Patent application title: RECOMBINANT HERPESVIRUS USEFUL IN VACCINE PRODUCTION
Inventors:
Mark D. Cochran (Carlsbad, CA, US)
Stephanie M. Cook (Omaha, NE, US)
Matha A. Wild (San Diego, CA, US)
IPC8 Class: AA61K39245FI
USPC Class:
4241991
Class name: Drug, bio-affecting and body treating compositions antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) recombinant virus encoding one or more heterologous proteins or fragments thereof
Publication date: 2010-01-14
Patent application number: 20100008948
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Patent application title: RECOMBINANT HERPESVIRUS USEFUL IN VACCINE PRODUCTION
Inventors:
Mark D. Cochran
Stephanie M. Cook
Matha A. Wild
Agents:
SCHERING-PLOUGH CORPORATION;PATENT DEPARTMENT (K-6-1, 1990)
Assignees:
Origin: KENILWORTH, NJ US
IPC8 Class: AA61K39245FI
USPC Class:
4241991
Patent application number: 20100008948
Abstract:
The present invention provides a novel avian herpesvirus (NAHV) vector and
recombinant vaccines made therefrom that are useful to immunize avian
species against Marek's disease, infectious laryngotracheitis and
Newcastle disease. Methods of immunizing an avian species against Marek's
disease, infectious laryngotracheitis and Newcastle disease are also
provided.Claims:
1. A recombinant avian herpesvirus comprising a herpes virus of turkeys
unique long and repeat viral genome region and a Marek's disease virus
unique short viral genome region wherein at least one foreign DNA
sequence is inserted within a US2 gene of the unique short region of the
recombinant avian herpesvirus and is capable of being expressed in a host
cell.
2. The recombinant avian herpesvirus of claim 1 wherein the foreign DNA sequence is selected from the group consisting of: a Newcastle disease virus fusion gene, an infectious laryngotracheitis virus glycoprotein D gene, or an infectious laryngotracheitis glycoprotein I gene.
3. The recombinant avian herpesvirus of claim 2 wherein the foreign DNA sequence is the Newcastle disease virus fusion gene.
4. The recombinant avian herpesvirus of claim 3 designated NAHV/NDV 295-93 (ATCC Accession number VR______).
5. The recombinant avian herpesvirus of claim 2 wherein the foreign DNA sequence is the infectious laryngotracheitis virus glycoprotein D gene.
6. The recombinant avian herpesvirus of claim 2 wherein the foreign DNA sequence is the infectious laryngotracheitis virus glycoprotein I gene.
7. The recombinant avian herpesvirus of claim 2 wherein the foreign DNA sequences are the infectious laryngotracheitis virus glycoprotein D gene and the infectious laryngotracheitis virus glycoprotein I gene.
8. The recombinant avian herpesvirus of claim 7 designated NAHV/ILT 295-149 (ATCC Accession number VR______).
9. The recombinant avian herpesvirus of claim 2 wherein the foreign DNA sequences are the Newcastle disease virus fusion gene, the infectious laryngotracheitis virus glycoprotein D gene and the infectious laryngotracheitis virus glycoprotein I gene.
10. The recombinant avian herpesvirus designated NAHV 295-01 (ATCC Accession number VR______).
11. A vaccine for protecting against Newcastle disease comprising an effective immunizing amount of the recombinant avian herpesvirus of claim 3 and a suitable carrier.
12. A vaccine for protecting against Newcastle disease comprising an effective immunizing amount of the recombinant avian herpesvirus of claim 4 and a suitable carrier.
13. A vaccine for protecting against infectious laryngotracheitis comprising an effective immunizing amount of the recombinant avian herpesvirus of claim 7 and a suitable carrier.
14. A vaccine for protecting against infectious laryngotracheitis comprising an effective immunizing amount of the recombinant avian herpesvirus of claim 8 and a suitable carrier.
15. A vaccine for protecting against Marek's disease comprising an effective immunizing amount of the recombinant avian herpesvirus of claim 10.
16. A multivalent vaccine for protecting against Marek's disease, infectious laryngotracheitis and Newcastle disease comprising an effective immunizing amount of the recombinant avian herpesvirus of claim 9 and a suitable carrier.
17. A multivalent vaccine for protecting against Marek's disease, infectious laryngotracheitis and Newcastle disease comprising, as a mixture, an effective immunizing amount of a first recombinant avian herpesvirus, designated NAHV/ILT 295-149 (ATCC Accession number VR______), an effective immunizing amount of a second recombinant avian herpesvirus, designated NAHV/NDV 295-93 (ATCC Accession number VR______), and a suitable carrier.
18. A method of immunizing an avian species against Newcastle disease comprising administering to the avian species an effective immunizing amount of the vaccine of claim 11.
19. A method of immunizing an avian species against Newcastle disease comprising administering to the avian species an effective immunizing amount of the vaccine of claim 12.
20. A method of immunizing an avian species against infectious laryngotracheitis comprising administering to the avian species an effective immunizing amount of the vaccine of claim 13.
21. A method of immunizing an avian species against infectious laryngotracheitis comprising administering to the avian species an effective immunizing amount of the vaccine of claim 14.
22. A method of immunizing an avian species against Marek's disease comprising administering to the avian species an effective immunizing amount of the vaccine of vaccine of claim 15.
23. A method of immunizing an avian species against Marek's disease, infectious laryngotracheitis and Newcastle disease comprising administering to the avian species an effective immunizing amount of the vaccine of claim 16.
24. The vaccine as in claim 11 wherein the suitable carrier is a physiologically balanced culture medium containing stabilizing agents.
25. The vaccine as in claim 13 wherein the suitable carrier is a physiologically balanced culture medium containing stabilizing agents.
26. The vaccine as in claim 15 wherein the suitable carrier is a physiologically balanced culture medium containing stabilizing agents.
27. The vaccine as in claim 16 wherein the suitable carrier is a physiologically balanced culture medium containing stabilizing agents.
28. The method of claim 18, wherein the vaccine is administered by injection.
29. The method of claim 20, wherein the vaccine is administered by injection.
30. The method of claim 22, wherein the vaccine is administered by injection.
31. The method of claim 23, wherein the vaccine is administered by injection.
32. The method of claim 18, wherein the vaccine is administered intraocularly.
33. The method of claim 20, wherein the vaccine is administered intraocularly.
34. The method of claim 22, wherein the vaccine is administered intraocularly.
35. The method of claim 23, wherein the vaccine is administered intraocularly.
36. The method of claim 18, wherein the vaccine is administered orally.
37. The method of claim 20, wherein the vaccine is administered orally.
38. The method of claim 22, wherein the vaccine is administered orally.
39. The method of claim 23, wherein the vaccine is administered orally.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of copending application U.S. Ser. No. 11/949,377 filed Dec. 3, 2007; which is a continuation of U.S. Ser. No. 11/126,465 filed May 11, 2005, now U.S. Pat. No. 7,314,715 granted Jan. 4, 2008; which is a continuation of U.S. Ser. No. 09/881,457, filed Jun. 14, 2001, now U.S. Pat. No. 6,913,751 granted Jul. 5, 2005, and claims priority under 35 U.S.C. § 120, the contents of which are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002]The present invention relates to recombinant herpesviruses and, more particularly to a novel avian herpesvirus (NAHV) suitable for use as a viral vector for vaccination of birds against disease.
BACKGROUND OF THE INVENTION
[0003]The ability to isolate DNA and clone such isolated DNA into bacterial plasmids has greatly expanded the approaches available to make viral vaccines. The methods used to make the present invention involve modifying cloned DNA sequences from various viral pathogens of animals, by insertions, deletions, single or multiple base changes, and subsequent insertions of these modified sequences into the genome of the virus. One utility of the addition of a foreign sequence is achieved when the foreign sequence encodes a foreign protein that is expressed during viral infection of the animal. The resulting live virus may then be used in a vaccine to elicit an immune response in a host animal and provide protection to the animal against disease. A virus with these characteristics is referred to as a viral vector, because it becomes a living vector that will carry and express the foreign protein in the host animal. In effect it becomes an elaborate delivery system for the foreign protein(s).
[0004]The application of recombinant DNA techniques to animal viruses in general has a recent history. The first viruses to be engineered have been those with the smallest genomes. For example, in the case of the papovaviruses, because these viruses are so small and cannot accommodate much extra DNA, their use in genetic engineering has been as defective replicons. Thus, foreign DNA sequence expression from these viruses requires a wild-type helper virus and is limited to cell culture systems. On the other hand, for adenoviruses, there is a small amount of nonessential DNA that can be replaced by foreign sequences limiting its utility as a vector.
[0005]Another group of viruses that have been engineered are the poxviruses. One member of this group, vaccinia, has been the subject of much research on foreign gene expression. Poxviruses are large DNA-containing viruses that replicate in the cytoplasm of the infected cell. They have a structure that is unique in that they do not contain any capsid that is based upon icosahedral symmetry or helical symmetry. The poxviruses are most likely to have evolved from bacterial-like microorganisms through the loss of function and degeneration. In part due to this uniqueness, the advances made in the genetic engineering of poxviruses cannot be directly extrapolated to other viral systems, including the avian herpesviruses. The utility of vaccinia as a vaccine vector is in question because of its close relationship to human smallpox and its known pathogenicity to humans. Thus, the use of host-specific avian herpesviruses is a preferred solution to vaccination of poultry. Viral vectoring techniques have been applied to the genomes of several avian herpesviruses (e.g. U.S. Pat. No. 6,121,043, U.S. Pat. No. 5,965,138, and WO061736A2).
[0006]Marek's disease virus (MDV) is the causative agent of Marek's disease, which encompasses fowl paralysis, a common lymphoproliferative disease of chickens. MDV, a naturally occurring herpesvirus, infects bursa-derived and thymus-derived lymphocytes in chickens, and may subsequently induce a lymphoma of thymus-derived lymphocytes. MDV is a designation of a family of avian herpesviruses. For example, MDV (MDV1) is a virulent strain of herpesvirus in chickens, SB-1 (MDV2) is a naturally attenuated herpesvirus strain in chickens, and HVT (MDV3) is a nonpathogenic herpesvirus of turkey.
[0007]Since Marek's disease is contagious, the virus has become an important pathogen of chickens, particularly in an environment of large scale breeding such as in the poultry industry. The disease occurs most commonly in young chickens between 2 and 5 months of age. The prominent clinical signs are progressive paralysis of one or more of the extremities, incoordination due to paralysis of legs, drooping of the limb due to wing involvement, and a lowered head position due to involvement of the neck muscles. In acute cases, severe depression may result. In the case of highly oncogenic strains, there is characteristic bursal and thymic atrophy. In addition, there are lymphoid tumors affecting the gonads, lungs, liver, spleen, kidney and thymus (Mohanty and Dutta, Veterinary Virology, Lea and Febiger, pubs., Philadelphia, 1981).
[0008]Currently, Marek's disease is controlled by vaccination of embryos at 17-19 days of incubation, or one day old chicks. The principal vaccination method for MDV involves using naturally occurring strains of turkey herpesvirus (HVT) or conventionally attenuated Marek's disease virus (MDV). It would be advantageous to incorporate other antigens into this vaccination, but efforts to combine conventional vaccines have not proven satisfactory due to competition and immunosuppression between pathogens. The multivalent NAHV based vaccines engineered in this invention represent a novel way to simultaneously vaccinate against a number of different pathogens.
[0009]A foreign gene of interest targeted for insertion into the genome of NAHV may be obtained from any pathogenic organism of interest. Typically, the gene of interest will be derived from pathogens that in poultry cause diseases that have an economic impact on the poultry industry. The genes may be derived from organisms for which there are existing vaccines, and because of the novel advantages of the vectoring technology, the NAHV derived vaccines will be superior. In addition, the gene of interest may be derived from pathogens for which there is currently no vaccine but where there is a requirement for control of the disease. Typically, the gene of interest encodes immunogenic polypeptides of the pathogen, and may represent surface proteins, secreted proteins and structural proteins.
[0010]An avian pathogen that is a target for NAHV vectoring is infectious laryngotracheitis virus (ILTV). ILTV is a member of the herpesviridiae family, and this pathogen causes an acute disease of chickens, which is characterized by respiratory depression, gasping, and expectoration of bloody exudate. Viral replication is limited to cells of the respiratory tract, where in the trachea the infection gives rise to tissue erosion and hemorrhage. In chickens, no drug has been effective in reducing the degree of lesion formation or in decreasing clinical signs. Vaccination of birds with various modified forms of the ILTV derived by cell passage and/or tedious regimes of administration have conferred acceptable protection in susceptible chickens. Because of the degree of attenuation of current ILT vaccines care must be taken to assure that the correct level of virus is maintained; enough to provide protection, but not enough to cause disease in the flock.
