyelitis virus 167Leu Glu Asn Pro
1 1687310DNASeneca Valley Virus
168tttgaaatgg ggggctgggc cctgatgccc agtccttcct ttccccttcc ggggggttaa
60ccggctgtgt ttgctagagg cacagagggg caacatccaa cctgcttttg cggggaacgg
120tgcggctccg attcctgcgt cgccaaaggt gttagcgcac ccaaacggcg cacctaccaa
180tgttattggt gtggtctgcg agttctagcc tactcgtttc tcccccgacc attcactcac
240ccacgaaaag tgtgttgtaa ccataagatt taacccccgc acgggatgtg cgataaccgt
300aagactggct caagcgcgga aagcgctgta accacatgct gttagtccct ttatggctgc
360aagatggcta cccacctcgg atcactgaac tggagctcga ccctccttag taagggaacc
420gagaggcctt cgtgcaacaa gctccgacac agagtccacg tgactgctac caccatgagt
480acatggttct cccctctcga cccaggactt ctttttgaat atccacggct cgatccagag
540ggtggggcat gacccctagc atagcgagct acagcgggaa ctgtagctag gccttagcgt
600gccttggata ctgcctgata gggcgacggc ctagtcgtgt cggttctata ggtagcacat
660acaaatatgc agaactctca tttttctttc gatacagcct ctggcacctt tgaagatgta
720accggaacaa aagtcaagat cgttgaatac cccagatcgg tgaacaatgg tgtttacgat
780tcgtctactc atttggagat actgaaccta cagggtgaaa ttgaaatttt aaggtctttc
840aatgaatacc aaattcgcgc cgccaaacaa caactcggac tggacatcgt gtacgaacta
900cagggtaatg ttcagacaac gtcaaagaat gattttgatt cccgtggcaa taatggtaac
960atgaccttca attactacgc aaacacttat cagaattcag tagacttctc gacctcctcg
1020tcggcgtcag gcgccggacc cgggaactcc cggggcggat tagcgggtct cctcacaaat
1080ttcagtggaa tcttgaaccc tcttggctac ctcaaagatc acaacaccga agaaatggaa
1140aactctgctg atcgagtcac aacgcaaacg gcgggcaaca ctgccataaa cacgcaatca
1200tcattgggtg tgttgtgtgc ctacgttgaa gacccgacca aatctgatcc tccgtccagc
1260agcacagatc aacccaccac cactttcact gccatcgaca ggtggtacac tggacgtctc
1320aattcttgga caaaagctgt aaaaaccttc tcttttcagg ccgtcccgct tcccggggcc
1380tttctgtcta ggcagggagg cctcaacgga ggggccttca cagctaccct acatagacac
1440tttttgatga agtgcgggtg gcaggtgcag gtccaatgta atttgacaca attccaccaa
1500ggcgctcttc ttgttgccat ggttcctgaa accacccttg atgtcaagcc cgacggtaag
1560gcaaagagct tacaggagct gaatgaagaa cagtgggtgg aaatgtctga cgattaccgg
1620accgggaaaa acatgccttt tcagtctctt ggcacatact atcggccccc taactggact
1680tggggtccca atttcatcaa cccctatcaa gtaacggttt tcccacacca aattctgaac
1740gcgagaacct ctacctcggt agacataaac gtcccataca tcggggagac ccccacgcaa
1800tcctcagaga cacagaactc ctggaccctc ctcgttatgg tgctcgttcc cctagactat
1860aaggaaggag ccacaactga cccagaaatt acattttctg taaggcctac aagtccctac
1920ttcaatgggc ttcgcaaccg ctacacggcc gggacggacg aagaacaggg gcccattcct
1980acggcaccca gagaaaattc gcttatgttt ctctcaaccc tccctgacga cactgtccct
2040gcttacggga atgtgcgtac ccctcctgtc aattacctcc ctggtgaaat aaccgacctt
2100ttgcaactgg cccgcatacc cactctcatg gcatttgagc gggtgcctga acccgtgcct
2160gcctcagaca catatgtgcc ctacgttgcc gttcccaccc agttcgatga caggcctctc
2220atctccttcc cgatcaccct ttcagatccc gtctatcaga acaccctggt tggcgccatc
2280agttcaaatt tcgccaatta ccgtgggtgt atccaaatca ctctgacatt ttgtggaccc
2340atgatggcga gagggaaatt cctgctctcg tattctcccc caaatggaac gcaaccacag
2400actctttccg aagctatgca gtgcacatac tctatttggg acataggctt gaactctagt
2460tggaccttcg tcgtccccta catctcgccc agtgactacc gtgaaactcg agccattacc
2520aactcggttt actccgctga tggttggttt agcctgcaca agttgaccaa aattactcta
2580ccacctgact gtccgcaaag tccctgcatt ctctttttcg cttctgctgg tgaggattac
2640actctccgtc tccccgttga ttgtaatcct tcctatgtgt tccactccac cgacaacgcc
2700gagaccgggg ttattgaggc gggtaacact gacaccgatt tctctggtga actggcggct
2760cctggctcta accacactaa tgtcaagttc ctgtttgatc gatctcgatt attgaatgta
2820atcaaggtac tggagaagga cgccgttttc ccccgccctt tccctacaca agaaggtgcg
2880cagcaggatg atggttactt ttgtcttctg accccccgcc caacagtcgc ttcccgaccc
2940gccactcgtt tcggcctgta cgccaatccg tccggcagtg gtgttcttgc taacacttca
3000ctggacttca atttttatag cttggcctgt ttcacttact ttagatcgga ccttgaggtt
3060acggtggtct cactagagcc ggatctggaa tttgctgtag ggtggtttcc ttctggcagt
3120gaataccagg cttccagctt tgtctacgac cagctgcatg tgcccttcca ctttactggg
3180cgcactcccc gcgctttcgc tagcaagggt gggaaggtat ctttcgtgct cccttggaac
3240tctgtctcgt ctgtgctccc cgtgcgctgg gggggggctt ccaagctctc ttctgctacg
3300cggggtctac cggcgcatgc tgattggggg actatttacg cctttgtccc ccgtcctaat
3360gagaagaaaa gcaccgctgt aaaacacgtg gccgtgtaca ttcggtacaa gaacgcacgt
3420gcctggtgcc ccagcatgct tccctttcgc agctacaagc agaagatgct gatgcaatct
3480ggcgatatcg agaccaatcc tggtcctgct tctgacaacc caattttgga gtttcttgaa
3540gcagaaaatg atctagtcac tctggcctct ctctggaaga tggtgcactc tgttcaacag
3600acctggagaa agtatgtgaa gaacgatgat ttttggccca atttactcag cgagctagtg
3660ggggaaggct ctgtcgcctt ggccgccacg ctatccaacc aagcttcagt aaaggctctt
3720ttgggcctgc actttctctc tcgggggctc aattacactg acttttactc tttactgata
3780gagaaatgct ctagtttctt taccgtagaa ccacctcctc caccagctga aaacctgatg
3840accaagccct cagtgaagtc gaaattccga aaactgttta agatgcaagg acccatggac
3900aaagtcaaag actggaacca aatagctgcc ggcttgaaga attttcaatt tgttcgtgac
3960ctagtcaaag aggtggtcga ttggctgcag gcctggatca acaaagagaa agccagccct
4020gtcctccagt accagttgga gatgaagaag ctcgggcctg tggccttggc tcatgacgct
4080ttcatggctg gttccgggcc ccctcttagc gacgaccaga ttgaatacct ccagaacctc
4140aaatctcttg ccctaacact ggggaagact aatttggccc aaagtctcac cactatgatc
4200aatgccaaac aaagttcagc ccaacgagtt gaacccgttg tggtggtcct tagaggcaag
4260ccgggatgcg gcaagagctt ggcctctacg ttgattgccc aggctgtgtc caagcgcctc
4320tatggctccc aaagtgtata ttctcttccc ccagatccag atttcttcga tggatacaaa
4380ggacagttcg tgaccttgat ggatgatttg ggacaaaacc cggatggaca agatttctcc
4440accttttgtc agatggtgtc gaccgcccaa tttctcccca acatggcgga ccttgcagag
