Patent application title: REOVIRUS COMPOSITIONS AND METHODS OF USE
Inventors:
Holly S. Sellers (Bishop, GA, US)
Assignees:
University of Georgia Research Foundation, Inc.
IPC8 Class: AA61K3915FI
USPC Class:
4241861
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from virus
Publication date: 2012-10-04
Patent application number: 20120251564
Abstract:
The present invention relates to novel strains of avian reovirus isolated
from severe cases of Runting Stunting Syndrome in young broiler chickens
in southeast United States. The invention is directed to avian reoviruses
that impair digestion in poultry, diagnostic assays using nucleotide- or
amino acid-specific components of such viruses, and to vaccines that
protect chickens from disease caused by such viruses. Nucleotide
sequences for the S1 gene, encoding the sigma C minor outer capsid
protein, were amplified, and the nucleotide and predicted amino acid
sequences were compared with sequences from other recently isolated
reovirus field isolates and vaccine strains. Antigenic and molecular
characterization of the newly isolated reoviruses revealed a lack of
homogeneity with current U.S. isolates, with less than 60% percent amino
acid similarity across the sigma C protein. Sequence comparisons with
previously reported malabsorption isolates from Europe and Asia revealed
a higher amino acid similarity, approaching 80%.Claims:
1. A vaccine comprising antigenic material derived from an avian
reovirus, wherein said reovirus comprises an S1 protein, wherein said S1
protein comprises an amino acid sequence set forth in the group
consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, and 30, and wherein said antigenic material is selected from the
group consisting of live virus, live attenuated virus, inactivated virus,
and one or more immunologically active subcomponents, thereof.
2. The vaccine of claim 1, wherein said reovirus comprises an S1 protein comprising the amino acid sequence set forth in SEQ ID NO: 6.
3. The vaccine of claim 1 further comprising one or more vaccines selected from the group consisting of infectious bronchitis virus vaccine, Newcastle disease virus vaccine, infectious bursal disease virus vaccine, fowl adenovirus vaccine, EDS virus vaccine, and turkey rhinotracheitis virus vaccine.
4. A method of eliciting an immune response in a chicken, the method comprising the step of administering to the chicken an amount of a composition comprising an isolated reovirus effective to elicit an immune response in the chicken, wherein the isolated reovirus comprises an S1 protein, and wherein said S1 protein comprises the amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30.
5. The method of claim 4, wherein the isolated reovirus is designated strain 40973 and has ATCC accession number PTA-7174.
6. The method of claim 4, wherein the composition further comprises a pharmaceutically acceptable carrier, adjuvant, or diluent.
7. The method of claim 4, wherein the isolated reovirus is an attenuated live reovirus.
8. The method of claim 4, wherein the isolated reovirus is an inactivated reovirus.
9. The method of claim 4, wherein the composition comprises at least 10.sup.2 titration units of the reovirus.
10. The method of claim 4, wherein the composition is introduced into the chicken on multiple occasions.
11. The method of claim 4, wherein the composition is introduced into the chicken during a time period extending from day 1 after hatching to week 18 after hatching.
12. The method of claim 4, wherein the composition further comprises additional isolated virus particles selected from the group consisting of infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, EDS virus, and turkey rhinotracheitis virus.
13. A method for measuring the amount of a reovirus in a chicken, the method comprising the steps of: (a) contacting a chicken sample with an antibody that selectively binds to a reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, and (b) measuring the amount of binding of the antibody to a component of the chicken sample.
14. The method of claim 13, wherein an ELISA is used to measure the amount of binding of the antibody to a component of the chicken sample.
Description:
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No. 12/440,762, filed Jun. 12, 2009, which is the National Stage of International Application No. PCT/US2007/082556, filed Oct. 25, 2007, which claims the benefit of U.S. Provisional Application No. 60/862,958, filed Oct. 25, 2006, the disclosures of which are incorporated herein by reference.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 39189_Sequence_FINAL--20120410.txt. The text file is 115 KB; was created on Apr. 10, 2012; and is being submitted via EFS-Web with the filing of the specification.