[0011]An additional target for the NAHV vectoring approach is Newcastle disease, an infectious, highly contagious and debilitating disease that is caused by the Newcastle disease virus (NDV). NDV is a single-stranded RNA virus of the paramyxovirus family. The various pathotypes of NDV (velogenic, mesogenic, lentogenic) differ with regard to the severity of the disease, the specificity and symptoms, but most types seem to infect the respiratory system and the nervous system. NDV primarily infects chickens, turkeys and other avian species. Historically vaccination has been used to prevent disease, but because of maternal antibody interferences, life-span of the bird and route of administration, the producer needs to adapt immunization protocols to fit specific needs.
SUMMARY OF THE INVENTION
[0012]The present invention is directed to a recombinant avian herpesvirus comprising a herpes virus of turkeys unique long and repeat viral genome region and a Marek's disease virus unique short viral genome region wherein at least one foreign DNA sequence is inserted within the US2 gene of the unique short region of the recombinant avian herpesvirus and wherein the foreign DNA sequence is capable of being expressed in a host cell. In a preferred embodiment, the foreign DNA sequence is selected from the group consisting of a Newcastle disease virus fusion gene, an infectious laryngotracheitis virus glycoprotein D gene, an infectious laryngotracheitis glycoprotein I gene, or combinations thereof.
[0013]In another embodiment, the present invention is directed to a vaccine against Marek's disease, Newcastle disease, and/or infectious laryngotracheitis. The vaccine comprises a recombinant avian herpesvirus comprising a herpes virus of turkeys unique long and repeat viral genome region and a Marek's disease virus unique short viral genome region wherein at least one foreign DNA sequence is inserted within the US2 gene of the unique short region of the recombinant avian herpesvirus and wherein the foreign DNA sequence is capable of being expressed in a host cell, and a suitable carrier. Preferably the foreign DNA sequence is selected from the group consisting of a Newcastle disease virus fusion gene, an infectious laryngotracheitis virus glycoprotein D gene, an infectious laryngotracheitis glycoprotein I gene, or combinations thereof.
[0014]The present invention is also directed to a method of immunizing an avian species against Marek's disease, Newcastle disease, and/or infectious laryngotracheitis by administering a vaccine of the present invention.
BRIEF DESCRIPTION OF FIGURES
[0015]FIG. 1 is a comparison of HVT, NAHV, and MDV BamHI endonuclease restriction enzyme maps. Restriction fragments are labeled alphabetically in decreasing order of size. The structure of each virus is indicated below the map. Repeats regions are shown as boxes (open=HVT derived, shaded=MDV derived) and the unique regions are shown as lines (single=HVT derived, double=MDV derived). TRL=terminal repeat long; IRL=internal repeat long; IRS=internal repeat short; TRS=terminal repeat short, UL=unique long region; US=unique short region
[0016]FIG. 2 is a BamHI endonuclease restriction enzyme map of NAHV and the positions of subgenomic clones used in the NAHV construction. Restriction fragments are label alphabetically in decreasing order of size. In the NAHV genome, the fragment corresponding to HVT fragment B is denoted as fragment A' and the fragment corresponding to MDV fragment A is denoted as fragment B'.
DETAILED DESCRIPTION OF THE INVENTION
[0017]The present invention is directed to a recombinant novel avian herpesvirus virus (NAHV) optionally comprising a foreign DNA sequence inserted into a non-essential site in the NAHV genome. The foreign DNA sequence is capable of being expressed in a host cell infected with the recombinant NAHV and its expression is under the control of a promoter located upstream of the foreign DNA sequence. The foreign DNA sequence encodes a polypeptide, which is antigenic in an animal into which the recombinant NAHV is introduced. More particularly, the foreign DNA sequence is from Newcastle disease virus (NDV) or infectious laryngotracheitis virus (ILTV) and the non-essential site in the NAHV genome is the US2 gene.
[0018]We have created recombinant organisms consisting of the unique long (UL) and repeat regions of the herpesvirus of turkeys (HVT) and the unique short (US) region of Marek's disease virus (MDV). The genome structure of these recombinant organisms and their parental viruses are compared in FIG. 1. Since these organisms are distinctly different from both of their parent organisms, they represent a completely new type of organism, a novel avian herpesvirus (NAHV).
[0019]These NAHV provide for highly efficacious and safe vaccines that protect poultry from Marek's disease. They combine the strong protective response provided by antigens from their Marek's disease virus parent with the established safety of their herpesvirus of turkeys parent. The NAHV-based vaccines exhibit increased protection against very virulent strains of MDV relative to HVT-based vaccines. However the NAHV-based vaccines retain the same non-pathogenic non-oncogenic safety profile of HVT.
[0020]The NAHV may also be used to create multivalent vaccines against Marek's disease, infectious laryngotracheitis, infectious bursal disease, Newcastle disease, or other poultry diseases. Multivalent viral vaccine strains are created by genetically engineering the NAHV to express antigens from the appropriate disease-causing organism. Several examples of NAHV-based vaccines are described below (examples 1-3).
[0021]As defined herein "a non-essential site in the NAHV genome" means a region in the NAHV viral genome, which is not necessary for the viral infection or replication. A "viral genome" or "genomic DNA" means the entire DNA, which the naturally occurring herpesvirus contains. As defined herein, "foreign DNA sequence" or "gene" means any DNA or gene that is exogenous to the genomic DNA. An "open reading frame" is a segment of DNA, which contains codons that can be transcribed into RNA which can be translated into an amino acid sequence and which does not contain a termination codon.
[0022]An "immunological composition" of the invention, as used herein, refers to any composition that elicits an immune response in an animal. An immune response is the reaction of the body to foreign substances, without implying a physiologic or pathologic consequence of such a reaction, i.e., without necessarily conferring protective immunity on the animal. An immune response may include one or more of the following: (a) a cell mediated immune response, which involves the production of lymphocytes by the thymus (T cells) in response to exposure to the antigen; and/or (b) a humoral immune response, which involves production of plasma lymphocytes (B cells) in response to antigen exposure with subsequent antibody production. The term "vaccine", as used herein, broadly refers to any compositions that may be administered to an animal to protect the animal against an infectious disease.
[0023]The invention further provides a recombinant NAHV suitable for use as a vaccine against Marek's disease. One example of such a virus is designated NAHV 295-01. This virus is also known as S-HVY-165. The recombinant avian herpesvirus designated NAHV 295-01 is a superior virus vaccine strain against very virulent Marek's disease, in chickens and turkeys, providing the safety of avirulent HVT, with the improved antigenicity of added MDV genes. The NAHV 295-01 recombinant virus vaccine is a superior virus vaccine because a single virus vaccine strain will protect against very virulent MDV. Currently the industry relies on combinations of vaccine strains. Since the NAHV 295-01 virus vaccine strain is genetically defined, it provides superior safety compared to conventional vaccine strains that risk reversion to virulence.
[0024]The present invention also provides a recombinant NAHV suitable for use as a vaccine containing a foreign DNA sequence encoding an antigenic polypeptide from NDV. In such case, it is preferred that the antigenic polypeptide is NDV fusion (F) protein. One example of such a virus is designated NAHV/NDV 295-93. This virus is also known as S-HVY-177. The recombinant avian herpesvirus designated NAHV/NDV 295-93 is a multivalent virus vaccine strain against Newcastle disease and very virulent Marek's disease in chickens. It contains a foreign gene encoding the fusion protein of the Newcastle disease virus inserted into the MDV US2 gene of the NAHV.
[0025]This recombinant virus vaccine has multiple advantages over conventional vaccines. The NAHV/NDV 295-93 vaccine can be administered in ovo without the interference often seen when conventional MDV and NDV vaccines are used. Since the vaccine lacks any NDV virulence genes there is no possibility of reversion to virulence or vaccine induced Newcastle disease. Additionally, the cell-associated nature of the NAHV backbone provides protection from NDV maternal antibody interference. The NAHV/NDV 295-93 recombinant virus vaccine is a superior Marek's disease virus vaccine because a single virus vaccine strain will protect against very virulent MDV. Currently the industry relies on combinations of vaccine strains. Since the NAHV/NDV 295-93 virus vaccine strain is genetically defined, it provides superior safety compared to conventional Marek's vaccine strains that risk reversion to virulence.
[0026]The invention further provides recombinant NAHV containing foreign DNA sequence encodes the antigenic polypeptide from an ILTV and encodes ILTV glycoprotein I and/or ILTV glycoprotein D. One example of such a virus is designated NAHV/ILT 295-149. This virus is also known as S-HVY-176.
[0027]The recombinant avian herpesvirus designated NAHV/ILT 295-149 is a multivalent virus vaccine strain against infectious laryngotracheitis and very virulent Marek's disease in chickens. It contains two foreign genes encoding glycoprotein D and glycoprotein I of the infectious laryngotracheitis virus inserted into the MDV US2 gene of the NAHV. This recombinant virus vaccine has multiple advantages over conventional vaccines. The NAHV/ILT 295-149 vaccine can be administered in ovo providing increased efficiency. Since the vaccine lacks any ILTV virulence genes there is no possibility of reversion to virulence or vaccine induced laryngotracheitis. The NAHV/ILT 295-149 recombinant virus vaccine is a superior Marek's disease virus vaccine because a single virus vaccine strain will protect against very virulent MDV. Currently the industry relies on combinations of vaccine strains. Since the NAHV/ILT 295-149 virus vaccine strain is genetically defined, it provides superior safety compared to conventional Marek's vaccine strains that risk reversion to virulence.
[0028]The novel recombinant avian herpesviruses of the present invention may be used as vaccines or immunological compositions against avian diseases which comprise an effective immunizing amount of a recombinant NAHV of the present invention and a suitable carrier. This invention provides a vaccine useful for immunizing an avian species against Marek's disease, which comprises an effective immunizing amount of the recombinant NAHV, and a suitable carrier.
[0029]This invention provides a vaccine useful for immunizing an avian species against Newcastle disease, which comprises an effective immunizing amount of the recombinant NAHV, and a suitable carrier.
[0030]This invention provides a vaccine useful for immunizing an avian species against infectious laryngotracheitis, which comprises an effective immunizing amount of the recombinant NAHV, and a suitable carrier.
[0031]This invention provides a multivalent vaccine useful for immunizing an avian species against Marek's disease and Newcastle disease, which comprises an effective immunizing amount of the recombinant NAHV and a suitable carrier.
[0032]This invention provides a multivalent vaccine useful for immunizing an avian species against Marek's disease and infectious laryngotracheitis, which comprises an effective immunizing amount of the recombinant NAHV and a suitable carrier.
[0033]Vaccines of the invention may be combined with other vaccines for other diseases to produce multivalent vaccines. For example, the present invention includes, a multivalent vaccine useful for immunizing an avian species against Marek's disease, Newcastle disease and infectious laryngotracheitis, which comprises a mixture of a first recombinant NAHV, a second recombinant NAHV, and a suitable carrier. One example of such a mixture comprises a first recombinant avian herpesvirus designated NAHV/ILT 295-149, a second recombinant avian herpesvirus designated NAHV/NDV 295-93, and a suitable carrier.
[0034]This invention provides an immunological composition which comprises at least one recombinant NAHV and a suitable carrier that elicits an immune response in a host avian species. The immune response can be local or systemic. The immune response can be protective or not be protective.
[0035]This invention provide recombinant NAHV, which express foreign DNA, sequences and are useful as vaccines in avian species including but not limited to chickens, turkeys, and ducks. These vaccines may contain either inactivated or live recombinant virus. These vaccines may contain infected cells. The vaccines of the present invention are administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneous, intraperitoneal, or intravenous injection. The vaccine can be administered in ovo. Additional methods for administration of the vaccine well known to those skilled in the art are, for example, intranasally, intraocularly or orally.
[0036]For purposes of this invention, the term an "effective immunizing amount" refers to the amount of a substance that is sufficient to produce or elicit an immune response. For the present invention, an "effective immunizing amount" of the recombinant NAHV within the range of 102 to 109 PFU/dose. In another embodiment the immunizing amount is 105 to 107 PFU/dose. In a preferred embodiment the immunizing amount is approximately 2000 PFU/dose.