4500aaagggcgtc cctttacctc caatctcatc attgcaacta caaatctccc ccacttcagt
4560cctgtcacca ttgctgatcc ttctgcagtc tctcgccgta tcaactacga tctgactcta
4620gaagtatctg aggcctacaa gaaacacaca cggctgaatt ttgacttggc tttcaggcgc
4680acagacgccc cccccattta tccttttgct gcccatgtgc cctttgtgga cgtagctgtg
4740cgcttcaaaa atggtcacca gaattttaat ctcctagagt tggtcgattc catttgtaca
4800gacattcgag ccaagcaaca aggtgcccga aacatgcaga ctctggttct acagagcccc
4860aacgagaatg atgacacccc cgtcgacgag gcgttgggta gagttctctc ccccgctgcg
4920gtcgatgagg cgcttgtcga cctcactcca gaggccgacc cggttggccg tttggctatt
4980cttgccaagc taggtcttgc cctagctgcg gtcacccctg gtctgataat cttggcagtg
5040ggactctaca ggtacttctc tggctctgat gcagaccaag aagaaacaga aagtgaggga
5100tctgtcaagg cacccaggag cgaaaatgct tatgacggcc cgaagaaaaa ctctaagccc
5160cctggagcac tctctctcat ggaaatgcaa cagcccaacg tggacatggg ctttgaggct
5220gcggtcgcta agaaagtggt cgtccccatt accttcatgg ttcccaacag accttctggg
5280cttacacagt ccgctcttct ggtgaccggc cggaccttcc taatcaatga acatacatgg
5340tccaatccct cctggaccag cttcacaatc cgcggtgagg tacacactcg tgatgagccc
5400ttccaaacgg ttcatttcac tcaccacggt attcccacag atctgatgat ggtacgtctc
5460ggaccgggca attctttccc taacaatcta gacaagtttg gacttgacca gatgccggca
5520cgcaactccc gtgtggttgg cgtttcgtcc agttacggaa acttcttctt ctctggaaat
5580ttcctcggat ttgttgattc catcacctct gaacaaggaa cttacgcaag actctttagg
5640tacagggtga cgacctacaa aggatggtgc ggctcggccc tggtctgtga ggccggtggc
5700gtccgacgca tcattggcct gcattctgct ggcgccgccg gtatcggcgc cgggacctat
5760atctcaaaat taggactaat caaagccctg aaacacctcg gtgaaccttt ggccacaatg
5820caaggactga tgactgaatt agagcctgga atcaccgtac atgtaccccg gaaatccaaa
5880ttgagaaaga cgaccgcaca cgcggtgtac aaaccggagt ttgagcctgc tgtgttgtca
5940aaatttgatc ccagactgaa caaggatgtt gacttggatg aagtaatttg gtctaaacac
6000actgccaatg tcccttacca acctcctttg ttctacacat acatgtcaga gtacgctcat
6060cgagtcttct ccttcttggg gaaagacaat gacattctga ccgtcaaaga agcaattctg
6120ggcatccccg gactagaccc catggatccc cacacagctc cgggtctgcc ttacgccatc
6180aacggccttc gacgtactga tctcgtcgat tttgtgaacg gtacagtaga tgcggcgctg
6240gctgtacaaa tccagaaatt cttagacggt gactactctg accatgtctt ccaaactttt
6300ctgaaagatg agatcagacc ctcagagaaa gtccgagcgg gaaaaacccg cattgttgat
6360gtgccctccc tggcgcattg cattgtgggc agaatgttgc ttgggcgctt tgctgccaag
6420tttcaatccc atcctggctt tctcctcggc tctgctatcg ggtctgaccc tgatgttttc
6480tggaccgtca taggggctca actcgagggg agaaagaaca cgtatgacgt ggactacagt
6540gcctttgact cttcacacgg cactggctcc ttcgaggctc tcatctctca ctttttcacc
6600gtggacaatg gttttagccc tgcgctggga ccgtatctca gatccctggc tgtctcggtg
6660cacgcttacg gcgagcgtcg catcaagatt accggtggcc tcccctccgg ttgtgccgcg
6720accagcctgc tgaacacagt gctcaacaat gtgatcatca ggactgctct ggcattgact
6780tacaaggaat ttgaatatga catggttgat atcatcgcct acggtgacga ccttctggtt
6840ggcacggatt acgatctgga cttcaatgag gtggcacgac gcgctgccaa gttggggtat
6900aagatgactc ctgccaacaa gggttctgtc ttccctccga cttcctctct ttccgatgct
6960gtttttctaa agcgcaaatt cgtccaaaac aacgacggct tatacaaacc agttatggat
7020ttaaagaatt tggaagccat gctctcctac ttcaaaccag gaacactact cgagaagctg
7080caatctgttt ctatgttggc tcaacattct ggaaaagaag aatatgatag attgatgcac
7140cccttcgctg actacggtgc cgtaccgagt cacgagtacc tgcaggcaag atggagggcc
7200ttgttcgact gacccagata gcccaaggcg cttcggtgct gccggcgatt ctgggagaac
7260tcagtcggaa cagaaaaggg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
73101692181PRTSeneca Valley Virus 169Met Gln Asn Ser His Phe Ser Phe Asp
Thr Ala Ser Gly Thr Phe Glu 1 5 10
15 Asp Val Thr Gly Thr Lys Val Lys Ile Val Glu Tyr Pro Arg
Ser Val 20 25 30
Asn Asn Gly Val Tyr Asp Ser Ser Thr His Leu Glu Ile Leu Asn Leu
35 40 45 Gln Gly Glu Ile
Glu Ile Leu Arg Ser Phe Asn Glu Tyr Gln Ile Arg 50
55 60 Ala Ala Lys Gln Gln Leu Gly Leu
Asp Ile Val Tyr Glu Leu Gln Gly 65 70
75 80 Asn Val Gln Thr Thr Ser Lys Asn Asp Phe Asp Ser
Arg Gly Asn Asn 85 90
95 Gly Asn Met Thr Phe Asn Tyr Tyr Ala Asn Thr Tyr Gln Asn Ser Val
100 105 110 Asp Phe Ser
Thr Ser Ser Ser Ala Ser Gly Ala Gly Pro Gly Asn Ser 115
120 125 Arg Gly Gly Leu Ala Gly Leu Leu
Thr Asn Phe Ser Gly Ile Leu Asn 130 135
140 Pro Leu Gly Tyr Leu Lys Asp His Asn Thr Glu Glu Met
Glu Asn Ser 145 150 155
160 Ala Asp Arg Val Thr Thr Gln Thr Ala Gly Asn Thr Ala Ile Asn Thr
165 170 175 Gln Ser Ser Leu
Gly Val Leu Cys Ala Tyr Val Glu Asp Pro Thr Lys 180
185 190 Ser Asp Pro Pro Ser Ser Ser Thr Asp
Gln Pro Thr Thr Thr Phe Thr 195 200
205 Ala Ile Asp Arg Trp Tyr Thr Gly Arg Leu Asn Ser Trp Thr
Lys Ala 210 215 220
Val Lys Thr Phe Ser Phe Gln Ala Val Pro Leu Pro Gly Ala Phe Leu 225
230 235 240 Ser Arg Gln Gly Gly
Leu Asn Gly Gly Ala Phe Thr Ala Thr Leu His 245
250 255 Arg His Phe Leu Met Lys Cys Gly Trp Gln
Val Gln Val Gln Cys Asn 260 265
270 Leu Thr Gln Phe His Gln Gly Ala Leu Leu Val Ala Met Val Pro
Glu 275 280 285 Thr
Thr Leu Asp Val Lys Pro Asp Gly Lys Ala Lys Ser Leu Gln Glu 290
295 300 Leu Asn Glu Glu Gln Trp
Val Glu Met Ser Asp Asp Tyr Arg Thr Gly 305 310
315 320 Lys Asn Met Pro Phe Gln Ser Leu Gly Thr Tyr
Tyr Arg Pro Pro Asn 325 330
335 Trp Thr Trp Gly Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe
340 345 350 Pro His
Gln Ile Leu Asn Ala Arg Thr Ser Thr Ser Val Asp Ile Asn 355
360 365 Val Pro Tyr Ile Gly Glu Thr
Pro Thr Gln Ser Ser Glu Thr Gln Asn 370 375
380 Ser Trp Thr Leu Leu Val Met Val Leu Val Pro Leu
Asp Tyr Lys Glu 385 390 395
400 Gly Ala Thr Thr Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser
405 410 415 Pro Tyr Phe