FIELD OF THE INVENTION
[0003] The present invention relates to novel strains of avian reovirus that were isolated from severe cases of Runting Stunting Syndrome in young broiler chickens in the southeast United States. The invention is directed to avian reoviruses that impair digestion in poultry, diagnostic assays using nucleotide- or amino acid-specific components of such viruses, such as the S1 gene encoding the sigma C minor outer capsid protein, and to vaccines that protect chickens from disease caused by such viruses.
BACKGROUND OF THE INVENTION
[0004] Avian and mammalian reoviruses comprise the genus Orthoreovirus in the family Reoviridae. These viruses contain 10 dsRNA genome segments enclosed within a non-enveloped, icosahedral double capsid of approximately 80 nm (Joklik, W. K., In: The reoviridae. W. K. Joklik, ed., Plenum Press, New York, pp. 9-78, 1983; Schnitzer, T. J. et al. J. Virol. 43:1006-1014, 1982). The genome segments can be separated based on electrophoretic mobility into three large (L1,2,3), three medium (M1,2,3) and four small (S1,2,3,4) segments which encode proteins λ1, λ2, λ3, μ1, μ2, μNS, σ3, σ1, σ2, σNS, respectively (Spandidos D. A., and Graham, A. F., J. Virol. 19: 968-976, 1976; Varela, R. and Benavente, J., J. Virol. 68: 6775-6777, 1994; Wu, W. Y. et al., J. Virol. Methods 48: 119-122, 1994). The σ2 protein is an outer capsid protein which carries group-specific neutralizing epitopes, binds double-stranded RNA, and has been identified as a zinc metalloprotein (Wickramsinghe et al., Virology 194:688-696, 1993; Nibert, M. L. and Schiff, L. A., In: Fields virology, 4th ed., vol. 2. Knipe, D. M., and Howley, P. M., eds. Lippincott Williams & Wilkins, Philadelphia, Pa., p. 3087, 2001).
[0005] Avian reoviruses (ARV) are a diverse group of poultry pathogens whose virulence varies greatly, among isolates within different hosts. ARVs have been isolated from turkeys with poult enteritis and mortality syndrome (PEMS) (Heggen-Peay, C. L., et al., Avian Dis. 46: 32-47, 2002) as well as from chickens and ducks. These isolates have been associated with enteric and respiratory disease (Fahey, J. E., and Crawley, J. F., Can. J. Comp. Med. 18: 13-21, 1954), viral arthritis/tenosynovitis (Glass, S. E., et al., Avian Dis. 17: 415-424, 1973), malabsorption and stunting syndrome (Rosenberger, J. K., In: Diseases of poultry, 11th ed. Barnes, H. J., et al., eds. Iowa State University Press, Ames Iowa, pp. 284-293, 2003). Not all reoviruses are highly virulent, as they can be isolated from chickens or turkeys exhibiting no clinical signs of disease.
[0006] RSS is a disease of domesticated chickens that causes a variety of symptoms, such as delayed growth, lack of flock uniformity, a small liver with an enlarged gall bladder, pale, thin, almost translucent, intestinal walls, large amounts of fluid inside the small and large intestines, occasional increased amount of pericardial fluid, and sporadic white, or cream-colored, plaques in individual proventricular glands. Various microscopic lesions have also been observed, and frequently noted as multiple cysts in the intestinal crypts. The early lesion is clinically considered to be a form of cystic enteropathy, and which evolves into an inflammatory lesion causing cystic enteritis. As the lesions progress, they may result in shortening and clubbing of the intestinal villi. Outbreaks of RSS reduce the growth rate, body weight, and size, and increase the mortality in affected chicken populations.
[0007] Runting-Stunting Syndrome can been reproduced in the clinic. Gavage inoculations with intestinal contents from affected chickens, for example, cause RSS in the inoculated chickens. Inoculation with filtered gut contents from affected chickens can also cause RSS, suggesting that a virus is responsibly for the syndrome, since the filters used to inoculate the gut contents are able to exclude bacteria. RSS has also been reproduced in experiments that place healthy broiler chickens on litter contaminated by affected chickens.