[0037]This invention provides methods for vaccination of avian species against disease. The method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention. This invention provides a method for vaccination of an avian species against Marek's disease. It provides a method for vaccination of an avian species Newcastle disease. The present invention provides a method for vaccination of an avian species against infectious laryngotracheitis.
[0038]This invention provides methods for the vaccination of an avian species against more than one disease. The diseases can be caused by more than one pathogen. A method is provided for the vaccination of an avian species against Marek's disease and Newcastle disease. The present invention provides a method for vaccination of an avian species against Marek's disease and infectious laryngotracheitis. It also provides a method for vaccination of an avian species against Marek's disease, Newcastle disease and infectious laryngotracheitis.
[0039]The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which a compound is administered. Suitable carriers for the recombinant virus are well known to those skilled in the art and include but are not limited to sterile water, aqueous saline solutions, aqueous dextrose or glycerol solutions, proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as dimethyl sulfoxide, hydrolyzed proteins, lactose, etc.
[0040]This invention is further illustrated in the Methods and Examples sections, which follow. These sections are set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.
Methods
[0041]Methods for constructing, selecting and purifying recombinant novel avian herpesviruses are detailed below in the materials, methods and examples. The following serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
Preparation of NAHV Vaccine and Viral Stocks
[0042]Novel avian herpesvirus stock samples were prepared by infecting tissue culture cells at a multiplicity of infection of approximately 0.01 PFU/cell in complete F10/199 medium. All incubations were carried out at 39° C. in a humidified incubator with 5% CO2 in air. This medium is composed of a 1:1 mixture of Medium 199 and Nutrient Mixture F10 Ham, 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, 1×MEM non-essential amino acids (these components are obtained from BioWhittaker or an equivalent supplier) plus 1% fetal bovine serum. After cytopathic effect was complete, the medium and cells were harvested and the cells were pelleted at 3000 rpm for 5 minutes in a clinical centrifuge. Infected cells were resuspended in complete medium containing 20% fetal bovine serum, 7% dimethyl sulfoxide (DMSO) and stored frozen at -70° C. For long term storage and shipping virus stocks were stored under liquid nitrogen or in the vapor phase of liquid nitrogen.
Preparation of Poultry Herpesvirus DNA
[0043]For the preparation of HVT, MDV and NAHV genomic DNA from the cytoplasm of infected cells, primary chicken embryo fibroblasts were infected at a MOI sufficient to cause extensive cytopathic effect before the cells overgrew. All incubations were carried out at 39° C. in a humidified incubator with 5% CO2 in air. Best DNA yields were obtained by harvesting monolayers maximally infected, but showing incomplete cell lysis (typically 5-7 days). Infected cells were harvested by scraping the cells into the medium using a cell scraper (Costar brand). The cell suspension was centrifuged at 3000 rpm for 10 minutes at 5° C. in a GS-3 rotor (Sorvall Instruments). The resultant pellet was resuspended in cold PBS (Dulbecco's Phosphate Buffered Saline, 10 ml/225 cm2 flask) and subjected to another centrifugation for 10 minutes at 3000 rpm in the cold. After decanting the PBS, the cellular pellet was resuspended in 2 ml/flask of cold RSB buffer (10 mM Tris pH 7.5, 1 mM ethylene diamine tetraacetic acid, disodium salt (EDTA), and 1.5 mM MgCl2). One hundred μl of 20% SDS (5 mM final) was added and mixed by rocking. The sample was poured into a 15 ml conical tube. Twenty-four μl of Proteinase K was added and mixed by rocking, then incubated at 50° C. for ≧1 hr. After this period, an equal volume of water-saturated phenol was added to the sample and gently mixed by hand. The sample was spun in a clinical centrifuge for 5 minutes at 3000 rpm to separate the phases. The aqueous layer containing the DNA was transferred to a fresh 15 ml conical tube and the phenol extraction repeated a second time. The aqueous layer containing the DNA was then transferred to a 15 ml corex tube and precipitated by adding one-tenth volume of 3 M sodium acetate (NaAC) and 2.5 volumes of cold 100% ethanol (EtOH). The DNA was pelleted by centrifugation (Sorval, HB4 swinging bucket rotor, 10,000 rpm, 20-30 minutes, 4° C.). The DNA pellet was washed with 80% EtOH and centrifuged again for 10 minutes. The pellet was air dried and resuspended in 300 μl TE. All viral DNA was stored at approximately 4° C.
Molecular Biological Techniques
[0044]Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels, ligation, phosphorylation with kinase, treatment with phosphatase, growth of bacterial cultures, transformation of bacteria with DNA, and other molecular biological methods are described by Maniatis et al (Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1982) and Sambrook et al (Molecular Cloning A Laboratory Manual Second Edition, Cold Spring Harbor Press, 1989). The polymerase chain reaction (PCR) was used to introduce restriction sites convenient for the manipulation of various DNAs. The procedures used are described by Innis et al (PCR Protocols A Guide to Methods and Applications, 84-91, Academic Press, Inc., San Diego, 1990). In general, amplified fragments were less than 500 base pairs in size and critical regions of amplified fragments were confirmed by DNA sequencing. Except as noted these techniques were used with minor variation.
DNA Sequencing
[0045]DNA sequencing was performed on the Applied Biosystems Automated Sequencer Model 373A (with XL upgrade) per instructions of the manufacturer. Subclones were made to facilitate sequencing. Internal primers were synthesized on an ABI 392 DNA synthesizer or obtained commercially (Genosys Biotechnologies, Inc., The Woodlands, Tex.). Larger DNA sequences were built utilizing consecutive overlapping primers. Assembly, manipulation and comparison of sequences were performed with DNASTAR programs.
Procedure for Cloning NAHV Subgenomic DNA Fragments
[0046]A library of subclones containing overlapping HVT subgenomic fragments was generated as follows. DNA was obtained from the FC-126 strain of HVT (American Type Culture Collection). It was sheared and then size selected on a glycerol gradient as described by van Zijl et al., (Journal of Virology 62, 2191-2195, 1988) with 40-50 kb fragments chosen as the insert population. The pooled fractions were diluted twofold with TE (10 mM Tris pH 7.5, 1 mM EDTA), one-tenth volume of 3M NaAc and 2.5 volumes of ethanol were added, and the DNA was precipitated at 30K rpm in a Beckman SW41 rotor for 1 hr. The sheared fragments were given blunt ends by initial treatment with T4 DNA polymerase, using low dNTP concentrations to promote 3' overhang removal, followed by treatment with Klenow polymerase to fill in recessed 3' ends. These insert fragments were then ligated to a pWE15 (Strategene) cosmid vector, which had been digested with BamHI, treated with calf intestinal phosphatase, and made blunt by treatment with Klenow polymerase. The ligated mixture was then packaged using Gigapack XL packaging extracts (Stratagene). Ligation and packaging was as recommended by the manufacturer.
[0047]Published restriction maps for the enzymes BamHI, HindIII, and XhoI permitted the use of subcloned fragments as specific probes to screen the cosmid library for subclones spanning the genome. Probes were generated from subcloned restriction fragments. The fragments were then labeled using a non-radioactive system (Genius, Boehringer Mannheim). Screening was facilitated by picking colonies into media, followed by growth overnight. Sets of five filters and a master plate were stamped from microtiter dish and again grown overnight. Glycerol was added to the wells to 15% and the plates were frozen at -20° C. to provide stock cultures of each colony. Filters were BioRad Colony Lift Membranes and were treated and hybridized per manufacturer's instructions, and washed in 0.1×SSC, 0.1% SDS, 65° C. Positive clones, which hybridized with the non-radioactive probe, were detected according to the Genius kit directions.
[0048]Colonies were selected for further analysis on the basis of their hybridization to two or more of the specific probes. These were then digested with BamHI, and compared to published maps of HVT (Buckmaster et al., J. Gen. Virol. 69:2033, 1988). The three cosmids (407-32.2C3, 407-32.1C1, and 407-32.5G6) were obtained in this manner. A detailed description of each clone is given below. It was found that chloramphenicol amplification (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, New York, 1982) was necessary to achieve reasonable yields of DNA from these clones. In addition, one cosmid clone (407-32.5G6) was unstable and had to be grown from the original frozen stock in order to obtain satisfactory DNA preparations.
[0049]The pWE15 vector allows the inserts to be excised with NotI. However, four NotI sites are present in the HVT genome, so that inserts spanning these sites cannot be excised with NotI. Two of the NotI sites are present in the BamHI B fragment of HVT, this fragment was cloned directly in pSP64 (clone 172-07.BA2). The other two sites are present in the unique short region within the BamHI A fragment. This fragment was cloned directly in the pWE15 vector. The three sheared cosmids and the two BamHI fragments cover all but a small portion of the ends of the HVT genome. Because these regions are repeated in the internal portions of the genome, all of the genetic information is available.
[0050]Marek's Disease Virus (MDV), GA strain, was obtained from the USDA (Agricultural Research Service Regional Poultry Laboratory, East Lansing, Mich.). In order to clone the short region, a partial SmaI digest of the DNA was performed and run out on a 0.6% low melt agarose gel. DNA fragments running greater than 24 kb were chosen as the insert population and excised from the gel. The DNA contained within the gel slice was extracted by using warm phenol, centrifugation, and then the aqueous phase was precipitated with one-tenth volume of 3M NaAc, an equal volume of isopropanol and centrifugation at 30K rpm in a Beckman SW41 rotor for 15 minutes. The pelleted DNA was then rinsed with 80% EtOH, air dried and resuspended in H2O, Since the SmaI enzyme leaves blunt ends on the isolated fragments, a blunt end pWE15 cosmid vector was prepared as above. The ligated mixture was then packaged using Gigapack Plus packaging extracts (Stratagene). Ligation and packaging was as recommended by the manufacturer. Colonies were selected for further analysis on the basis of their hybridization to a MDV gD specific probe, and comparison to the published restriction digestion maps.
Procedure for Generating Novel Avian Herpesvirus from Overlapping Subgenomic Fragments
[0051]Overlapping subgenomic fragments were cotransfected into chicken embryo fibroblast (CEF) cells by calcium phosphate precipitation, resulting in the regeneration of the NAHV genome. The regeneration was mediated by homologous recombination across the overlapping regions of the fragments. First, cosmid and plasmid DNAs were linearized using an appropriate enzyme, and purified by phenol extraction and ethanol precipitation. Then, approximately one microgram of each linear fragment was combined in H2O (300 μl final volume) and 37 μl of a 2.5M CaCl2 stock was added. Next, 340 μl of a 2× Hepes buffered saline solution (140 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), pH 7.05) was added and gently mixed for one minute at room temperature, to allow a fine precipitate to form. The precipitated DNA was added to a subconfluent monolayer of secondary CEF cells, plate in a 6 cm dish and allowed to absorb to the cells for approximately three hours at 39° C. Media was removed and cells were exposed to a 15% (v/v) Glycerol/PBS solution for one minute. The glycerol solution was removed and the monolayer was washed 3 times with PBS. Monolayers were fed with maintenance media (F10/199, 1% fetal bovine serum (FBS), 2% glutamine, 1% non-essential amino acids (NEAA), 1% penicillin/streptomycin (P/S)), and incubated at 39° C. The next day media was replaced and incubation at 39° C. continued. Plaques were visible in three to four days. When cells became overcrowded, infected monolayers were passed up to a larger size dish to allow spread of the virus. For passage, media was aspirated, cells were rinsed twice with PBS, aspirated again, then 0.5 ml trypsin-EDTA was added and incubated for 1-3 minutes at room temperature. Cells were pipetted up and down and diluted to 5 ml with maintenance media. The mixture was transferred to a larger dish and incubated at 39° C. When approximately 70 to 80% cytopathic effect (CPE) was achieved the infected cells were harvested by trypsinizing the monolayer as described above, except the cells were resuspended in F10/M199 Complete Freezing media. Infected cells were placed on ice and DMSO was added to 7%. After 15 minutes on ice, the cells were frozen at -70° C.