Asn Gly Leu Arg Asn Arg Tyr Thr Ala Gly Thr Asp Glu 420
425 430 Glu Gln Gly Pro Ile Pro Thr Ala
Pro Arg Glu Asn Ser Leu Met Phe 435 440
445 Leu Ser Thr Leu Pro Asp Asp Thr Val Pro Ala Tyr Gly
Asn Val Arg 450 455 460
Thr Pro Pro Val Asn Tyr Leu Pro Gly Glu Ile Thr Asp Leu Leu Gln 465
470 475 480 Leu Ala Arg Ile
Pro Thr Leu Met Ala Phe Glu Arg Val Pro Glu Pro 485
490 495 Val Pro Ala Ser Asp Thr Tyr Val Pro
Tyr Val Ala Val Pro Thr Gln 500 505
510 Phe Asp Asp Arg Pro Leu Ile Ser Phe Pro Ile Thr Leu Ser
Asp Pro 515 520 525
Val Tyr Gln Asn Thr Leu Val Gly Ala Ile Ser Ser Asn Phe Ala Asn 530
535 540 Tyr Arg Gly Cys Ile
Gln Ile Thr Leu Thr Phe Cys Gly Pro Met Met 545 550
555 560 Ala Arg Gly Lys Phe Leu Leu Ser Tyr Ser
Pro Pro Asn Gly Thr Gln 565 570
575 Pro Gln Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp
Asp 580 585 590 Ile
Gly Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro 595
600 605 Ser Asp Tyr Arg Glu Thr
Arg Ala Ile Thr Asn Ser Val Tyr Ser Ala 610 615
620 Asp Gly Trp Phe Ser Leu His Lys Leu Thr Lys
Ile Thr Leu Pro Pro 625 630 635
640 Asp Cys Pro Gln Ser Pro Cys Ile Leu Phe Phe Ala Ser Ala Gly Glu
645 650 655 Asp Tyr
Thr Leu Arg Leu Pro Val Asp Cys Asn Pro Ser Tyr Val Phe 660
665 670 His Ser Thr Asp Asn Ala Glu
Thr Gly Val Ile Glu Ala Gly Asn Thr 675 680
685 Asp Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly
Ser Asn His Thr 690 695 700
Asn Val Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys 705
710 715 720 Val Leu Glu
Lys Asp Ala Val Phe Pro Arg Pro Phe Pro Thr Gln Glu 725
730 735 Gly Ala Gln Gln Asp Asp Gly Tyr
Phe Cys Leu Leu Thr Pro Arg Pro 740 745
750 Thr Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr
Ala Asn Pro 755 760 765
Ser Gly Ser Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr 770
775 780 Ser Leu Ala Cys
Phe Thr Tyr Phe Arg Ser Asp Leu Glu Val Thr Val 785 790
795 800 Val Ser Leu Glu Pro Asp Leu Glu Phe
Ala Val Gly Trp Phe Pro Ser 805 810
815 Gly Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu
His Val 820 825 830
Pro Phe His Phe Thr Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly
835 840 845 Gly Lys Val Ser
Phe Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu 850
855 860 Pro Val Arg Trp Gly Gly Ala Ser
Lys Leu Ser Ser Ala Thr Arg Gly 865 870
875 880 Leu Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala
Phe Val Pro Arg 885 890
895 Pro Asn Glu Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Ile
900 905 910 Arg Tyr Lys
Asn Ala Arg Ala Trp Cys Pro Ser Met Leu Pro Phe Arg 915
920 925 Ser Tyr Lys Gln Lys Met Leu Met
Gln Ser Gly Asp Ile Glu Thr Asn 930 935
940 Pro Gly Pro Ala Ser Asp Asn Pro Ile Leu Glu Phe Leu
Glu Ala Glu 945 950 955
960 Asn Asp Leu Val Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val
965 970 975 Gln Gln Thr Trp
Arg Lys Tyr Val Lys Asn Asp Asp Phe Trp Pro Asn 980
985 990 Leu Leu Ser Glu Leu Val Gly Glu
Gly Ser Val Ala Leu Ala Ala Thr 995 1000
1005 Leu Ser Asn Gln Ala Ser Val Lys Ala Leu Leu
Gly Leu His Phe 1010 1015 1020
Leu Ser Arg Gly Leu Asn Tyr Thr Asp Phe Tyr Ser Leu Leu Ile
1025 1030 1035 Glu Lys Cys
Ser Ser Phe Phe Thr Val Glu Pro Pro Pro Pro Pro 1040
1045 1050 Ala Glu Asn Leu Met Thr Lys Pro
Ser Val Lys Ser Lys Phe Arg 1055 1060
1065 Lys Leu Phe Lys Met Gln Gly Pro Met Asp Lys Val Lys
Asp Trp 1070 1075 1080
Asn Gln Ile Ala Ala Gly Leu Lys Asn Phe Gln Phe Val Arg Asp 1085
1090 1095 Leu Val Lys Glu Val
Val Asp Trp Leu Gln Ala Trp Ile Asn Lys 1100 1105
1110 Glu Lys Ala Ser Pro Val Leu Gln Tyr Gln
Leu Glu Met Lys Lys 1115 1120 1125
Leu Gly Pro Val Ala Leu Ala His Asp Ala Phe Met Ala Gly Ser
1130 1135 1140 Gly Pro
Pro Leu Ser Asp Asp Gln Ile Glu Tyr Leu Gln Asn Leu 1145
1150 1155 Lys Ser Leu Ala Leu Thr Leu
Gly Lys Thr Asn Leu Ala Gln Ser 1160 1165
1170 Leu Thr Thr Met Ile Asn Ala Lys Gln Ser Ser Ala
Gln Arg Val 1175 1180 1185
Glu Pro Val Val Val Val Leu Arg Gly Lys Pro Gly Cys Gly Lys 1190
1195 1200 Ser Leu Ala Ser Thr
Leu Ile Ala Gln Ala Val Ser Lys Arg Leu 1205 1210
1215 Tyr Gly Ser Gln Ser Val Tyr Ser Leu Pro
Pro Asp Pro Asp Phe 1220 1225 1230
Phe Asp Gly Tyr Lys Gly Gln Phe Val Thr Leu Met Asp Asp Leu
1235 1240 1245 Gly Gln
Asn Pro Asp Gly Gln Asp Phe Ser Thr Phe Cys Gln Met 1250
1255 1260 Val Ser Thr Ala Gln Phe Leu
Pro Asn Met Ala Asp Leu Ala Glu 1265 1270
1275 Lys Gly Arg Pro Phe Thr Ser Asn Leu Ile Ile Ala
Thr Thr Asn 1280 1285 1290
Leu Pro His Phe Ser Pro Val Thr Ile Ala Asp Pro Ser Ala Val 1295
1300 1305 Ser Arg Arg Ile Asn
Tyr Asp Leu Thr Leu Glu Val Ser Glu Ala 1310 1315
1320 Tyr Lys Lys His Thr Arg Leu Asn Phe Asp
Leu Ala Phe Arg Arg 1325 1330 1335
Thr Asp Ala Pro Pro Ile Tyr Pro Phe Ala Ala His Val Pro Phe
1340 1345 1350 Val Asp
Val Ala Val Arg Phe Lys Asn Gly His Gln Asn Phe Asn 1355
1360 1365 Leu Leu Glu Leu Val Asp Ser
Ile Cys Thr Asp Ile Arg Ala Lys 1370 1375
1380 Gln Gln Gly Ala Arg Asn Met Gln Thr Leu Val Leu
Gln Ser Pro 1385 1390 1395
Asn Glu Asn Asp Asp Thr Pro Val Asp Glu Ala Leu Gly Arg Val 1400
1405 1410 Leu Ser Pro Ala Ala
Val Asp Glu Ala Leu Val Asp Leu Thr Pro 1415 1420
1425 Glu Ala Asp Pro Val Gly Arg Leu Ala Ile
Leu Ala Lys Leu Gly 1430 1435 1440
Leu Ala Leu Ala Ala Val Thr Pro Gly Leu Ile Ile Leu Ala Val
1445 1450 1455 Gly Leu
Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp Gln Glu Glu 1460
1465 1470 Thr Glu Ser Glu Gly Ser Val
Lys Ala Pro Arg Ser Glu Asn Ala 1475 1480