[0008] Runting-Stunting Syndrome has a significant economic impact on the chicken farming industry. Delayed growth during the first few weeks of age, for example, increases the cost of feed conversion. In 2004, broiler companies across the southeast United States began seeing more cases of Runting-Stunting Syndrome (RSS) during the winter and spring seasons. Losses due to RSS at one company were estimated to be over $100,000 per week per million birds. These impacts demonstrate a continuing need for compositions and methods for ameliorating at one or more symptoms of RSS, for identifying and characterizing the causative agent(s) of RSS, and for preventing the onset of RSS in chickens.
[0009] While viral agents, environmental conditions, and genetic factors are all believed to play a role in determining the susceptibility of chickens to RSS, vaccines against various infectious agents hold much promise in containing this and related diseases. Vaccines can be divided into two general groups: live (attenuated) vaccines, and inactivated vaccines. Live vaccines can present of all the relevant immunogenic determinants of an infectious agent in their natural forms to the host's immune system, and because they can multiply in the vaccinated host, only require a small amount of the immunizing agent. Safety is often a concern with live vaccines, however, as they may induce disease in immuno-compromised animals, or they may revert to a virulent form, leading to more serious infections. Live vaccines may also impair reproductive function. Inactivated vaccines by comparison are generally far safer than live vaccines. One major disadvantage, however, is their intrinsically low immunogenicity (i.e., ability to trigger the host's immune system). Adjuvants with significant immunostimulatory capabilities are often necessary, then, to augment the immunogenicity of inactivated immunogens to reach a minimum potential suitable for preventing disease in a vaccinated bird or preventing the spread of infection and disease to unvaccinated birds.
[0010] Vaccination of poultry is useful for several reasons. One purpose is to prevent egg production losses in commercial layers. Vaccines may also be used to reduce the level of transmission by eggs in breeding stock or as a tool for eradicating disease in infected flocks on multi-age production sites. Thus there remains a need for safe, efficacious, and inexpensive vaccines that sufficient to protect poultry susceptible to etiological agents, such as avian reoviruses, that may cause Runting Shunting Syndrome or related diseases. Vaccines effective against avian reoviruses, for example, may optionally be combined with vaccines that protect poultry from other commercially-troublesome infectious diseases. The formulation should also be stable, and provide lasting protection without undesirable side effects.
SUMMARY OF THE INVENTION
[0011] The present invention relates to a composition comprising an isolated avian reovirus comprising an S1 protein comprising the amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30.
[0012] The invention also relates to a vaccine comprising antigenic material derived from an avian reovirus wherein said reovirus comprises an S1 protein comprising an amino acid sequence set forth in the group consisting of SEQ ID NOS: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, wherein said antigenic material is selected from the group consisting of live virus, live attenuated virus, inactivated virus, and one or more immunologically active subcomponents, thereof.
[0013] The invention also relates to a method of eliciting an immune response in a chicken, the method comprising the step of introducing into a chicken a composition comprising an isolated reovirus comprising an S1 protein comprising the amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30.
[0014] The invention also relates to a method for measuring the amount of a reovirus in a chicken, the method comprising the steps of: (a) contacting a chicken sample with an antibody that selectively binds to a reovirus that comprises an S1 protein that comprises an amino acid sequence selected from the group set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, and (b) measuring the amount of binding of the antibody to a component of the chicken sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
[0016] FIGS. 1A-1F sets forth a sequence alignment among avian reoviruses based on their sigma C (S1) amino acid sequences. The alignment was performed using ClustalW software.
[0017] FIG. 2 sets forth the phylogenetic relationships among avian reoviruses based on the sigma C(S1) amino acid sequences. The phylogenetic tree was constructed following amino acid alignment by neighbor-joining method using maximum parsimony analysis. The nomenclature for naming of the reoviruses is typically State/Species/Case ID/Year of Isolation. Reoviruses isolated by the inventors from clinical cases of Runting and Stunting Syndrome are indicated with an asterisk. U.S. vaccine viruses are circled.