[0052]Stocks were then plaque purified two times. For each purification, stocks were diluted 1:100,000 in maintenance media and plated on several 10 cm dishes of secondary CEFs. After allowing the infected cells to sit down overnight, the infecting media was removed and replaced with 10 ml of nutrient agarose (0.8% low melt agarose, Media 199, 1% FBS, 2% glutamine, 1% NEAA 1% P/S), melted and cooled to 42° C., then allowed to harden at room temperature. Plates were then incubated 5 days at 39° C., until plaques formed. Isolated plaques were then picked using a glass Pasteur pipette to make a plug in the agarose. The plug containing the plaque was transferred into 24-well dish of CEFs. The dish was incubated 3 days, then each well was passed to a 6-well dish, and then to a 6 cm dish in 3 more days. Cells were grown until a 50-75% cytopathic effect was seen, then harvested as above and frozen at -70° C. A second purification was the performed as described above to obtain the final stock.
Southern Blot Analysis of Novel Avian Herpesviruses
[0053]Total DNA was isolated from a virus stock as described above. One tenth of the resuspended DNA isolated from a flask, 30 μl, was digested in 60 μl volume. Digestions with appropriate restriction endonucleases were performed as directed by the manufacturer. Digested DNA was loaded into a single well on a 25 cm long 0.7% agarose gel, and run overnight at 45 volts. Gel buffer was 0.5×TBE (a 1:10 dilution of 5×TBE).
[0054]Southern blots were performed using Zetaprobe blotting membranes. The alkaline blotting technique for DNA capillary transfer was used exactly as described in the Zetaprobe instruction manual (Section 2.3). The standard hybridization protocol and subsequent washes are also described in the same manual (Section 4.1) except that the membranes are not dried after the final wash. The probe was labeled using the Genius® non-radioactive DNA labeling and detection kit. Labeling was performed as described in the detection kit instruction manual under section I, "DNA labeling". One half of the labeled material was denatured by boiling, and added to the hybridization buffer. After the hybridization washes described above, the Zetaprobe filters were treated as described in section III, "Immunological Detection", of the Genius® labeling kit protocol.
Black Plaque Assay for Foreign Gene Expression in Novel Avian Herpesvirus
[0055]To analyze expression of foreign antigens expressed by recombinant NAHV viruses, monolayers of CEF cells were infected with recombinant NAHV, overlaid with nutrient agarose media and incubated for 4-5 days at 39° C. Once plaques developed, the agarose overlay was removed from the dish, the monolayer rinsed 1× with PBS, fixed with 100% methanol for 10 minutes at room temperature and the cells air-dried. After re-hydrating the plate with PBS, the primary antibody was diluted to the appropriate dilution with Blotto (5% non-fat milk/50 mM Tris pH 7.5/154 mM NaCl) and incubated with the cell monolayer for 2 hours to overnight at room temperature. Unbound antibody was then removed from the cells by washing three times with PBS at room temperature. An alkaline phosphatase conjugated secondary antibody was diluted with Blotto and incubated with the cells for 2 hours at room temperature. Unbound secondary antibody was then removed by washing the cells three times with PBS at room temperature. Next, the monolayer was rinsed in color development buffer (100 mM Tris pH 9.5/100 mM NaCl/50 mM MgCl2), and then incubated 10 minutes to overnight at room temperature with freshly prepared substrate solution (0.3 mg/ml Nitro Blue tetrazolium+0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphatase in color development buffer.) Finally, the reaction was stopped by replacing the substrate solution with TE (10 mM Tris pH 7.5/1 mM EDTA). Plaques expressing the correct antigen stained a purplish-black color.
Newcastle Disease Virus (NDV) Fusion Gene cDNA Cloning
[0056]cDNA cloning refers to the methods used to convert RNA molecules into DNA molecules. These methods are described in (U. Gubler and B. J Hoffman, Gene 25, 263-269, 1983). These methods may also be accomplished through the use of various commercially available cDNA cloning kits.
[0057]For cloning NDV mRNA, primary chicken embryo fibroblast (CEF) cells were infected at 5-10 plaque forming units per cell with the NDV B1 Hitchner strain (American Tissue Type Culture). When cytopathic effect was evident, but before total destruction, the medium was removed and the cells were lysed in 10 ml lysis buffer (4 M guanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0, 0.5% N-lauroyl sarcosine, 0.1 M beta-metcaptoethanol). The cell lysate was poured into a sterilized Dounce homogenizer and homogenized on ice 8-10 times until the solution was homogenous. For RNA purification, 8 ml of cell lysate were gently layered over 3.5 ml of CsCl solution (5.7 M CsCl, 25 mM sodium citrate pH 7.0) in Beckman SW41 centrifuge tube. The samples were centrifuged for 18 hrs at 20° C. at 36000 rpm in a Beckman SW41 rotor. The tubes were put on ice and the supernatants from the tubes were carefully removed by aspiration to leave the RNA pellet undisturbed. The pellet was resuspended in 400 μl glass distilled water, and 2.6 ml of guanidine solution (7.5 M guanidine-HCL, 25 mM sodium citrate pH 7.0, 5 mM dithiothreitol) were added. The 0.37 volumes of 1 M acetic acid were added, followed by 0.75 volumes of cold ethanol and the sample was put at -20° C. for 18 hrs to precipitate RNA. The precipitate was collected by centrifugation in a Sorvall centrifuge for 10 min a 4° C. at 10000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, centrifuged at 13000 rpm, and the supernatant saved. RNA was re-extracted from the pellet 2 more times as above with 0.5 ml distilled water, and the supernatants were pooled. A 0.1 volume of 2 M potassium acetate solution was added to the sample followed by 2 volumes of cold ethanol and the sample was put at -20° C. for 18 hrs. The precipitated RNA was collected by centrifugation in the SS34 rotor at 4° C. for 10 min at 10000 rpm. The pellet was dissolved in 1 ml distilled water and the concentration taken by absorption at A260/280. The RNA was stored at -70° C.
[0058]mRNA containing polyadenylate tails (poly-A) was selected using oligo-dT cellulose (Pharmacia #27 5543-0). Three mg of total RNA was boiled and chilled and applied to the 100 mg oligo-dT cellulose column in binding buffer (0.1 M Tris pH 7.5, 0.5 M LiCl, 5 mM EDTA pH 8.0, 0.1% lithium dodecyl sulfate). The retained poly-A RNA was eluted from the column with elution buffer (5 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 0.1% sodium dodecyl sulfate). This mRNA was reapplied to an oligo-dT column in binding buffer and eluted again in elution buffer. The sample was precipitated with 200 mM sodium acetate and 2 volumes cold ethanol at -20° C. for 18 hrs. The RNA was resuspended in 50 μl distilled water.
[0059]Ten μg poly-A RNA was denatured in 20 mM methyl mercury hydroxide for 6 min at 22° C. β-mercaptoethanol was added to 75 mM and the sample was incubated for 5 min at 22° C. The reaction mixture for first strand cDNA synthesis in 0.25 ml contained 1 μg oligo-dT primer (P-L Bio-chemicals) or 1 μg synthetic primer, 28 units placental ribonuclease inhibitor (Bethesda Research Labs #5518SA), 100 mM Tris pH 8.3, 140 mM KCl, 10 mM MgCl2, 0.8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia), 100 microcuries 32p-labeled dCTP (New England Nuclear #NEG-013H), and 180 units AMV reverse transcriptase (Molecular Genetics Resources #MG 101). The reaction was incubated at 42° C. for 90 min, and then was terminated with 20 mM EDTA pH 8.0. The sample was extracted with an equal volume of phenol/chloroform (1:1) and precipitated with 2 M ammonium acetate and 2 volumes of cold ethanol -20° C. for 3 hrs. After precipitation and centrifugation, the pellet was dissolved in 100 μl distilled water. The sample was loaded onto a 15 ml G-100 Sephadex column (Pharmacia) in buffer (100 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 100 mM NaCl). The leading edge of the eluted DNA fractions was pooled, and DNA was concentrated by lyophilization until the volume was about 100 μl, then the DNA was precipitated with ammonium acetate plus ethanol as above.
[0060]The entire first strand sample was used for second strand reaction which followed the Gubler and Hoffman (1983) method except that 50 μg/ml dNTP's, 5.4 units DNA polymerase I (Boerhinger Mannheim #642-711), and 100 units/ml E. coli DNA ligase (New England Biolabs #205) in a total volume of 50 microliters were used. After second strand synthesis, the cDNA was phenol/chloroform extracted and precipitated. The DNA was resuspended in 10 μl distilled water, treated with 1 μg RNase A for 10 min at 22° C., and electrophoresed through a 1% agarose gel (Sigma Type II agarose) in 40 mM Tris-acetate pH 6.85. The gel was stained with ethidium bromide, and DNA in the expected size range was excised from the gel and electroeluted in 8 mM Tris-acetate pH 6.85. Electroeluted DNA was lyophilized to about 100 microliters, and precipitated with ammonium acetate and ethanol as above. The DNA was resuspended in 20 μl water.
[0061]Oligo-dC tails were added to the DNA to facilitate cloning. The reaction contained the DNA, 100 mM potassium cacodylate pH 7.2, 0.2 mM dithiothreitol, 2 mM CaCl2, 80 μmoles dCTP, and 25 units terminal deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 μl. After 30 min at 37° C., the reaction was terminated with 10 mM EDTA, and the sample was phenol/chloroform extracted and precipitated as above.
[0062]The dC-tailed DNA sample was annealed to 200 ng plasmid vector pBR322 that contained oligo-dG tails (Bethesda Research Labs #5355 SA/SB) in 200 μl of 0.01 M Tris pH 7.5, 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65° C. for 2 min and then 57° C. for 2 hrs. Fresh competent E. coli DH-1 cells were prepared and transformed as described by Hanahan (Molecular Biology 166, 557-580, 1983) using half the annealed cDNA sample in twenty 200 μl aliquots of cells. Transformed cells were plated on L-broth agar plates plus 10 μg/ml tetracycline. Colonies were screened for the presence of inserts into the ampicillin gene using Ampscreen (Bethesda Research Labs #5537 UA), and the positive colonies were picked for analysis. Resulting positive clones were screened for homology to paramyxovirus fusion gene sequences. A clone containing the complete coding sequence of the NDV fusion gene was identified. The sequence of this clone is given in SEQ ID NO: 1.
Subgenomic Clone 407-32.2C3
[0063]Cosmid 407-32.2C3 contains an approximately 40,000 base pair region of genomic HVT DNA (from the left terminus to position 39,750 GenBank Accession No. AF291866, see FIG. 2). This region includes NAHV BamHI fragments F', L, P, N1, E, D, and 2,092 base pairs of fragment A'. Note: NAHV BamHI fragment A', is called fragment B in HVT. This cosmid may be constructed as described above in the Procedure for Cloning NAHV Subgenomic DNA Fragments. It was isolated from the sheared DNA library by screening with the probes P1 (HVT BamHI fragment F, position 116,948 to 125,961, Genbank Accession No. AF291866) and P2 (HVT BamHI fragment B, 37,663 to 63,593, Genbank Accession No. AF291866). A bacterial strain containing this cosmid has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20010-2209 U.S.A. under ATCC Accession No. 75430.
Subgenomic Clone 172-07.BA2
[0064]Plasmid 172-07.BA2 contains a 25,947 base pair region of genomic HVT DNA. This plasmid was constructed using standard recombinant DNA techniques joining two restriction fragments from the following sources. The first fragment is a 2999 base pair BamHI to BamHI restriction fragment of pSP64 (Promega). The second fragment is the 25,947 base pair BamHI B fragment of HVT (position 37,663 to 63,593, GenBank Accession No. AF291866). Note: In the NAHV genome, this fragment is denoted as fragment A', due to size considerations.
Subgenomic Clone 407-32.5G6
[0065]Cosmid 407-32.5G6 contains a 39,404 base pair region of genomic HVT DNA (position 61,852 to 101,255, Genbank Accession No. AF291866). This region includes NAHV BamHI fragments H, C, Q, K1, M, K2, plus 1,742 base pairs of fragment A', and 3,880 base pairs of fragment J. Note: NAHV BamHI fragment A', is called fragment B in HVT. This cosmid was constructed as described above in the Procedure for Cloning NAHV Subgenomic DNA Fragments. It was isolated from the sheared DNA library by screening with the probes P2 (HVT BamHI fragment B, 37,663 to 63,593, Genbank Accession No. AF291866) and P3 (HVT BamHI fragment J, position 97,376 to 102,720, Genbank Accession No. AF291866). A bacterial strain containing this cosmid has been deposited on Mar. 3, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20010-2209 U.S.A. under ATCC Accession No. 75427.