1485 Tyr Asp Gly Pro Lys Lys Asn Ser Lys Pro Pro Gly
Ala Leu Ser 1490 1495 1500
Leu Met Glu Met Gln Gln Pro Asn Val Asp Met Gly Phe Glu Ala 1505
1510 1515 Ala Val Ala Lys Lys
Val Val Val Pro Ile Thr Phe Met Val Pro 1520 1525
1530 Asn Arg Pro Ser Gly Leu Thr Gln Ser Ala
Leu Leu Val Thr Gly 1535 1540 1545
Arg Thr Phe Leu Ile Asn Glu His Thr Trp Ser Asn Pro Ser Trp
1550 1555 1560 Thr Ser
Phe Thr Ile Arg Gly Glu Val His Thr Arg Asp Glu Pro 1565
1570 1575 Phe Gln Thr Val His Phe Thr
His His Gly Ile Pro Thr Asp Leu 1580 1585
1590 Met Met Val Arg Leu Gly Pro Gly Asn Ser Phe Pro
Asn Asn Leu 1595 1600 1605
Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg Asn Ser Arg Val 1610
1615 1620 Val Gly Val Ser Ser
Ser Tyr Gly Asn Phe Phe Phe Ser Gly Asn 1625 1630
1635 Phe Leu Gly Phe Val Asp Ser Ile Thr Ser
Glu Gln Gly Thr Tyr 1640 1645 1650
Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys Gly Trp Cys
1655 1660 1665 Gly Ser
Ala Leu Val Cys Glu Ala Gly Gly Val Arg Arg Ile Ile 1670
1675 1680 Gly Leu His Ser Ala Gly Ala
Ala Gly Ile Gly Ala Gly Thr Tyr 1685 1690
1695 Ile Ser Lys Leu Gly Leu Ile Lys Ala Leu Lys His
Leu Gly Glu 1700 1705 1710
Pro Leu Ala Thr Met Gln Gly Leu Met Thr Glu Leu Glu Pro Gly 1715
1720 1725 Ile Thr Val His Val
Pro Arg Lys Ser Lys Leu Arg Lys Thr Thr 1730 1735
1740 Ala His Ala Val Tyr Lys Pro Glu Phe Glu
Pro Ala Val Leu Ser 1745 1750 1755
Lys Phe Asp Pro Arg Leu Asn Lys Asp Val Asp Leu Asp Glu Val
1760 1765 1770 Ile Trp
Ser Lys His Thr Ala Asn Val Pro Tyr Gln Pro Pro Leu 1775
1780 1785 Phe Tyr Thr Tyr Met Ser Glu
Tyr Ala His Arg Val Phe Ser Phe 1790 1795
1800 Leu Gly Lys Asp Asn Asp Ile Leu Thr Val Lys Glu
Ala Ile Leu 1805 1810 1815
Gly Ile Pro Gly Leu Asp Pro Met Asp Pro His Thr Ala Pro Gly 1820
1825 1830 Leu Pro Tyr Ala Ile
Asn Gly Leu Arg Arg Thr Asp Leu Val Asp 1835 1840
1845 Phe Val Asn Gly Thr Val Asp Ala Ala Leu
Ala Val Gln Ile Gln 1850 1855 1860
Lys Phe Leu Asp Gly Asp Tyr Ser Asp His Val Phe Gln Thr Phe
1865 1870 1875 Leu Lys
Asp Glu Ile Arg Pro Ser Glu Lys Val Arg Ala Gly Lys 1880
1885 1890 Thr Arg Ile Val Asp Val Pro
Ser Leu Ala His Cys Ile Val Gly 1895 1900
1905 Arg Met Leu Leu Gly Arg Phe Ala Ala Lys Phe Gln
Ser His Pro 1910 1915 1920
Gly Phe Leu Leu Gly Ser Ala Ile Gly Ser Asp Pro Asp Val Phe 1925
1930 1935 Trp Thr Val Ile Gly
Ala Gln Leu Glu Gly Arg Lys Asn Thr Tyr 1940 1945
1950 Asp Val Asp Tyr Ser Ala Phe Asp Ser Ser
His Gly Thr Gly Ser 1955 1960 1965
Phe Glu Ala Leu Ile Ser His Phe Phe Thr Val Asp Asn Gly Phe
1970 1975 1980 Ser Pro
Ala Leu Gly Pro Tyr Leu Arg Ser Leu Ala Val Ser Val 1985
1990 1995 His Ala Tyr Gly Glu Arg Arg
Ile Lys Ile Thr Gly Gly Leu Pro 2000 2005
2010 Ser Gly Cys Ala Ala Thr Ser Leu Leu Asn Thr Val
Leu Asn Asn 2015 2020 2025
Val Ile Ile Arg Thr Ala Leu Ala Leu Thr Tyr Lys Glu Phe Glu 2030
2035 2040 Tyr Asp Met Val Asp
Ile Ile Ala Tyr Gly Asp Asp Leu Leu Val 2045 2050
2055 Gly Thr Asp Tyr Asp Leu Asp Phe Asn Glu
Val Ala Arg Arg Ala 2060 2065 2070
Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala Asn Lys Gly Ser Val
2075 2080 2085 Phe Pro
Pro Thr Ser Ser Leu Ser Asp Ala Val Phe Leu Lys Arg 2090
2095 2100 Lys Phe Val Gln Asn Asn Asp
Gly Leu Tyr Lys Pro Val Met Asp 2105 2110
2115 Leu Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys
Pro Gly Thr 2120 2125 2130
Leu Leu Glu Lys Leu Gln Ser Val Ser Met Leu Ala Gln His Ser 2135
2140 2145 Gly Lys Glu Glu Tyr
Asp Arg Leu Met His Pro Phe Ala Asp Tyr 2150 2155
2160 Gly Ala Val Pro Ser His Glu Tyr Leu Gln
Ala Arg Trp Arg Ala 2165 2170 2175
Leu Phe Asp 2180 1701560DNASeneca Valley Virus
170aatgggcttc gcaaccgcta cacggccggg acggacgaag aacaggggcc cattcctacg
60gcacccagag aaaattcgct tatgtttctc tcaaccctcc ctgacgacac tgtccctgct
120tacgggaatg tgcgtacccc tcctgtcaat tacctccctg gtgaaataac cgaccttttg
180caactggccc gcatacccac tctcatggca tttgagcggg tgcctgaacc cgtgcctgcc
240tcagacacat atgtgcccta cgttgccgtt cccacccagt tcgatgacag gcctctcatc
300tccttcccga tcaccctttc agatcccgtc tatcagaaca ccctggttgg cgccatcagt
360tcaaatttcg ccaattaccg tgggtgtatc caaatcactc tgacattttg tggacccatg
420atggcgagag ggaaattcct gctctcgtat tctcccccaa atggaacgca accacagact
480ctttccgaag ctatgcagtg cacatactct atttgggaca taggcttgaa ctctagttgg
540accttcgtcg tcccctacat ctcgcccagt gactaccgtg aaactcgagc cattaccaac
600tcggtttact ccgctgatgg ttggtttagc ctgcacaagt tgaccaaaat tactctacca
660cctgactgtc cgcaaagtcc ctgcattctc tttttcgctt ctgctggtga ggattacact
720ctccgtctcc ccgttgattg taatccttcc tatgtgttcc actccaccga caacgccgag
780accggggtta ttgaggcggg taacactgac accgatttct ctggtgaact ggcggctcct
840ggctctaacc acactaatgt caagttcctg tttgatcgat ctcgattatt gaatgtaatc
900aaggtactgg agaaggacgc cgttttcccc cgccctttcc ctacacaaga aggtgcgcag
960caggatgatg gttacttttg tcttctgacc ccccgcccaa cagtcgcttc ccgacccgcc
1020actcgtttcg gcctgtacgc caatccgtcc ggcagtggtg ttcttgctaa cacttcactg
1080gacttcaatt tttatagctt ggcctgtttc acttacttta gatcggacct tgaggttacg
1140gtggtctcac tagagccgga tctggaattt gctgtagggt ggtttccttc tggcagtgaa
1200taccaggctt ccagctttgt ctacgaccag ctgcatgtgc ccttccactt tactgggcgc
1260actccccgcg ctttcgctag caagggtggg aaggtatctt tcgtgctccc ttggaactct
1320gtctcgtctg tgctccccgt gcgctggggg ggggcttcca agctctcttc tgctacgcgg
1380ggtctaccgg cgcatgctga ttgggggact atttacgcct ttgtcccccg tcctaatgag
1440aagaaaagca ccgctgtaaa acacgtggcc gtgtacattc ggtacaagaa cgcacgtgcc
1500tggtgcccca gcatgcttcc ctttcgcagc tacaagcaga agatgctgat gcaatctggc