DEFINITIONS
[0018] The following is a list of terms and their definitions used throughout the specification and the claims:
[0019] The term "isolated" when used with respect to a reovirus, means a reovirus that is substantially free from the cellular components that are associated with the reovirus as it is found in nature. In this context, "substantially free from cellular components" means that the reovirus is purified to a level of greater than 80% (such as greater than 90%, greater than 95%, or greater than 99%).
[0020] The term "isolated reovirus" means reoviruses that have been isolated from cellular components and then combined with pharmaceutically-acceptable components (e.g., carriers, adjuvants, and/or diluents) to facilitate the administration of the reovirus to a chicken for the subsequent production of antibodies against the reovirus.
[0021] The term "Galliformes" refers to an order of birds containing turkeys, grouse, chickens, quails, and pheasants. About 256 species are found worldwide.
[0022] Abbreviations and their corresponding meanings include: aa or AA=amino acid; ARV=avian reovirus; MAS=malabsorption syndrome mg=milligram(s); ml or mL=milliliter(s); mm=millimeter(s); mM=millimolar; nmol=nanomole(s); pmol=picomole(s); ppm=parts per million; RSS=Runting Stunting Syndrome; RT=room temperature; U=units; ug, μg=microgram(s); ul, μl=microliter(s); uM, μM=micromolar.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0023] The present invention relates to compositions and vaccines comprising antigenic components capable of protecting birds of the order Galliformes, including chickens, against Runting Stunting Syndrome. The compositions and vaccines may comprise an avian reovirus, or one or more of its subcomponents, capable of stimulating a protective immune response. The invention also relates to methods of stimulating an immune response by administering said compositions or vaccines to a susceptible bird.
[0024] The invention relates to a composition comprising an isolated avian reovirus comprising an S1 protein comprising the amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30.
[0025] The composition may comprise the isolated reovirus designated as strain 40973, or a progeny thereof. Reovirus strain 40973 has been deposited in the American Type Culture Collection, 10001 University Boulevard, Manassas, Va., on Oct. 20, 2005, and accorded has ATCC accession number PTA-7174. Compositions of the invention may further comprising a pharmaceutically acceptable carrier, adjuvant, or diluent. The compositions may comprise an isolated live reovirus, a live reovirus that is attenuated, or an isolated reovirus that is inactivated. The invention also relates to compositions comprising at least 102 titration units of the reovirus. The invention also relates to compositions that further comprise additional isolated virus particles that are different from the isolated reovirus comprising an S1 protein comprising the amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. Examples of additional isolated virus particles include infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, EDS virus, and turkey rhinotracheitis virus.
[0026] The invention also relates to a method of eliciting an immune response in a chicken, the method comprising the step of introducing into a chicken a composition comprising an isolated reovirus comprising an S1 protein comprising the amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. One aspect of the invention relates to a method wherein the isolated reovirus is reovirus strain 40973, ATCC accession number PTA-7174. Another aspect of the invention relates to a method wherein the composition further comprises a pharmaceutically acceptable carrier, adjuvant, or diluent. The composition may comprise an attenuated live reovirus or an inactivated reovirus. Another aspect of the invention relates to a method wherein the composition comprises at least 102 titration units of the reovirus. The compositions of the invention may be introduced into the chicken on multiple occasions, such as during a time period extending from day 1 after hatching to week 18 after hatching. The invention also relates to method of eliciting an immune response in a chicken, using compositions comprising an isolated reovirus, as described above, wherein the composition further comprises additional isolated virus particles selected from the group consisting of infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, EDS virus, and turkey rhinotracheitis virus.
[0027] The invention also relates to a vaccine comprising antigenic material derived from an avian reovirus wherein said reovirus comprises an S1 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, wherein said antigenic material is selected from the group consisting of live virus, live attenuated virus, inactivated virus, and one or more immunologically active subcomponents, thereof. One aspect of the invention relates to a vaccine wherein said reovirus comprises an S1 protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
[0028] Another aspect of the invention relates to a combination vaccine, comprising antigenic material derived from an avian reovirus and one or more vaccines selected from the group consisting of infectious bronchitis virus vaccine, Newcastle disease virus vaccine, infectious bursal disease virus vaccine, fowl adenovirus vaccine, EDS virus vaccine, and turkey rhinotracheitis virus vaccine.