Subgenomic Clone 407-32.1C1
[0066]Cosmid 407-32.1C1 contains a 37,444 base pair region of genomic HVT DNA (position 96,095 to 133,538, GenBank Accession No. AF291866, see FIG. 2). This region includes NAHV BamHI fragments J, G, I, F, O, plus 1,281 base pairs of fragment K2, and 6,691 base pairs of fragment B'. Note: NAHV BamHI fragment B', is called fragment A in HVT. This cosmid was constructed as described above in the Procedure for Cloning NAHV Subgenomic DNA Fragments. It was isolated from the sheared DNA library by screening with the probes P1 (HVT BamHI fragment F, position 116,948 to 125,961, Genbank Accession No. AF291866) and P4 (4169 base pair BgIII to StuI sub-fragment (position 132,088 to 136,256, GenBank Accession No. AF291866) of HVT XhoI fragment #5 (position 128,950 to 136,510, GenBank Accession No. AF291866)). Note: an internal StuI site occurs within the 4169 base pair sub-fragment (position 134,083, GenBank Accession No. AF291866). However this site is methylated and does not cleave in plasmid DNA prepared from standard cloning strains of bacteria. A bacterial strain containing this cosmid has been deposited on Mar. 3, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20010-2209 U.S.A. under ATCC Accession No. 75428.
Subgenomic Clone 989-72.8#1
[0067]The cosmid 989-72-8#1 contains the NAHV short region cloned into the cosmid pWE15 (Stratagene). To create a short region cosmid for the NAHV, the US region of MDV was joined with the short repeat regions of HVT by PCR amplification and standard cloning techniques. In this engineered cosmid, the complete MDV US region was used, but the short repeat regions of HVT were shortened to remove the US8 (gE) sequence. This avoids the inclusion of sequence homologous to the US8 gene within the MDV US. Cosmid 989-72.8-1 contains the following DNAs: 9,193 bp of the short repeat region from HVT BamHI B (position 126,848 to 136,040; GenBank Accession No. AF291866), an 8 bp synthetic Pac-I linker, 11,156 bp from the MDV US (position 66 to 11,221, GenBank Accession L22174), an 8 bp synthetic Pac-I linker, and a second HVT short repeat (position 136,040 to 126,848, GenBank Accession No. AF291866), inverted relative to the other repeat. The pWE15 cosmid vector, used to clone these DNAs, was modified by replacing the 64 bp multiple cloning site (EcoRI to EcoRI), with a 68 bp synthetic linker (EcoRI, I-SceI, NotI, BamHI, NotI, I-SceI, and EcoRI), to allow excision of the insert with the I-SceI enzyme.
[0068]Foreign DNA sequences are added into the NAHV genome at a KpnI site with in the MDV US2 gene (position 4646, GenBank Accession L22174). The KpnI site interrupts this 270 amino acid coding region at approximately amino acid 85. Cloning the appropriate foreign DNA sequence into the NAHV short region cosmid, 989-72.8#1 at this KpnI site, creates Subgenomic clones used to introduce foreign genes.
Subgenomic Clone 1002-75.4
[0069]The cosmid 1002-75.4 contains a foreign gene encoding the fusion protein of the Newcastle disease virus inserted within the MDV US2 gene of the NAHV short region cosmid, 989-72.8#1. The NDV fusion gene (F) is under the control of the human cytomegalovirus immediate early (HCMV IE) promoter and utilizes the herpes simplex virus thymidine kinase (HSV tk) poly adenylation signal (pA). This cosmid was created using standard DNA cloning techniques. The sequence of the foreign DNA inserted into cosmid 989-72.8#1 is given in SEQ ID NO: 1. This sequence was inserted such that the NDV F and MDV US2 genes are transcribed in the same direction. The source of each region of the insert is indicated in the following table.
TABLE-US-00001 TABLE 1 Source of foreign DNAs inserted into Subgenomic Clone 1002-75.4 Region Starta Endb Source 1 1 36 Synthetic Linker 2 37 1189 HCMV genomic DNA (IE promoter cloned as described in U.S. Pat. No. 5,830,745 and sequenced as described above) 3 1190 1200 Synthetic linker 4 1201 3004 NDV cDNA (F gene cloned and sequenced as described above) 5 3005 3025 Synthetic linker 6 3026 3548 HSV genomic DNA (tk pA position 37,694 to 37,172 GenBank Accession No. D10879) 7 3549 3570 Synthetic linker aStarting position of the region in SEQ. ID NO: 1 bEnding position of the region in SEQ. ID NO: 1
Subgenomic Clone Vector 1012-89.2
[0070]The cosmid 1012-89.2 contains two foreign genes encoding the glycoprotein D and glycoprotein I of the infectious laryngotracheitis virus (ILTV) inserted in to the MDV US2 gene of the NAHV short region cosmid, 989-72.8#1. The ILTV genes are under the control of their endogenous promoters. This cosmid was created using standard DNA cloning techniques. The sequence of the foreign DNA inserted into cosmid 989-72.8#1 is given in SEQ ID NO: 3. This sequence was inserted such that the ILTV gD gene and ILTV gI gene are transcribed in the opposite direction of the MDV US2 genes. The source of each region of the insert is indicated in the following table.
TABLE-US-00002 TABLE 2 Source of foreign DNAs inserted into Subgenomic Clone 1012-89.2 Region Starta Stopb Source 1 1 18 Synthetic Linker 2 19 3581 ILTV genomic DNA (gD and gI genes (position 10,532 to 14,094 Genbank Accession No. U28832) 3 3582 3605 Synthetic linker aStarting position of the region in SEQ. ID NO: 3 bEnding position of the region in SEQ. ID NO: 3
EXAMPLES
Example 1
The NAHV Designated NAHV 295-01 and the Marek's Disease Recombinant Vaccine (NAHV 295-01)
[0071]The NAHV 295-01 recombinant virus was generated according to the Procedure for Generating Novel Avian Herpesvirus from Overlapping Subgenomic Fragments. The following combination of subgenomic clones and enzymes were used: 989-72.8#1 with I-SceI, 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, and 407-32.1C1 with NotI. (The location of subgenomic clones on the resulting NAHV genome is indicated in FIG. 2.) The NAHV was shown to have the correct genomic structure using the Southern Blot Analysis of Novel Avian Herpesviruses. Stability of the NAHV 295-01 virus vaccine strain was demonstrated by serial passage 12 times in tissue culture followed by a second Southern blot analysis. This virus strain has been deposited on Jun. 13, 2001 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20010-2209 U.S.A. under ATCC Accession No. PTA-3451.
[0072]The following studies were conducted to demonstrate the safety of the NAHV 295-01 vaccine, and its effectiveness in protecting against challenge with very virulent Marek's disease virus. In study 1, 18-day-old specific pathogen free (SPF) embryos, or one-day-old chicks were vaccinated with the NAHV 295-01 vaccine. As controls, additional groups of one-day-old chicks were vaccinated with one of two USDA-licensed, conventional vaccines comprised of either HVT or MDV-1/Rispens. Five days post-hatch, vaccinated chicks, and non-vaccinated, control chicks were challenged with virulent MDV/RB1B. Birds were then observed for clinical signs of disease for 7 weeks, then necropsied to examine for gross lesions. The results, in Table 3, show the NAHV 295-01 vaccine gave greater protection against very virulent Marek's disease challenge than either commercial vaccine.
TABLE-US-00003 TABLE 3 Efficacy of the NAHV 295-01 Recombinant Vaccine Against Virulent Marek's Disease Virus Challenge Protection % Group Route Dosea Challengeb Ratioc Protected Non-vac. -- -- -- 25/25 -- Non-vac. -- -- RB1B 1/30 3% NAHV in ovo 895 pfu RB1B 28/30 93% 295-01 NAHV SC 940 pfu RB1B 29/30 97% 295-01 HVT SC As per label RB1B 18/30 60% Rispens SC As per label RB1B 27/30 90% ain ovo dose: PFU/0.05 ml; SC dose: PFU/0.2 ml bChallenge 5 days post-vaccination, intra-abdominal cNo. protected/Total on day 54
[0073]In the second study, 18-day-old embryos or one-day-old SPF chicks were vaccinated with ten times the maximum dose of the NAHV 295-01 vaccine. The chicks were observed for 120 days for clinical signs of Marek's disease, then necropsied and examined for Marek's lesions. As controls, a third group of birds remained un-vaccinated, and a fourth group of un-vaccinated birds was challenged on day 4 with virulent MDV/RB1B to demonstrate that the birds were susceptible to Marek's disease. The results, in Table 4, demonstrate the safety of the NAHV 295-01 vaccine given in ovo (18-day-old embryos) or at one day-of-age.
TABLE-US-00004 TABLE 4 Safety the NAHV 295-01 Recombinant Vaccine Following in ovo or Subcutaneous Injection with 10x Dose. Group Route Dosea Challengeb % MDc Non-vac. -- -- -- -- Non-vac. -- -- RB1B 100% NAHV 295-01 in ovo 20,000 pfu -- 0% NAHV 295-01 SC 20,000 pfu -- 0% adose: PFU/0.05 ml (In ovo) or PFU/0.2 ml (SC). bChallenge 5 days post-vaccination; intra-abdominal. cPercentage MD positive/Total by day 120.
Example 2
The NAHV Designated NAHV/NDV 295-93 and the Multivalent Marek's Disease/Newcastle Disease Recombinant Vaccine (NAHV/NDV 295-93)
[0074]The NAHV/NDV 295-93 recombinant virus was generated according to the Procedure for Generating Novel Avian Herpesvirus from Overlapping Subgenomic Fragments. The following combination of subgenomic clones and enzymes were used: 1002-75.4 with I-SceI, 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, and 407-32.1C1 with NotI. (The location of subgenomic clones on the resulting NAHV genome is indicated in FIG. 2.) The NAHV was shown to have the correct genomic structure using the Southern Blot Analysis of Novel Avian Herpesviruses. Stability of the NAHV/NDV 295-93 virus vaccine strain was demonstrated by serial passage 12 times in tissue culture followed by a second Southern blot analysis. This virus strain has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20010-2209 U.S.A. under ATCC Accession No. PTA-3453.
[0075]The Black Plaque Assay for Foreign Gene Expression in Novel Avian Herpesvirus was used to demonstrate expression of the NDV fusion gene. The assay used a monoclonal antibody directed to the NDV fusion gene (3-1G5) diluted 1:100 as the primary antibody. A goat anti-mouse alkaline phosphatase conjugated antibody diluted 1:1000 was used as the secondary antibody. Purity of the virus was demonstrated by assay of serial passage 12 stocks, 97.6% (1029/1054) of the viral plaques were black plaque positive.
[0076]The following studies were conducted to demonstrate the effectiveness of the NAHV/NDV 295-93 vaccine, in protecting against challenge with either virulent Newcastle disease virus, or very virulent Marek's disease virus. In Study 1, 18-day-old specific pathogen free (SPF) embryos, or one-day-old chicks were vaccinated with the NAHV/NDV 295-93 vaccine. As a control, a third group of one-day-old chicks remained un-vaccinated Twenty-one days post-hatch, vaccinated chicks, and non-vaccinated, control chicks were challenged with virulent NDV/Texas GB strain. Birds were then observed for clinical signs of disease for fourteen days. The results, in Table 5, show the NAHV/NDV 295-93 vaccine gave better than 90% protection against virulent NDV.
TABLE-US-00005 TABLE 5 Efficacy of the NAHV/NDV 295-93 Recombinant Vaccine Against Virulent Newcastle Disease Virus Challenge Group Route Dosea Challengeb % Protectedc Non-vac. -- -- -- -- Non-vac. -- -- Texas GB 0% NAHV/NDV in ovo 525 pfu Texas GB 90% 295-93 NAHV/NDV SC 707 pfu Texas GB 97% 295-93 aIn ovo dose: PFU/0.05 ml; SC dose: PFU/0.2 ml. bChallenge 21 days post-vaccination, intra-ocular. cPercentage Protected/Total; 14 days post-challenge.
[0077]In study two, 18-day-old embryos, or one-day-old SPF chicks were vaccinated with the NAHV/NDV 295-93 vaccine. As controls, additional groups of one-day-old chicks were vaccinated with one of two USDA-licensed, conventional vaccines comprised of either HVT or MDV-1/Rispens. Five days post-hatch, vaccinated chicks, and non-vaccinated, control chicks were challenged with virulent MDV/RB1B. Birds were observed for clinical signs of disease for 7 weeks, then necropsied to examine for gross lesions. The results, in Table 6, show the NAHV/NDV 295-93 vaccine protected against very virulent Marek's disease challenge.