15601711560DNAPicornavirusmisc_feature(8)..(8)n is a, c, g, or t
171aatgggcntc gcaaccgcta catggccggg acggacgaag aacaggggcc nattcccacg
60gcacccagag agaattcgct tatgtttctt tcaactctgc ctgacgacac agttcctgct
120tacgggaatg tgcgcacccc tcctgtcaat tacctccctg gtgaaataac cgacctcttg
180caactggccc gcatacccac tctcatggca tttgagcggg tgcccgaacc cgtgcctgcc
240tcagatacgt atgtgcccta cgttgccgtt cccacccaat tcgctgacag gcctctcatc
300tccttcccga tcaccctttc agaccctgtc tatcaaaaca ccctggttgg cgccatcagt
360tcttactttg ccaattaccg tgggtgtatc cagatcactc tgacgttttg tggacccatg
420atggcgagag ggaaattcct actgttatat tctcccccaa atggaacgca accacagact
480ctttccgaag ctatgcagtg cacatactct atttgggaca taggcttgaa ctcgagttgg
540accttcgtcg tcccctacat ctcgcccagt gactaccgtg aaacccgggc cattaccaac
600tcggtttact ccgctgatgg ttggtttagc ctgcacaagt tgaccaaaat tactctccca
660cctgactgtc cgcaaagtcc ctgcgttctc tttttcgctt ctgctggtga ggattacact
720ctccgtctcc ccattgattg taatccttcc tacgtgttcc actccaccga caacgccgaa
780actggggttg ttgaggcggg taacactgac accgatttct ctggtgaact agcggctcct
840ggctccaacc acactaatgt caagttcctg tttgatcgat ctcgattatt gaatgtaatt
900aaggtactgg agaaagacgc cgttttcccc caccctttcc ctacgctaga aggtgcgcaa
960caggatgatg gttacttttg tcttctgacc ccccgcccaa cagtcgcttc ccgacccgcc
1020actcgtttcg gcctgtacgc caatccgtcc ggcagcggtg ttcttgctaa tacttcattg
1080gactttaact tttatagctt ggcctgtttc acttacttta gatcggacct cgaggttacg
1140gtggtctcac tggagccaga tctggaattt gctgtagggt ggtttccttc cggcagtgaa
1200tatcaggctt ccagctttgt ctacgaccag ctgcacgtgc ccttccactt cactggacgc
1260accccccgcg cttttgctag caagggtggg aaggtatcct ttgtgctccc ttggaactct
1320gtctcatctg tgctccccgt gcgctggggg ggggcttcca agctctcttc tgccacgcgg
1380ggtctaccgg cgcatgctga ctgggggact atctacgcct ttgtcccccg tcccaatgaa
1440aagaaaagca ccgctgcaaa acacgtggcc gtgtacattc ggtacaagaa cgcacgcgcc
1500tggtgcccca gcatgcttcc ctttcgcagc tataagcaga agatgctgat gcaatctggc
15601721560DNAPicornavirus 172aatgggcttc gcaaccgcta cacgaccggg acggacgagg
aacaggggcc cattcccacg 60gcacccagag aacattcgct tatgtttctc tcaaccctcc
ctgacgacac tgtccctgct 120tacgggaatg tgcgtacccc tcctgtcaat tacctccctg
gtgaaataac cgaccttttg 180caactggccc gcatacccac tctcatggcg tttgagcggg
tgcctgaacc cgtgcctgcc 240tcagacacat atgtgcccta cgttgccgtt cccacccagt
ttgatgacaa gcctctcatc 300tccttcccga tcaccctttc agatcctgtc tatcagaaca
ccctggtagg cgccatcagt 360tcaaatttcg ccaactaccg tgggtgtatc caaatcactc
tgacattttg tggacccatg 420atggcaagag ggaaattcct actctcgtat tctcccccaa
atggaacgca accacagact 480ctttccgaag ctatgcagtg cacatactct atttgggaca
taggcttgaa ctctagttgg 540accttcgtcg tcccctacat ctcgcccagt gactaccgtg
aaactcgggc cattaccaac 600tcggtttact ccgctgatgg ttggtttagt ctgcacaagt
tgaccaaaat tactctacca 660cctgactgcc cgcaaagtcc ctgtattctc tttttcgctt
ctgctggtga ggattacacc 720ctccgtctcc ccgttgattg taatccttcc tatgtgttcc
actccaccga caacgccgag 780actggggtta ttgaggcggg taacactgac accgatttct
ctggtgaact ggcggctcct 840ggctctaacc acactaatgt caagttcctg tttgatcgat
ctcgattact gaatgtaatt 900aaggtactgg agaaggacgc cgttttcccc cgtcctttcc
ctacaaaaga aggtgcgcag 960caggacgatg gttacttttg tcttctgaca ccccgcccaa
cagtcgcctc ccgacccgcc 1020actcgtttcg gcctgtacgt caatccgtcc ggcagtggtg
ttctcgccaa cacttcactg 1080gacttcaatt tttatagctt ggcctgtttc acttacttta
gatcggacct tgaagttacg 1140gtggtctcac tggagccaga tctggaattt gctgtagggt
ggtttccttc tggcagtgaa 1200taccaggctt ccagctttgt ctacggccag ctgcatgtac
ccttccactt tactgggcgc 1260actccccgcg ctttcgccag caagggtggg aaggtatctt
tcgtgctccc ttggaactct 1320gtctcatctg tgctccccgt gcgctggggg ggcgcttcca
agctctcttc tgccacgcgg 1380ggtctaccgg cgcatgctga ctgggggact atttacgcct
ttgtcccccg tcctaatgag 1440aagaaaagca ccgctgtaaa gcacgtggcc gtgtacgttc
ggtacaagaa cgcacgtgcc 1500tggtgcccca gcatgcttcc ttttcgcagc tacaagcaga
agatgctgat gcaatctggc 1560173936DNAPicornavirus 173tttagcctgc
acaagttgac caaaattact ctaccacctg actgcccgca aaatccctgc 60attctctttt
tcgcttctgc tggtgaggat tacactctcc gtctccccat tgattgcaat 120ccttcctacg
tgttccactc caccgacaac gccgaaactg gggttgtcga ggcgggtaac 180actgacaccg
atttctctgg tgaactagcg gctcctggct ccaaccacac taatgtcaag 240ttcctgtttg
atcgatctcg gttattgaat gtaattaagg tactggagaa agacgctgtt 300ttccctcacc
ctttccctac gctagaaggt gtgcaacagg atgatggcta cttttgtctt 360ctgacccccc
gcccaacagt cgcttcccgg cctgccactc gtttcggcct gtacgccaat 420ccgtccggca
gcggtgttct tgctaatact tcattggact ttaactttta tagcttggcc 480tgtttcactt
actttagatc ggacctcgag gttacggtgg tctcactaga gccggatctg 540gaatttgctg
tggggtggtt tccttccggc agtgaatatc aggcttccag ctttgtctac 600gaccagctgc
acgtgccctt ccacttcact ggacgcaccc cccgcgcttt tgctagcaag 660ggtgggaagg
tatcctttgt gctcccttgg aactctgtct catctgtgct ccccgtgcgc 720tgggggggag
cttccaagct ctcttctgcc acgcggggtc taccggtgca cgctgactgg 780gggactatct
acgcctttgt cccccgtccc aatgaaaaga aaagcaccgc tgcaaaacat 840gtggccgtgt
acattcggta caagaacgca cgcgcctggt gtcccaacat gctccccttt 900cgcagctata
agcagaagat gctgatgcaa tctggc
936174936DNAPicornavirus 174tttagcctgc acaagttgac caaaataact ctaccacctg
actgcccgca aaatccctgc 60attctctttt tcgcttctgc tggtgaggat tacactctcc
gtctccccat tgattgcaat 120ccttcctacg tgttccactc caccgacaac gccgaaactg
gggttgtcga ggcgggtaac 180actgacaccg atttctctgg tgaactagcg gctcctggct
ccaaccacac taatgtcaag 240ttcctgtttg atcgatctcg gttattgaat gtaattaagg
tactggagaa agacgctgtt 300ttccctcacc ctttccctac gctagaaggt gtgcaacagg
atgatggcta cttttgtctt 360ctgacccccc gcccaacagt cgcttcccgg cctgccactc