[0029] The invention also relates to a method for measuring the amount of a reovirus in a chicken, the method comprising the steps of: (a) contacting a chicken sample with an antibody that selectively binds to a reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, and (b) measuring the amount of binding of the antibody to a component of the chicken sample. One aspect of the invention relates to a method of the invention wherein an ELISA is used to measure the amount of binding of the antibody to a component of the chicken sample.
[0030] The present invention also relates to an isolated reovirus strain, exemplified by the strain designated 40973 and related viruses, which can be identified by the amino acid sequence of their small segment 1 (S1, sigma C, or σC) outer capsid protein, set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. Strain 40973 and these related viruses, for example, causes at least some of the symptoms of Runting-Stunting Syndrome, when the virus is administered to healthy chickens.
[0031] The present invention provides compositions that comprise an isolated reovirus comprising an S1 protein comprising the amino acid sequence set forth in SEQ ID NO: 2. The invention also provides a novel isolated reovirus designated strain 40973. The invention also provides methods of eliciting an immune response in a chicken against a reovirus.
[0032] One aspect of the invention relates to compositions that each comprise an isolated reovirus, wherein the isolated reovirus comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The composition can be, for example, a liquid, gel or solid (e.g., powder). The compositions of this aspect of the invention can be used, for example, to store strain 40973 or related viruses for future use or characterization (e.g., as a dry powder); to characterize strain 40973 or related viruses (e.g., in a liquid composition to assess the specificity of antibodies directed against strain 40973); to propagate strain 40973 or related viruses (e.g., as inoculum used to inoculate growth media to grow more virus particles).
[0033] Compositions of the invention can also be used to raise antibodies against strain 40973, or related viruses, such as those described in Table 1. For example, in some embodiments, compositions of the present invention comprise strain 40973 particles, or particles from related viruses, and at least one pharmaceutically acceptable carrier (e.g., saline) and, optionally, an adjuvant or diluent. These compositions can be administered to a chicken in order to raise antibodies in the chicken against strain 40973 or related viruses.
[0034] Some compositions of the present invention are vaccines that cause the production of antibodies against strain 40973 or related viruses in vaccinated chickens. The antibodies protect the chickens from disease caused by subsequent infection by reovirus strain 40973 or related viruses.
[0035] Compositions of the invention can also, for example, be attached to a solid substrate and used to purify anti-40973 antibodies, or antibodies from related viruses, which releasably bind to the immobilized composition.
[0036] The present invention provides compositions that each comprise an isolated reovirus, wherein the isolated reovirus comprises an S1 protein that comprises an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The isolated reovirus can be in a live, live attenuated, or inactivated form.
[0037] An example of an isolated reovirus that comprises an S1 protein that comprises the amino acid sequence set forth in SEQ ID NO: 2 is avian reovirus strain 40973. SEQ ID NO: 2 shows the amino acid sequence of the S1 protein from the avian reovirus strain designated 40973.
[0038] An isolated reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 can be isolated from an infected chicken. A typical broiler to be used for this purpose shows the following signs: the secretion of too liquid faeces and/or maldigested feed resulting in growth retardation.
[0039] By way of example, the intestine is isolated from the affected chicken followed by homogenization of the organ in a suitable buffer. Thereafter, the homogenized tissue is clarified by centrifugation and the supernatant is filtrated through filters with a pore size of 0.2 μm. A sample of the filtrate is added to freshly prepared primary chicken cells, preferably chicken embryo liver (CEL) cells, prepared from specific pathogen free (SPF) embryos, and observed daily for the presence of a cytopathic effect (CPE). If no CPE is present after 5 to 6 days, a freeze/thawed suspension of the first monolayer is added to freshly prepared CEL cells. If after the first passage or the second passage CPE is observed, then the virus is further characterized by its in vivo properties in broilers to induce decreased body weight and lack of uniformity, and by determining the amino acid sequence of its S1 protein. A more detailed method for the isolation of an avian reovirus that comprises an S1 protein that comprises the amino acid sequence set forth in SEQ ID NO: 2 is disclosed in Example 1.