TABLE-US-00006 TABLE 6 Efficacy of the NAHV/NDV 295-93 Recombinant Vaccine Against Virulent Marek's Disease Virus Challenge Protection % Group Route Dosea Challengeb Ratioc Protected Non-vac. -- -- -- 25/25 -- Non-vac. -- -- RB1B 1/30 3% NAHV/ in ovo 675 pfu RB1B 28/30 93% NDV 295-93 NAHV/ SC 680 pfu RB1B 26/30 87% NDV 295-93 HVT SC As per label RB1B 18/30 60% Rispens SC As per label RB1B 27/30 90% aIn ovo dose: PFU/0.05 ml; SC dose: PFU/0.2 ml. bChallenge 5 days post-vaccination, intra-abdominal. cNo. protected/Total on day 54.
Example 3
The NAHV Designated NAHV/ILT 295-149 and the Multivalent Marek's Disease/Infectious Laryngotrachetitis Recombinant Vaccine (NAHV/ILT 295-149)
[0078]The NAHV/ILT 295-149 recombinant virus was generated according to the Procedure for Generating Novel Avian Herpesvirus from Overlapping Subgenomic Fragments. The following combination of subgenomic clones and enzymes were used: 1012-89.2 with I-SceI, 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, and 407-32.1C1 with NotI. (The location of subgenomic clones on the resulting NAHV genome is indicated in FIG. 2.) The NAHV was shown to have the correct genomic structure using the Southern Blot Analysis of Novel Avian Herpesviruses. Stability of the NAHV/ILT 295-149 virus vaccine strain was demonstrated by serial passage 12 times in tissue culture followed by a second Southern blot analysis. This virus strain has been deposited on Jun. 13, 2001 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20010-2209 U.S.A. under ATCC Accession No. PTA-3452.
[0079]The Black Plaque Assay for Foreign Gene Expression in Novel Avian Herpesvirus was used to demonstrate expression of the ILT glycoproteins. The assay used a convalescent ILT chicken sera (SPAFAS, Inc.) diluted 1:100 as the primary antibody. A goat anti-chicken alkaline phosphatase conjugated antibody diluted 1:1000 was used as the secondary antibody. Purity of the virus was demonstrated by assay of serial passage 12 stocks. 99.4% (1043/1049) of the viral plaques were black plaque positive.
[0080]The following studies were conducted to demonstrate the effectiveness of the NAHV/ILT 295-149 vaccine, in protecting against challenge with either virulent infectious laryngotracheitis virus, or very virulent Marek's disease virus. In study 1, 18-day-old specific pathogen free (SPF) embryos, or one-day-old chicks were vaccinated with the NAHV/ILT 295-149 vaccine. As controls, additional groups of one-day-old chicks either remained un-vaccinated, or were vaccinated with a USDA-licensed, conventional vaccine comprised of attenuated, live ILTV (LT-Ivax). Twenty-five days post-hatch, vaccinated chicks, and non-vaccinated, control chicks were challenged with virulent ILT/USDA LT-96-3. Birds were observed for clinical signs of disease for ten days, and then necropsied to examine for gross lesions. The results, in Table 7, show the NAHV/ILT 295-149 vaccine gave better protection against virulent ILT than the commercial vaccine.
TABLE-US-00007 TABLE 7 Efficacy of the NAHV/ILT 295-149 Recombinant Vaccine Against Virulent Infectious Laryngotracheitis Virus Challenge Group Route Dosea Challengeb % Protectedc Non-vac. -- -- -- -- Non-vac. -- -- ILT (USDA) 0% NAHV/ILT in ovo 750 pfu ILT (USDA) 100% 295-149 NAHV/ILT SC 750 pfu ILT (USDA) 100% 295-149 LT-Ivax Per label Per label ILT (USDA) 60% aIn ovo dose: PFU/0.05 ml; SC dose: PFU/0.2 ml. bChallenge 25 days post-vaccination, intra-tracheal. cPercentage Protected/Total; 10 days post-challenge
[0081]In study two, 18-day-old embryos, or one-day-old SPF chicks were vaccinated with NAHV/ILT 295-149. As controls, additional groups of one-day-old chicks were vaccinated with a USDA-licensed, conventional vaccine comprised of MDV-1/Rispens, or left un-vaccinated. Five days post-hatch, vaccinated chicks, and non-vaccinated, control chicks were challenged with virulent MDV/RB1B. Birds were observed for clinical signs of disease for 7 weeks, then necropsied to examine for gross lesions. The results, in Table 8, show the NAHV/ILT 295-149 vaccine protected better against virulent Marek's disease challenge, than the commercial vaccine.
TABLE-US-00008 TABLE 8 Efficacy of the NAHV/ILT 295-149 Recombinant Vaccine Against Virulent Marek's Disease Virus Challenge Protection % Group Route Dosea Challengeb Ratioc Protected Non-vac. -- -- -- 33/33 -- Non-vac. -- -- RB1B 2/35 6% NAHV/ in ovo 1500 pfu RB1B 25/26 96% ILT 295-149 NAHV/ SC 1500 pfu RB1B 32/34 94% ILT 295-149 Rispens SC As per label RB1B 26/33 79% aIn ovo dose: PFU/0.05 ml; SC dose: PFU/0.2 ml. bChallenge 5 days post-vaccination, intra-abdominal
[0082]Although certain presently preferred embodiments of the invention have been disclosed herein, it will be apparent to those skilled in the art to which the invention pertains that variation and modification of described embodiments may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited to the extent required by the appended claims and the applicable rules of law.
Sequence CWU
1
513570DNANewcastle disease virusCDS(1194)..(2888)NDV Fusion Protein
1gtacgttaat taacccggga agcttgcatg cctgcagtga ataataaaat gtgtgtttgt
60ccgaaatacg cgttttgaga tttctgtcgc cgactaaatt catgtcgcgc gatagtggtg
120tttatcgccg atagagatgg cgatattgga aaaatcgata tttgaaaata tggcatattg
180aaaatgtcgc cgatgtgagt ttctgtgtaa ctgatatctg gcgatagcgc ttatatcgtt
240tacgggggat ggcgatagac gactttggcg acttgggcga ttctgtgtgt cgcaaatatc
300gcagtttcga tataggtgac agacgatatg aggctatatc gccgatagag gcgacatcaa
360gctggcacat ggccaatgca tatcgatcta tacattgaat caatattggc aattagccat
420attagtcatt ggttatatag cataaatcaa tattggctat tggccattgc atacgttgta
480tctatatcat aatatgtaca tttatattgg ctcatgtcca atatgaccgc catgttgaca
540ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
600tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
660cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
720ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
780gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
840ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt
900catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt
960tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
1020ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg
1080cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
1140cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg acc atg
1196 Met
1gat cga tcc cgg ttg
gcg ccc tcc agg tgc agg atg ggc tcc aga cct 1244Asp Arg Ser Arg Leu
Ala Pro Ser Arg Cys Arg Met Gly Ser Arg Pro 5
10 15tct acc aag aac cca gca cct atg atg ctg act atc
cgg gtc gcg ctg 1292Ser Thr Lys Asn Pro Ala Pro Met Met Leu Thr Ile
Arg Val Ala Leu 20 25 30gta ctg
agt tgc atc tgt ccg gca aac tcc att gat ggc agg cct ctt 1340Val Leu
Ser Cys Ile Cys Pro Ala Asn Ser Ile Asp Gly Arg Pro Leu 35
40 45gca gct gca gga ctn tgg tta cag gag aca aag
caa tca aca tat aca 1388Ala Ala Ala Gly Xaa Trp Leu Gln Glu Thr Lys
Gln Ser Thr Tyr Thr50 55 60
65cct cat ccc aga cag gtc aat cat att aag ctc ctc ccg aat ctg cca
1436Pro His Pro Arg Gln Val Asn His Ile Lys Leu Leu Pro Asn Leu Pro
70 75 80aag gat aag gag gca
tgt gcg aaa gcc ccc ttg gat gca tac aac agg 1484Lys Asp Lys Glu Ala
Cys Ala Lys Ala Pro Leu Asp Ala Tyr Asn Arg 85
90 95aca ttg acc act ttg ctc acc ccc ctt ggt gac tct
atc cgt agg ata 1532Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly Asp Ser
Ile Arg Arg Ile 100 105 110caa gag
tct gtg act aca tct gga ggg ggg aga cag ggg cgc ctt ata 1580Gln Glu
Ser Val Thr Thr Ser Gly Gly Gly Arg Gln Gly Arg Leu Ile 115
120 125ggc gcc att att ggc ggt gtg gct ctt ggg gtt
gca act gcc gca caa 1628Gly Ala Ile Ile Gly Gly Val Ala Leu Gly Val
Ala Thr Ala Ala Gln130 135 140
145ata aca gcg gcc gca gct ctg ata caa gcc aaa caa aat gct gcc aac
1676Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala Lys Gln Asn Ala Ala Asn
150 155 160atc ctc cga ctt aaa
gag agc att gcc gca acc aat gag gct gtg cat 1724Ile Leu Arg Leu Lys
Glu Ser Ile Ala Ala Thr Asn Glu Ala Val His 165
170 175gag gtc act gac gga tta tcg caa cta gca gtg gca
gtt ggg aag atg 1772Glu Val Thr Asp Gly Leu Ser Gln Leu Ala Val Ala
Val Gly Lys Met 180 185 190cag cag
ttc gtt aat gac caa ttt aat aaa aca gct cag gaa tta gac 1820Gln Gln
Phe Val Asn Asp Gln Phe Asn Lys Thr Ala Gln Glu Leu Asp 195
200 205tgc atc aaa att gca cag caa gtt ggt gta gag
ctc aac ctg tac cta 1868Cys Ile Lys Ile Ala Gln Gln Val Gly Val Glu
Leu Asn Leu Tyr Leu210 215 220
225acc gaa tcg act aca gta ttc gga cca caa atc act tca cct gcc tta
1916Thr Glu Ser Thr Thr Val Phe Gly Pro Gln Ile Thr Ser Pro Ala Leu
230 235 240aac aag ctg act att
cag gca ctt tac aat cta gct ggt ggg aat atg 1964Asn Lys Leu Thr Ile
Gln Ala Leu Tyr Asn Leu Ala Gly Gly Asn Met 245
250 255gat tac tta ttg act aag tta ggt ata ggg aac aat
caa ctc agc tca 2012Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly Asn Asn
Gln Leu Ser Ser 260 265 270tta atc
ggt agc ggc tta atc acc ggt aac cct att cta tac gac tca 2060Leu Ile
Gly Ser Gly Leu Ile Thr Gly Asn Pro Ile Leu Tyr Asp Ser 275
280 285cag act caa ctc ttg ggt ata cag gta act cta
cct tca gtc ggg aac 2108Gln Thr Gln Leu Leu Gly Ile Gln Val Thr Leu
Pro Ser Val Gly Asn290 295 300
305cta aat aat atg cgt gcc acc tac ttg gaa acc tta tcc gta agc aca
2156Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu Thr Leu Ser Val Ser Thr
310 315 320acc agg gga ttt gcc