gtttcggcct gtacgccaat 420ccgtccggca gcggtgttct tgctaatact tcattggact
ttaactttta tagcttggcc 480tgtttcactt actttagatc ggacctcgag gttacggtgg
tctcactaga gccggatctg 540gaatttgctg tggggtggtt tccttccggc agtgaatatc
aggcttccag ctttgtctac 600gaccagctgc acgtgccctt ccacttcact ggacgcaccc
cccgcgcttt tgctagcaag 660ggtgggaagg tatcctttgt gctcccttgg aactctgtct
catctgtgct ccccgtgcgc 720tgggggggag cttccaagct ctcttctgcc acgcggggtc
taccggtgca cgctgactgg 780gggactatct acgcctttgt cccccgtccc aatgaaaaga
aaagcaccgc tgcaaaacat 840gtggccgtgt acattcggta caagaacgca cgcgcctggt
gtcccaacat gctccccttt 900cgcagctata agcagaagat gctgatgcaa tctggc
936175936DNAPicornavirus 175tttagcctgc acaagttgac
caaaattact ytaccacctg actgtccgca aagtccctgc 60attctctttt tcgcttctgc
tggtgaggat tacactctcc gtctccccat tgattgtaat 120ccttcctacg tgttccactc
caccgacaac gccgaaactg gggttgttga ggcgggtaac 180actgacaccg atttctctgg
tgaactagcg gctcctggct ccaaccacac taatgtcaag 240ttcctgtttg atcgatctcg
attattgaat gtaattaagg tactggagaa agacgccgtt 300ttcccccacc ctttccctac
gctagaaggt gcgcaacagg atgatggtta cttttgtctt 360ctgacccccc gcccaacagt
cgcttcccga cccgccactc gtttcggcct gtacgccaat 420ccgtccggca gcggtgttct
tgctaatact tcattggact ttaactttta tagcttggcc 480tgcttcactt actttagatc
ggacctcgag gttacggtgg tctcactgga gccagatctg 540gaatttgctg tagggtggtt
tccttccggc agtgaatatc aggcttccag ctttgtctac 600gaccagctgc acgtgccctt
ccacttcact ggacgcaccc cccgcgcttt tgctagcaag 660ggtgggaagg tatcctttgt
gctcccttgg aactctgtct catctgtgct ccccgtgcgc 720tggggggggg cttccaagct
ctcttctgcc acgcggggtc taccggcgca tgctgactgg 780gggactatct acgcctttgt
cccccgtccc aatgaaaaga aaagcaccgc tgcaaaacac 840gtggccgtgt acattcggta
caagaacgca cgcgcctggt gccccagcat gcttcccttt 900cgcagctata agcagaagat
gctgatgcaa tctggc 936176936DNAPicornavirus
176tttagcctgc acaagttgac aaaaattact ctaccacctg actgtccgca aagtccctgc
60attctctttt tcgcttctgc tggtgaggat tacactctcc gtctccccgt tgattgtaat
120ccttcttacg tgttccactc caccgacaac gccgaaactg gggttgttga ggcgggtaac
180actgacaccg atttctctgg tgaactagcg gctcctggtt ccaaccacac taacgtcaag
240ttcctgtttg atcgatctcg attattgaat gtaattaagg tactggagaa agacgccgtt
300ttccctcacc ctttccctac gctagaaggt gcgcaacagg atgatggtta cttttgtctt
360ctgacccccc gcccaacagt cgcttcccga cccgccactc gtttcggcct gtacgccaat
420ccgtccggca gtggtgttct tgctaatact tcattggact ttaactttta tagcttggcc
480tgtttcactt actttagatc ggacctcgag gttacggtgg tctcactgga gccagatctg
540gaatttgctg taggatggtt tccttccggc agtgaatacc aggcttccag ctttgtctac
600gaccagctgc acgtgccttt ccacttcact gggcgcactc cccgcgcttt tgctagcaag
660ggtgggaagg tatccttcgt gctcccttgg aactctgttt cgtctgtgct ccccgtgcgc
720tggggggggg cttccaagct ctcttctgcc acgcggggtc taccggcgca tgctgactgg
780gggactatct acgcctttgt cccccgtccc aatgaaaaga aaagcaccgc tgcaaaacac
840gtggccgtgt acattcggta caagaacgca cgcgcctggt gccccagcat gcttcccttt
900cgcagctata agcagaagat gctgatgcaa tctggc
936177936DNAPicornavirus 177tttagcctgc acaagttgac caaaattact ctaccacctg
actgtccgca aagtccctgc 60attctctttt tcgcttctgc tggtgaggat tacactctcc
gtctccccgt tgattgtaat 120ccttcctatg tgttccactc caccgacaac gccgagactg
gggttattga ggcgggtaac 180actgacaccg atttctctgg tgaactggcg gctcctggct
ctaaccacac taatgtcaag 240ttcctgtttg atcgatctcg attattgaat gtaattaagg
tactggagaa ggacgccgtt 300ttcccccgcc ctttccctac acaagaaggt gcgcagcagg
acgatggtta cttttgtctt 360ctgacccccc gcccaacagt cgcttcccga cccgccactc
gtttcggcct gtacgccaat 420ccgtccggca gtggtgttct cgccaacact tcactggact
tcaattttta tagcttggcc 480tgtttcactt actttagatc ggaccttgag gttacggtgg
tctcactgga gccaaatctc 540gaatttgctg tagggtggtt tccttccggt agtgaatacc
aggcttccag ttttgtctac 600gaccagctgc acgtgccctt ccacttcact gggcgcactc
cccgcgcttt cgctagcaag 660ggtgggaagg tgtccttcgt gctcccttgg aactctgtct
cgtctgtgct ccccgtgcgc 720tggggggggg cttccaagct ctcttctgcc acacggggtc
taccagtgca tgctgactgg 780gggactattt acgcctttgt cccccgtccc aatgaaaaga
aaagcactgc tgtaaaacac 840gtggccgtgt acattcggta caagaacgca cgcgcctggt
gccccagcat gcttcctttt 900cgcagctaca agcagaagat gctgatgcaa tctggc
9361781421DNAArtificial SequenceConsensus Sequence
from SVV and SVV-like Picornaviruses in a coding region
sequence for VP2(partial), VP3, VP1, and 2A (partial) 178aatgggctcg
caaccgctac agccgggacg gacgagaaca ggggccattc cacggcaccc 60agagaattcg
cttatgtttc ttcaacctcc tgacgacacg tcctgcttac gggaatgtgc 120gacccctcct
gtcaattacc tccctggtga aataaccgac ctttgcaact ggcccgcata 180cccactctca
tggctttgag cgggtgccga acccgtgcct gcctcagaac tatgtgccct 240acgttgccgt
tcccacccat tgtgacagcc tctcatctcc ttcccgatca ccctttcaga 300ccgtctatca
aacaccctgg tggcgccatc agttcattgc caataccgtg ggtgtatcca 360atcactctga
cttttgtgga cccatgatgg cagagggaaa ttcctcttta ttctccccca 420aatggaacgc
aaccacagac tctttccgaa gctatgcagt gcacatactc tatttgggac 480ataggcttga
actcagttgg accttcgtcg tcccctacat ctcgcccagt gactaccgtg 540aaaccggcca
ttaccaactc ggtttactcc gctgatggtt ggtttagctg cacaagttga 600caaaatactt
ccacctgact gccgcaaatc cctgttctct ttttcgcttc tgctggtgag 660gattacacct
ccgtctcccc ttgattgaat ccttctagtg ttccactcca ccgacaacgc 720cgaacggggt
ttgaggcggg taacactgac accgatttct ctggtgaact gcggctcctg 780gtcaaccaca
ctaagtcaag ttcctgtttg atcgatctcg ttatgaatgt aataaggtac 840tggagaagac
gcgttttccc ccctttccct acagaaggtg gcacaggaga tggtactttt 900gtcttctgac
ccccgcccaa cagtcgctcc cgccgccact cgtttcggcc tgtacgcaat 960ccgtccggca
gggtgttctg caaacttcat ggacttaatt ttatagcttg gcctgttcac 1020ttactttaga
tcggacctga gttacggtgg tctcactgag ccatctgaat ttgctgtggt 1080ggtttccttc