[0040] An avian reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 can be isolated from other parts of an infected chicken, such as the liver of affected broiler chickens or from the faeces excreted by such a broiler chicken.
[0041] Compositions of the present invention that are formulated to be administered to a chicken (e.g., vaccines) may comprise physiologically-acceptable carrier. Examples of physiologically-acceptable carriers include, but are not limited to, sterile water, aqueous saline solutions (e.g., a 0.9% NaCl solution), phosphate buffers, aqueous dextrose or glycerol solutions, proteins, and sugars.
[0042] Compositions of the present invention that are formulated to be administered to a chicken to elicit an immune response in the chicken can optionally comprise an adjuvant. Examples of adjuvants include, but are not limited, to aluminum hydroxide, aluminum phosphate, aluminum oxide, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol F° or Marcol 52® or a vegetable oil such as vitamin E acetate, and saponins.
[0043] Compositions of the present invention include, for example, compositions that elicit an immune response in a chicken. The immune response may, or may not, confer protective immunity on the chicken. An immune response may, for example, include one or more of the following: (a) a cell mediated immune response, which involves the production of lymphocytes 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.
[0044] Compositions of the present invention include, for example, vaccines. The term "vaccine", as used herein, broadly refers to any composition that may be administered to a chicken to cause the production of antibodies that protect the animal against disease caused by a reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. A vaccine of the present invention comprises an isolated reovirus (that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30), a physiologically acceptable carrier, and optionally, an adjuvant and/or diluent.
[0045] The isolated reovirus can be incorporated into the vaccine as a live attenuated or inactivated virus. The ability of the avian reovirus to induce some, or all, of the symptoms of Runting-Stunting Syndrome is significantly reduced, or completely absent, if the avian reovirus is in a live attenuated or inactivated form.
[0046] Attenuation of an avian reovirus useful in the practice of the present invention can be achieved by methods well known in the art for this purpose, such as disclosed in Gouvea et al. (Virology 126:240-247, 1983). Briefly, and by way of example, after the isolation of the virus from a target animal, a viral suspension is inoculated onto primary chicken embryo liver cells. If the isolate is not able to produce cytopathic effects (CPE), then the virus is passaged repeatedly (e.g., 3-10 times) until CPE is observed. As soon as CPE is visible, cells and cell culture fluids are collected, frozen and thawed, clarified by centrifugation and the supernatant containing the avian reovirus isolate is aliquoted and stored at -20° C. This process may be repeated (e.g., 10-100 times) to further attenuate the virus.
[0047] A vaccine according to the invention can be prepared by conventional methods such as, for example, commonly used for the commercially available live and inactivated reovirus vaccines. The preparation of veterinary vaccine compositions is described, for example, in Vaccines for Veterinary Applications (Peters, A. R., et al., eds., Butterworth-Heinemann Ltd, 1993). In brief, a susceptible substrate is inoculated with an avian reovirus according to the invention in a live or live attenuated form, and propagated until the virus is replicated to a desired infectious titer, or antigen mass content. Reovirus containing material is then harvested and formulated to a pharmaceutical composition with prophylactic activity.
[0048] Any substrate which is able to support the replication of an isolated avian reovirus of the present invention, if necessary after adaptation of the avian reovirus to the substrate, can be used to produce a vaccine according to the present invention. Suitable substrates include primary (avian) cell cultures, such as chicken embryo liver cells (CEL), chicken embryo fibroblasts (CEF) or chicken kidney cells (CK), mammalian cell lines such as the VERO cell line or the BGM-70 cell line, or avian cell lines such as QT-35, QM-7 or LMH. Usually, after inoculation of the cells, the virus is propagated for 3-10 days, after which the cell culture supernatant is harvested and, if desired, filtered or centrifuged in order to remove cell debris. Alternatively, an isolated avian reovirus according to the invention can be propagated in embrocated chicken eggs followed by harvesting the avian reovirus material by routine methods.