tcg gca ctt gtc cca aaa gtg gtg aca cgg gtc 2204Thr Arg Gly Phe Ala
Ser Ala Leu Val Pro Lys Val Val Thr Arg Val 325
330 335ggt tct gtg ata gaa gaa ctt gac acc tca tac tgt
ata gaa act gac 2252Gly Ser Val Ile Glu Glu Leu Asp Thr Ser Tyr Cys
Ile Glu Thr Asp 340 345 350tta gat
tta tat tgt aca aga ata gta acg ttc cct atg tcc cct ggt 2300Leu Asp
Leu Tyr Cys Thr Arg Ile Val Thr Phe Pro Met Ser Pro Gly 355
360 365att tac tcc tgc ttg agc ggc aat aca tcg gcc
tgt atg tac tca aag 2348Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser Ala
Cys Met Tyr Ser Lys370 375 380
385acc gaa ggc gca ctt act aca cca tat atg act atc aaa ggc tca gtc
2396Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met Thr Ile Lys Gly Ser Val
390 395 400atc gct aac tgc aag
atg aca aca tgt aga tgt gta aac ccc ccg ggt 2444Ile Ala Asn Cys Lys
Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly 405
410 415atc ata tcg caa aac tat gga gaa gcc gtg tct cta
ata gat aaa caa 2492Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val Ser Leu
Ile Asp Lys Gln 420 425 430tca tgc
aat gtt tta tcc tta ggc ggg ata act tta agg ctc agt ggg 2540Ser Cys
Asn Val Leu Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser Gly 435
440 445gaa ttc gat gta act tat cag aag aat atc tca
ata caa gat tct caa 2588Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile Ser
Ile Gln Asp Ser Gln450 455 460
465gta ata ata aca ggc aat ctt gat atc tca act gag ctt ggg aat gtc
2636Val Ile Ile Thr Gly Asn Leu Asp Ile Ser Thr Glu Leu Gly Asn Val
470 475 480aac aac tcg atc agt
aat gcc ttg aat aag tta gag gaa agc aac aga 2684Asn Asn Ser Ile Ser
Asn Ala Leu Asn Lys Leu Glu Glu Ser Asn Arg 485
490 495aaa cta gac aaa gtc aat gtc aaa ctg acc agc aca
tct gct ctc att 2732Lys Leu Asp Lys Val Asn Val Lys Leu Thr Ser Thr
Ser Ala Leu Ile 500 505 510acc tat
atc gtt ttg act atc ata tct ctt gtt ttt ggt ata ctt agc 2780Thr Tyr
Ile Val Leu Thr Ile Ile Ser Leu Val Phe Gly Ile Leu Ser 515
520 525ctg att cta gca tgc tac cta atg tac aag caa
aag gcg caa caa aag 2828Leu Ile Leu Ala Cys Tyr Leu Met Tyr Lys Gln
Lys Ala Gln Gln Lys530 535 540
545acc tta tta tgg ctt ggg aat aat acc cta gat cag atg aga gcc act
2876Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu Asp Gln Met Arg Ala Thr
550 555 560aca aaa atg tga
acacagatga ggaacgaagg tttccctaat agtaatttgt 2928Thr Lys
Metgtgaaagttc tggtagtctg tcagttcgga gagttaagaa aaaaaaaaaa cccccccccc
2988cccccccccc cccccctggg tacgatcctc tagagtcggg agatggggga ggctaactga
3048aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
3108taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
3168actctgtcga taccccaccg agaccccatt gggaccaata cgcccgcgtt tcttcctttt
3228ccccacccca acccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
3288caagccctgc catagccacg ggccccgtgg gttagggacg gggtccccca tggggaatgg
3348tttatggttc gtgggggtta ttattttggg cgttgcgtgg ggtcaggtcc acgactggac
3408tgagcagaca gacccatggt ttttggatgg cctgggcatg gaccgcatgt actggcgcga
3468cacgaacacc gggcgtctgt ggctgccaaa cacccccgac ccccaaaaac caccgcgcgg
3528atttctggcg ccgccggacg tcgacttaat taacaagctt ag
35702564PRTNewcastle disease virusmisc_feature(54)Xaa = any amino acid
2Met Asp Arg Ser Arg Leu Ala Pro Ser Arg Cys Arg Met Gly Ser Arg1
5 10 15Pro Ser Thr Lys Asn Pro
Ala Pro Met Met Leu Thr Ile Arg Val Ala 20 25
30Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile Asp
Gly Arg Pro 35 40 45Leu Ala Ala
Ala Gly Xaa Trp Leu Gln Glu Thr Lys Gln Ser Thr Tyr 50
55 60Thr Pro His Pro Arg Gln Val Asn His Ile Lys Leu
Leu Pro Asn Leu65 70 75
80Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro Leu Asp Ala Tyr Asn
85 90 95Arg Thr Leu Thr Thr Leu
Leu Thr Pro Leu Gly Asp Ser Ile Arg Arg 100
105 110Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly Arg
Gln Gly Arg Leu 115 120 125Ile Gly
Ala Ile Ile Gly Gly Val Ala Leu Gly Val Ala Thr Ala Ala 130
135 140Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala
Lys Gln Asn Ala Ala145 150 155
160Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala Thr Asn Glu Ala Val
165 170 175His Glu Val Thr
Asp Gly Leu Ser Gln Leu Ala Val Ala Val Gly Lys 180
185 190Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys
Thr Ala Gln Glu Leu 195 200 205Asp
Cys Ile Lys Ile Ala Gln Gln Val Gly Val Glu Leu Asn Leu Tyr 210
215 220Leu Thr Glu Ser Thr Thr Val Phe Gly Pro
Gln Ile Thr Ser Pro Ala225 230 235
240Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn Leu Ala Gly Gly
Asn 245 250 255Met Asp Tyr
Leu Leu Thr Lys Leu Gly Ile Gly Asn Asn Gln Leu Ser 260
265 270Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly
Asn Pro Ile Leu Tyr Asp 275 280
285Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr Leu Pro Ser Val Gly 290
295 300Asn Leu Asn Asn Met Arg Ala Thr
Tyr Leu Glu Thr Leu Ser Val Ser305 310
315 320Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro Lys
Val Val Thr Arg 325 330
335Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser Tyr Cys Ile Glu Thr
340 345 350Asp Leu Asp Leu Tyr Cys
Thr Arg Ile Val Thr Phe Pro Met Ser Pro 355 360
365Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser Ala Cys Met
Tyr Ser 370 375 380Lys Thr Glu Gly Ala
Leu Thr Thr Pro Tyr Met Thr Ile Lys Gly Ser385 390
395 400Val Ile Ala Asn Cys Lys Met Thr Thr Cys
Arg Cys Val Asn Pro Pro 405 410
415Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val Ser Leu Ile Asp Lys
420 425 430Gln Ser Cys Asn Val
Leu Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser 435
440 445Gly Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile Ser
Ile Gln Asp Ser 450 455 460Gln Val Ile
Ile Thr Gly Asn Leu Asp Ile Ser Thr Glu Leu Gly Asn465
470 475 480Val Asn Asn Ser Ile Ser Asn
Ala Leu Asn Lys Leu Glu Glu Ser Asn 485
490 495Arg Lys Leu Asp Lys Val Asn Val Lys Leu Thr Ser
Thr Ser Ala Leu 500 505 510Ile
Thr Tyr Ile Val Leu Thr Ile Ile Ser Leu Val Phe Gly Ile Leu 515
520 525Ser Leu Ile Leu Ala Cys Tyr Leu Met
Tyr Lys Gln Lys Ala Gln Gln 530 535
540Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu Asp Gln Met Arg Ala545
550 555 560Thr Thr Lys Met
33605DNAInfectious Laryngotracheitis VirusCDS(585)..(1889)ILTV
glycoprotein D 3ctaagcttgt taattaagtc gacggcagag tcgcagacgc ccctattgga
cgtcaaaatt 60gtagaggtga agttttcaaa cgatggcgaa gtaacggcga cttgcgtttc
caccgtcaaa 120tctccctata gggtagaaac taattggaaa gtagacctcg tagatgtaat
ggatgaaatt 180tctgggaaca gtcccgccgg ggtttttaac agtaatgaga aatggcagaa
acagctgtac 240tacagagtaa ccgatggaag aacatcggtc cagctaatgt gcctgtcgtg
cacgagccat 300tctccggaac cttactgtct tttcgacacg tctcttatag cgagggaaaa
agatatcgcg 360ccagagttat actttacctc tgatccgcaa acggcatact gcacaataac
tctgccgtcc 420ggcgttgttc cgagattcga atggagcctt aataatgttt cactgccgga
atatttgacg 480gccacgaccg ttgtttcgca taccgctggc caaagtacag tgtggaagag
cagcgcgaga 540gcaggcgagg cgtggatttc tggccgggga ggcaatatat acga atg cac
cgt cct 596 Met His
Arg Pro 1cat ctc aga cgg
cac tcg cgt tac tac gcg aaa gga gag gtg ctt aac 644His Leu Arg Arg
His Ser Arg Tyr Tyr Ala Lys Gly Glu Val Leu Asn5 10
15 20aaa cac atg gat tgc ggt gga aaa cgg
tgc tgc tca ggc gca gct gta 692Lys His Met Asp Cys Gly Gly Lys Arg
Cys Cys Ser Gly Ala Ala Val 25 30
35ttc act ctt ttc tgg act tgt gtc agg att atg cgg gag cat atc
tgc 740Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg Glu His Ile
Cys 40 45 50ttt gta cgc aac
gct atg gac cgc cat tta ttt ttg agg aat gct ttt 788Phe Val Arg Asn
Ala Met Asp Arg His Leu Phe Leu Arg Asn Ala Phe 55
60 65tgg act atc gta ctg ctt tct tcc ttc gct agc cag
agc acc gcc gcc 836Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gln
Ser Thr Ala Ala 70 75 80gtc acg tac
gac tac att tta ggc cgt cgc gcg ctc gac gcg cta acc 884Val Thr Tyr
Asp Tyr Ile Leu Gly Arg Arg Ala Leu Asp Ala Leu Thr 85
90 95 100ata ccg gcg gtt ggc ccg tat aac
aga tac ctc act agg gta tca aga 932Ile Pro Ala Val Gly Pro Tyr Asn
Arg Tyr Leu Thr Arg Val Ser Arg 105 110
115ggc tgc gac gtt gtc gag ctc aac ccg att tct aac gtg gac
gac atg 980Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn Val Asp
Asp Met 120 125 130ata tcg gcg
gcc aaa gaa aaa gag aag ggg ggc cct ttc gag gcc tcc 1028Ile Ser Ala
Ala Lys Glu Lys Glu Lys Gly Gly Pro Phe Glu Ala Ser 135
140 145gtc gtc tgg ttc tac gtg att aag ggc gac gac
ggc gag gac aag tac 1076Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp
Gly Glu Asp Lys Tyr 150 155 160tgt cca
atc tat aga aaa gag tac agg gaa tgt ggc gac gta caa ctg 1124Cys Pro
Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly Asp Val Gln Leu165
170 175 180cta tct gaa tgc gcc gtt caa
tct gca cag atg tgg gca gtg gac tat 1172Leu Ser Glu Cys Ala Val Gln
Ser Ala Gln Met Trp Ala Val Asp Tyr 185
190 195gtt cct agc acc ctt gta tcg cga aat ggc gcg gga
ctg act ata ttc 1220Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly
Leu Thr Ile Phe 200 205 210tcc
ccc act gct gcg ctc tct ggc caa tac ttg ctg acc ctg aaa atc 1268Ser
Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu Thr Leu Lys Ile 215
220 225ggg aga ttt gcg caa aca gct ctc gta
act cta gaa gtt aac gat cgc 1316Gly Arg Phe Ala Gln Thr Ala Leu Val
Thr Leu Glu Val Asn Asp Arg 230 235
240tgt tta aag atc ggg tcg cag ctt aac ttt tta ccg tcg aaa tgc tgg
1364Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro Ser Lys Cys Trp245
250 255 260aca aca gaa cag
tat cag act gga ttt caa ggc gaa cac ctt tat ccg 1412Thr Thr Glu Gln
Tyr Gln Thr Gly Phe Gln Gly Glu His Leu Tyr Pro 265
270 275atc gca gac acc aat aca cga cac gcg gac