ggagtgaata caggcttcca gtttgtctac gccagctgca gtccttccac 1140ttactggcgc
acccccgcgc tttgcagcaa gggtgggaag gttcttgtgc tcccttggaa 1200ctctgttctc
tgtgctcccc gtgcgctggg gggggcttcc aagctctctt ctgcaccggg 1260gtctaccggc
agctgatggg ggactattac gcctttgtcc cccgtccaat gaaagaaaag 1320cacgctgaaa
cagtggccgt gtacttcggt acaagaacgc acggcctggt gcccacatgc 1380tcctttcgca
gctaaagcag aagatgctga tgcaatctgg c
1421179551DNASeneca Valley Virus 179aaactgttta agatgcaagg acccatggac
aaagtcaaag actggaacca aatagctgcc 60ggcttgaaga attttcaatt tgttcgtgac
ctagtcaaag aggtggtcga ttggctgcag 120gcctggatca acaaagagaa agccagccct
gtcctccagt accagttgga gatgaagaag 180ctcgggcctg tggccttggc tcatgacgct
ttcatggctg gttccgggcc ccctcttagc 240gacgaccaga ttgaatacct ccagaacctc
aaatctcttg ccctaacact ggggaagact 300aatttggccc aaagtctcac cactatgatc
aatgccaaac aaagttcagc ccaacgagtt 360gaacccgttg tggtggtcct tagaggcaag
ccgggatgcg gcaagagctt ggcctctacg 420ttgattgccc aggctgtgtc caagcgcctc
tatggctccc aaagtgtata ttctcttccc 480ccagatccag atttcttcga tggatacaaa
ggacagttcg tgaccttgat ggatgatttg 540ggacaaaacc c
551180551DNAPicornavirus 180aaactgttta
agatgcaagt acccatggac aaagtcaaag actggaacca aatagccgcc 60ggcttgaaga
actttcaatt tgttcgtgac ctagtcaaag aggtggtcga ctggctgcag 120gcctggatca
acaaggagaa agccagccct gtcctccaat accagttgga gatgaagaag 180ctcgggcctg
tggctttggc tcatgacgct tttatggctg gttccgggcc ccctcttagc 240gacgaccaga
ttgagtatct ccagaacctc aaatctcttg ccctaacact agggaagact 300aatttggccc
aaagtctcac cactatgatc aatgccaaac aaagttccgc ccaacgagtt 360gaacccgttg
tggtggtcct tagaggtaag cctggatgtg gcaagagctt ggcctctacg 420ctgattgctc
aggctgtgtc caagcgcctc tatggctccc aaagtgtata ttccctcccc 480ccagacccag
atttctttga tggatacaaa ggacaattcg tgaccttgat ggatgatttg 540ggacaaaacc c
551181551DNAPicornavirus 181aaactgttta agatgcaagt acccatggac aaagtcaaag
actggaacca aatagccgcc 60ggcttgaaga attttcaatt tgttcgtgac ctagtcaaag
aggtggtcga ctggctgcag 120gcctggatca acaaagagaa agccagccct gtcctccagt
accagttgga gatgaagaag 180ctcgggcccg tggctttggc tcatgacgct ttcatggctg
gttccgggcc ccctcttagc 240gacgaccaga ttgaatacct ccagaacctc aaatctcttg
ccctaacact ggggaagact 300aatttggccc aaagtctcac cactatgatc aatgccaaac
aaagttccgc ccaacgagtt 360gaacccgttg tggtggtcct tagaggcaag ccgggatgcg
gcaagagctt ggcctccacg 420ttgattgccc aggctgtgtc caagcgtctc tatggctccc
aaagtgtgta ttctcttccc 480ccggatccag atttcttcga tggatacaaa ggacagtttg
tgaccttgat ggatgatttg 540ggacaaaacc c
551182523DNAArtificial SequenceConsensus Sequence
from SVV and SVV-like Picornaviruses in a partial coding region
sequence for 2C 182aaactgttta agatgcaaga cccatggaca aagtcaaaga ctggaaccaa
atagcgccgg 60cttgaagaat ttcaatttgt tcgtgaccta gtcaaagagg tggtcgatgg
ctgcaggcct 120ggatcaacaa gagaaagcca gccctgtcct ccataccagt tggagatgaa
gaagctcggg 180ccgtggcttg gctcatgacg ctttatggct ggttccgggc cccctcttag
cgacgaccag 240attgatactc cagaacctca aatctcttgc cctaacactg ggaagactaa
tttggcccaa 300agtctcacca ctatgatcaa tgccaaacaa agttcgccca acgagttgaa
cccgttgtgg 360tggtccttag aggaagccgg atgggcaaga gcttggcctc acgtgattgc
caggctgtgt 420ccaagcgctc tatggctccc aaagtgttat tcctcccccg accagatttc
ttgatggata 480caaaggacat tgtgaccttg atggatgatt tgggacaaaa ccc
523183460DNASeneca Valley Virus 183cttctggttg gcacggatta
cgatctggac ttcaatgagg tggcacgacg cgctgccaag 60ttggggtata agatgactcc
tgccaacaag ggttctgtct tccctccgac ttcctctctt 120tccgatgctg tttttctaaa
gcgcaaattc gtccaaaaca acgacggctt atacaaacca 180gttatggatt taaagaattt
ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc
tatgttggct caacattctg gaaaagaaga atatgataga 300ttgatgcacc ccttcgctga
ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg
acccagatag cccaaggcgc ttcggtgctg ccggcgattc 420tgggagaact cagtcggaac
agaaaaggga aaaaaaaaaa
460184460DNAPicornavirusmisc_feature(19)..(19)n is a, c, g, or t
184cttctggttg gcacggatna cnatctggac ttcaatgagg tggcgcggcg cgctgccaaa
60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt
120tccaatgctg tttttctaaa acgtaaattc gtccaaaaca atgacggctt gtacaagcca
180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc
240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atacgataga
300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga
360tggagggcct tgttcgattg acccagatag cccaaggcgc ttcggtgctg acggtgattc
420tgggagaact cagtcggaac aaaaagggga aaaaaaaaaa
460185460DNAPicornavirusmisc_feature(40)..(40)n is a, c, g, or t
185cttctggttg gcacggatta cgatctggac ttcaatgagn tggcgcggcg cgctgccaaa
60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt
120tccaatgctg tttttctaaa acgtaaattc gtccaaaaca atgacggctt gtacaagcca
180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc
240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atacgataga
300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga
360tggagggcct tgttcgattg acccaganag cccaaggcgc ttnggtgctg acggtgattc
420tgggagaact cagtcggaac aaaaagggga aaaaaaaaaa
460186460DNAPicornavirus 186cttctggttg gcacggatga cgatctggac ttcaatgagg
tggcgcggcg cgctgccaaa 60ttggggtata agatgacgcc tgccaacaag ggttccgtct
tccctccgac ttcctctctt 120tccgatgctg tttttctaaa acgcaaattc gtccaaaaca
acgacggctt gtacaaacca 180gttatggatt caaagaattt ggaagccatg ctctcctact
tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg
gaaaagaaga atatgataga 300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc
acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc
ttcggtgctg acggtgattc 420tgggagaact cagtcggaac agaaaagggg aaaaaaaaaa
460187460DNAPicornavirusmisc_feature(5)..(5)n is
a, c, g, or t 187cttcnggttg gcacggatga cgatctggac ttcaatgagg tggcgcggcg
cgctgccaaa 60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac
ttcctctctt 120tccgatgctg tttttctaaa acgcaaattc gtccaaaaca acgacggctt
gtacaaacca 180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg
aacactactc 240gagaagctgc aatctgtctc tatgttggct caacattctg gaaaagaaga
atatgataga 300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct
gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc ttcggtgctg
acggtgattc 420tgggagaact cagtcggaac agaaaaggga aaaaaaaaaa
460188460DNAPicornavirusmisc_feature(11)..(11)n is a, c, g,
or t 188cttctggttg ncacggatna cgatctgnac ttcaatgagg tggcgcggcg cgctgccaaa
60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt
120tccgatgctg tttttctaaa acgcaaattc gtccaaaaca acgacggctt gtacaaacca
180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc
240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atatgataga
300ctgatgcacc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga
360tggagggcct tgttcgactg acccagatag cccaaggcgc ctcggtgctg ccggtgattn
420tnggagaact cagtcggaac agaaaaggga gaaaaaaaaa
460189460DNAPicornavirus 189cttctggttg gcacggatta cgatctggac ttcaatgagg
tggcgcgacg cgctgccaaa 60ttggggtata agatgactcc tgccaacaag ggttccgtct
tcccttcgac ttcctctctt 120tccgacgctg tttttctaaa acgcaaattc gtccaaaaca
acgacggctt atacaaacca 180gttatggatt taaagaattt ggaagccatg ctctcctact
tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg
gaaaagaaga atatgataga 300ttgatgcacc ccttcgctga ctacggtgcc gtaccgagtc
acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc
ttcggtgctg ccggcgattc 420tgggagaact cagtcggaac agaaagggga aaaaaaaaaa
460190460DNAPicornavirus 190cttctggttg gtacggatta
cgatctggac ttcaatgagg tggcgcgacg cgctgccaag 60ctggggtata agatgactcc
tgccaacaag ggttccgtct tccctccgac ttcctctctc 120tccgatgctg ttttcctaaa
acgcaaattc gtccaaaaca acgacggctt atacaaacca 180gttatggatt taaagaattt
ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc
tatgttggct caacattctg gaaaagaaga atatgataga 300ttgatgcacc ccttcgctga
ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg
acccagatag cccaaggcgc ttcggtgctg ccggcgattc 420tgggagaact cagtcggaac
agaaaagggg aaaaaaaaaa 460191420DNAArtificial
SequenceConsensus Sequence from SVV and SVV-like Picornaviruses in a
partial coding region sequence for the 3D polymerase and the
3'UTR region 191cttcggttga cggatacatc tgacttcaat gagtggccgc gcgctgccaa
tggggtataa 60gatgaccctg ccaacaaggg ttcgtcttcc ctcgacttcc tctcttccag
ctgttttcta 120aacgaaattc gtccaaaaca agacggcttt acaaccagtt atggattaaa
gaatttggaa 180gccatgctct cctacttcaa accaggaaca ctactcgaga agctgcaatc
tgttctatgt 240tggctcaaca ttctggaaaa gaagaataga tagatgatgc acccttcgct
gactacggtg 300ccgtaccgag tcacgagtac ctgcaggcaa gatggagggc cttgttcgat
gacccagaag 360cccaaggcgc tggtgctgcg ggatttggag aactcagtcg gaacaaaagg
gaaaaaaaaa 4201924PRTPicornavirus 192Leu Gln Gly Asn 1
1934PRTPicornavirus 193Pro Gln Gly Asn 1
1944PRTPicornavirus 194Leu Lys Asp His 1
1954PRTPicornavirus 195Leu Ala Asp Gln 1
1964PRTPicornavirus 196Leu Leu Asp Gln 1
1974PRTPicornavirus 197Leu Met Asp Gln 1
1984PRTPicornavirus 198Leu Leu Asp Glu 1
1994PRTPicornavirus 199Arg Gln Ser Pro 1
2004PRTPicornavirus 200Ala Gln Ser Pro 1
2014PRTPicornavirus 201Pro Gln Ser Pro 1
2024PRTPicornavirus 202Pro Gln Gly Val 1
2034PRTPicornavirus 203Pro Gln Gly Ile 1
2044PRTPicornavirus 204Pro Gln Gly Ser 1
2054PRTPicornavirus 205Met Gln Ser Gly 1
2064PRTPicornavirus 206Leu Glu Ser Pro 1
2074PRTPicornavirus 207Leu Glu Asn Pro 1
2084PRTPicornavirus 208Leu Gln Asn Pro 1
2094PRTPicornavirus 209Gln Gln Ser Pro 1
2104PRTPicornavirus 210Pro Gln Gly Pro 1
2114PRTPicornavirus 211Ala Gln Gly Pro 1
2124PRTPicornavirus 212Ala Gln Ala Pro 1
2134PRTPicornavirus 213Glu Gln Gly Pro 1
2144PRTPicornavirus 214Glu Gln Ala Ala 1
2154PRTPicornavirus 215Ile Gln Gly Pro 1
2164PRTPicornavirus 216Val Gln Gly Pro 1
2174PRTPicornavirus 217Ile Gln Gly Gly 1
2184PRTPicornavirus 218Pro Gln Gly Ala 1 21920DNAArtificial
SequencePrimer sequence for Seneca Valley Virus 219tttgaaatgg ggggctgggc
2022019DNAArtificial
SequencePrimer Sequence for Seneca Valley Virus 220gaggagaccc gctaatccg
1922154DNAArtificial
SequencePrimer Sequence for generating an infectious plasmid clone
of Seneca Valley Virus 221tatgggtacc tgtaatacga ctcactatag
ggctttgaaa tggggggctg ggcc 5422224DNAArtificial SequencePrimer
sequence for generating an infectious plasmid clone of Seneca
Valley Virus 222ccgtcaaaga agcaattctg ggca
2422360DNAArtificial SequencePrimer sequence for generating
an infectious plasmid clone of Seneca Valley Virus
223gcatgcattt tttttttttt tttttttttt tttttttccc ttttctgttc cgactgagtt
6022430DNAArtificial SequencePrimer sequence for generating an infectious
clone of Seneca Valley Virus 224ggtaacatga ccttcaatta
ctacgcaaac 3022523DNAArtificial
SequencePrimer sequence for generating an infectious plasmid clone
of Seneca Valley Virus 225gatcagtacg tcgaaggccg ttg
2322676DNAArtificial SequencePrimer sequence
for generating an infectious plasmid clone of Seneca Valley
Virus 226gcttgcatgc atttaaattt tttttttttt tttttttttt ttttttttcc
cttttctgtt 60ccgactgagt tctccc
7622747DNAArtificial SequencePrimer sequence for generating
an infectious plasmid clone of Seneca Valley Virus
227caattgtgta atacgactca ctatagtttg aaatgggggg ctgggcc
47
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