gac gta tat cgg gga tac 1460Ile Ala Asp Thr Asn Thr Arg His Ala Asp
Asp Val Tyr Arg Gly Tyr 280 285
290gaa gat att ctg cag cgc tgg aat aat ttg ctg agg aaa aag aat cct
1508Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg Lys Lys Asn Pro
295 300 305agc gcg cca gac cct cgt cca
gat agc gtc ccg caa gaa att ccc gct 1556Ser Ala Pro Asp Pro Arg Pro
Asp Ser Val Pro Gln Glu Ile Pro Ala 310 315
320gta acc aag aaa gcg gaa ggg cgc acc ccg gac gca gaa agc agc gaa
1604Val Thr Lys Lys Ala Glu Gly Arg Thr Pro Asp Ala Glu Ser Ser Glu325
330 335 340aag aag gcc cct
cca gaa gac tcg gag gac gac atg cag gca gag gct 1652Lys Lys Ala Pro
Pro Glu Asp Ser Glu Asp Asp Met Gln Ala Glu Ala 345
350 355tct gga gaa aat cct gcc gcc ctc ccc gaa
gac gac gaa gtc ccc gag 1700Ser Gly Glu Asn Pro Ala Ala Leu Pro Glu
Asp Asp Glu Val Pro Glu 360 365
370gac acc gag cac gat gat cca aac tcg gat cct gac tat tac aat gac
1748Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp Tyr Tyr Asn Asp
375 380 385atg ccc gcc gtg atc ccg gtg
gag gag act act aaa agt tct aat gcc 1796Met Pro Ala Val Ile Pro Val
Glu Glu Thr Thr Lys Ser Ser Asn Ala 390 395
400gtc tcc atg ccc ata ttc gcg gcg ttc gta gcc tgc gcg gtc gcg ctc
1844Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys Ala Val Ala Leu405
410 415 420gtg ggg cta ctg
gtt tgg agc atc gta aaa tgc gcg cgt agc taa 1889Val Gly Leu Leu
Val Trp Ser Ile Val Lys Cys Ala Arg Ser 425
430tcgagcctag aataggtggt ttcttcctac atgccacgcc tcacgctcat aatataaatc
1949acatggaata gcataccaat gcctattcat tgggacgttc gaaaagc atg gca tcg
2005 Met Ala Ser
435cta ctt gga act ctg gct
ctc ctt gcc gcg acg ctc gca ccc ttc ggc 2053Leu Leu Gly Thr Leu Ala
Leu Leu Ala Ala Thr Leu Ala Pro Phe Gly 440 445
450gcg atg gga atc gtg atc act gga aat cac gtc tcc gcc agg
att gac 2101Ala Met Gly Ile Val Ile Thr Gly Asn His Val Ser Ala Arg
Ile Asp 455 460 465gac gat cac atc gtg
atc gtc gcg cct cgc ccc gaa gct aca att caa 2149Asp Asp His Ile Val
Ile Val Ala Pro Arg Pro Glu Ala Thr Ile Gln470 475
480 485ctg cag cta ttt ttc atg cct ggc cag aga
ccc cac aaa ccc tac tca 2197Leu Gln Leu Phe Phe Met Pro Gly Gln Arg
Pro His Lys Pro Tyr Ser 490 495
500gga acc gtc cgc gtc gcg ttt cgg tct gat ata aca aac cag tgc tac
2245Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile Thr Asn Gln Cys Tyr
505 510 515cag gaa ctt agc gag gag
cgc ttt gaa aat tgc act cat cga tcg tct 2293Gln Glu Leu Ser Glu Glu
Arg Phe Glu Asn Cys Thr His Arg Ser Ser 520 525
530tct gtt ttt gtc ggc tgt aaa gtg acc gag tac acg ttc tcc
gcc tcg 2341Ser Val Phe Val Gly Cys Lys Val Thr Glu Tyr Thr Phe Ser
Ala Ser 535 540 545aac aga cta acc gga
cct cca cac ccg ttt aag ctc act ata cga aat 2389Asn Arg Leu Thr Gly
Pro Pro His Pro Phe Lys Leu Thr Ile Arg Asn550 555
560 565cct cgt ccg aac gac agc ggg atg ttc tac
gta att gtt cgg cta gac 2437Pro Arg Pro Asn Asp Ser Gly Met Phe Tyr
Val Ile Val Arg Leu Asp 570 575
580gac acc aaa gaa ccc att gac gtc ttc gcg atc caa cta tcg gtg tat
2485Asp Thr Lys Glu Pro Ile Asp Val Phe Ala Ile Gln Leu Ser Val Tyr
585 590 595caa ttc gcg aac acc gcc
gcg act cgc gga ctc tat tcc aag gct tcg 2533Gln Phe Ala Asn Thr Ala
Ala Thr Arg Gly Leu Tyr Ser Lys Ala Ser 600 605
610tgt cgc acc ttc gga tta cct acc gtc caa ctt gag gcc tat
ctc agg 2581Cys Arg Thr Phe Gly Leu Pro Thr Val Gln Leu Glu Ala Tyr
Leu Arg 615 620 625acc gag gaa agt tgg
cgc aac tgg caa gcg tac gtt gcc acg gag gcc 2629Thr Glu Glu Ser Trp
Arg Asn Trp Gln Ala Tyr Val Ala Thr Glu Ala630 635
640 645acg acg acc agc gcc gag gcg aca acc ccg
acg ccc gtc act gca acc 2677Thr Thr Thr Ser Ala Glu Ala Thr Thr Pro
Thr Pro Val Thr Ala Thr 650 655
660agc gcc tcc gaa ctt gaa gcg gaa cac ttt acc ttt ccc tgg cta gaa
2725Ser Ala Ser Glu Leu Glu Ala Glu His Phe Thr Phe Pro Trp Leu Glu
665 670 675aat ggc gtg gat cat tac
gaa ccg aca ccc gca aac gaa aat tca aac 2773Asn Gly Val Asp His Tyr
Glu Pro Thr Pro Ala Asn Glu Asn Ser Asn 680 685
690gtt act gtc cgt ctc ggg aca atg agc cct acg cta att ggg
gta acc 2821Val Thr Val Arg Leu Gly Thr Met Ser Pro Thr Leu Ile Gly
Val Thr 695 700 705gtg gct gcc gtc gtg
agc gca acg atc ggc ctc gtc att gta att tcc 2869Val Ala Ala Val Val
Ser Ala Thr Ile Gly Leu Val Ile Val Ile Ser710 715
720 725atc gtc acc aga aac atg tgc acc ccg cac
cga aaa tta gac acg gtc 2917Ile Val Thr Arg Asn Met Cys Thr Pro His
Arg Lys Leu Asp Thr Val 730 735
740tcg caa gac gac gaa gaa cgt tcc caa act aga agg gaa tcg cga aaa
2965Ser Gln Asp Asp Glu Glu Arg Ser Gln Thr Arg Arg Glu Ser Arg Lys
745 750 755ttt gga ccc atg gtt gcg
tgc gaa ata aac aag ggg gct gac cag gat 3013Phe Gly Pro Met Val Ala
Cys Glu Ile Asn Lys Gly Ala Asp Gln Asp 760 765
770agt gaa ctt gtg gaa ctg gtt gcg att gtt aac ccg tct gcg
cta agc 3061Ser Glu Leu Val Glu Leu Val Ala Ile Val Asn Pro Ser Ala
Leu Ser 775 780 785tcg ccc gac tca ata
aaa atg tga ttaagtctga atgtggctct ccaatcattt 3115Ser Pro Asp Ser Ile
Lys Met790 795cgattctcta atctcccaat cctctcaaaa ggggcagtat
cggacacgga ctgggagggg 3175cgtacacgat agttatatgg tacagcagag gcctctgaac
acttaggagg agaattcagc 3235cggggagagc ccctgttgag taggcttggg agcatattgc
aggatgaaca tgttagtgat 3295agttctcgcc tcttgtcttg cgcgcctaac ttttgcgacg
cgacacgtcc tctttttgga 3355aggcactcag gctgtcctcg gggaagatga tcccagaaac
gttccggaag ggactgtaat 3415caaatggaca aaagtcctgc ggaacgcgtg caagatgaag
gcggccgatg tctgctcttc 3475gcctaactat tgctttcatg atttaattta cgacggagga
aagaaagact gcccgcccgc 3535gggacccctg tctgcaaacc tggtaatttt actaaagcgc
ggcgaaagct tcccgggtta 3595attaacgtac
36054434PRTInfectious Laryngotracheitis Virus 4Met
His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly1
5 10 15Glu Val Leu Asn Lys His Met
Asp Cys Gly Gly Lys Arg Cys Cys Ser 20 25
30Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile
Met Arg 35 40 45Glu His Ile Cys
Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu 50 55
60Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe
Ala Ser Gln65 70 75
80Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu
85 90 95Asp Ala Leu Thr Ile Pro
Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr 100
105 110Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn
Pro Ile Ser Asn 115 120 125Val Asp
Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro 130
135 140Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile
Lys Gly Asp Asp Gly145 150 155
160Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly
165 170 175Asp Val Gln Leu
Leu Ser Glu Cys Ala Val Gln Ser Ala Gln Met Trp 180
185 190Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser
Arg Asn Gly Ala Gly 195 200 205Leu
Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu 210
215 220Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr
Ala Leu Val Thr Leu Glu225 230 235
240Val Asn Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu
Pro 245 250 255Ser Lys Cys
Trp Thr Thr Glu Gln Tyr Gln Thr Gly Phe Gln Gly Glu 260
265 270His Leu Tyr Pro Ile Ala Asp Thr Asn Thr
Arg His Ala Asp Asp Val 275 280
285Tyr Arg Gly Tyr Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg 290
295 300Lys Lys Asn Pro Ser Ala Pro Asp
Pro Arg Pro Asp Ser Val Pro Gln305 310
315 320Glu Ile Pro Ala Val Thr Lys Lys Ala Glu Gly Arg
Thr Pro Asp Ala 325 330
335Glu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Glu Asp Asp Met
340 345 350Gln Ala Glu Ala Ser Gly
Glu Asn Pro Ala Ala Leu Pro Glu Asp Asp 355 360
365Glu Val Pro Glu Asp Thr Glu His Asp Asp Pro Asn Ser Asp
Pro Asp 370 375 380Tyr Tyr Asn Asp Met
Pro Ala Val Ile Pro Val Glu Glu Thr Thr Lys385 390
395 400Ser Ser Asn Ala Val Ser Met Pro Ile Phe
Ala Ala Phe Val Ala Cys 405 410
415Ala Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala
420 425 430Arg
Ser5362PRTInfectious Laryngotracheitis Virus 5Met Ala Ser Leu Leu Gly Thr
Leu Ala Leu Leu Ala Ala Thr Leu Ala1 5 10
15Pro Phe Gly Ala Met Gly Ile Val Ile Thr Gly Asn His
Val Ser Ala 20 25 30Arg Ile
Asp Asp Asp His Ile Val Ile Val Ala Pro Arg Pro Glu Ala 35
40 45Thr Ile Gln Leu Gln Leu Phe Phe Met Pro
Gly Gln Arg Pro His Lys 50 55 60Pro
Tyr Ser Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile Thr Asn65
70 75 80Gln Cys Tyr Gln Glu Leu
Ser Glu Glu Arg Phe Glu Asn Cys Thr His 85
90 95Arg Ser Ser Ser Val Phe Val Gly Cys Lys Val Thr
Glu Tyr Thr Phe 100 105 110Ser
Ala Ser Asn Arg Leu Thr Gly Pro Pro His Pro Phe Lys Leu Thr 115
120 125Ile Arg Asn Pro Arg Pro Asn Asp Ser
Gly Met Phe Tyr Val Ile Val 130 135
140Arg Leu Asp Asp Thr Lys Glu Pro Ile Asp Val Phe Ala Ile Gln Leu145
150 155 160Ser Val Tyr Gln
Phe Ala Asn Thr Ala Ala Thr Arg Gly Leu Tyr Ser 165
170 175Lys Ala Ser Cys Arg Thr Phe Gly Leu Pro
Thr Val Gln Leu Glu Ala 180 185
190Tyr Leu Arg Thr Glu Glu Ser Trp Arg Asn Trp Gln Ala Tyr Val Ala
195 200 205Thr Glu Ala Thr Thr Thr Ser
Ala Glu Ala Thr Thr Pro Thr Pro Val 210 215
220Thr Ala Thr Ser Ala Ser Glu Leu Glu Ala Glu His Phe Thr Phe
Pro225 230 235 240Trp Leu
Glu Asn Gly Val Asp His Tyr Glu Pro Thr Pro Ala Asn Glu
245 250 255Asn Ser Asn Val Thr Val Arg
Leu Gly Thr Met Ser Pro Thr Leu Ile 260 265
270Gly Val Thr Val Ala Ala Val Val Ser Ala Thr Ile Gly Leu
Val Ile 275 280 285Val Ile Ser Ile
Val Thr Arg Asn Met Cys Thr Pro His Arg Lys Leu 290
295 300Asp Thr Val Ser Gln Asp Asp Glu Glu Arg Ser Gln
Thr Arg Arg Glu305 310 315
320Ser Arg Lys Phe Gly Pro Met Val Ala Cys Glu Ile Asn Lys Gly Ala
325 330 335Asp Gln Asp Ser Glu
Leu Val Glu Leu Val Ala Ile Val Asn Pro Ser 340
345 350Ala Leu Ser Ser Pro Asp Ser Ile Lys Met
355 360
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