[0049] Vaccines of the present invention comprising a live attenuated virus can be prepared and marketed in the form of a (frozen) suspension or in a lyophilized form. The vaccine additionally contains a pharmaceutically acceptable carrier or diluent customarily used for such compositions. Carriers include stabilizers, preservatives and buffers. Suitable stabilizers are, for example, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, merthiolate, and gentamicin. Diluents include water, aqueous buffer (such as buffered saline), alcohols, and polyols (such as glycerol).
[0050] Although administration by injection (e.g., intramuscular, subcutaneous) of the live vaccine is possible, the live vaccine is preferably administered by the inexpensive mass application techniques commonly used for avian reovirus vaccination. These techniques include introducing the vaccine into drinking water imbibed by chickens, and spray vaccination. Alternative methods for the administration of the live vaccine include in ovo, eye drop, and beak dipping administration.
[0051] Vaccines of the present invention can also comprise an inactive form of an isolated reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. An advantage of an inactivated vaccine is the elevated levels of protective antibodies of long duration that can be obtained. This property makes such an inactivated vaccine particularly suited for breeder vaccination.
[0052] The aim of inactivation of the viruses after the propagation step is to prevent the viruses from replicating. In general, this can be achieved by chemical or physical means. Chemical inactivation can be effected by treating the viruses with, for example, enzymes, formaldehyde, β-propiolactone, ethylene-imine or a derivative thereof. If necessary, the inactivating compound is neutralized afterwards. Material inactivated with formaldehyde can, for example, be neutralized with thiosulphate. Physical inactivation can preferably be carried out by subjecting the viruses to energetic radiation, such as UV light or γ-rays. If desired, after treatment, the pH can be adjusted to a value of about 7.
[0053] A vaccine comprising the inactivated avian reovirus can, for example, comprise one or more of the above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose.
[0054] Typically, an inactivated vaccine according to the invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminum hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol F° or Marcol 52® or a vegetable oil such as vitamin E acetate, and saponins. Inactivated vaccines are usually administered parenterally (e.g., intramuscularly or subcutaneously).
[0055] A vaccine according to the invention has an effective dosage of the avian reovirus as the active component, i.e., an amount of immunizing avian reovirus material that will induce immunity in the vaccinated birds or their progeny against challenge by a virulent reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. Immunity is defined herein as the induction of a significant higher level of protection in a population of birds after vaccination compared to an unvaccinated group.
[0056] Typically, the live vaccine according to the invention can be administered in a dose of not less than 102 titration units (wherein titration units are defined in Title 9, Section 113.332, Code of Federal Regulations) per bird, and an inactivated vaccine may contain the antigenic equivalent of 104-1010 TCID50 per bird (wherein TCID is an abbreviation for Tissue Culture Infective Dose).
[0057] The avian reovirus vaccine according to the present invention may be used effectively in chickens. Chickens include broilers, reproduction stock, and laying stock. Chickens can be vaccinated at any suitable age, and are usually about one to three days old before first vaccination. The chickens may be vaccinated only once. Optionally, when two doses of vaccine are used, the first is given, for example, when the chickens are 3 days to a week old and subsequently after a further 1-10 weeks.
[0058] Multiple doses of the composition can be administered throughout the life of the chicken. As maternal immunity is a primary source of providing protection to broiler progeny, breeder chickens are typically vaccinated, although broiler chickens can be vaccinated if so desired.
[0059] The following, exemplary, vaccination regime can be applied to breeders, grandparents, and to great-grandparents: [0060] Day 1: Mild live tissue culture attenuated (subcutaneous). [0061] Weeks 3-4: Attenuated live (oral in the drinking water). [0062] Weeks 6-7: Attenuated live (oral in the drinking water). [0063] Week 12: Killed vaccine (subcutaneous or intramuscular). [0064] Week 18: Killed vaccine (subcutaneous or intramuscular).
[0065] Broilers may, for example, be vaccinated directly at one day of age with the live attenuated vaccine according to the invention. Vaccination of parent stock, such as broilers breeders, can be done with a live mild attenuated or inactivated vaccine according to the invention, or with combinations of both. The advantages of this type of immunization program includes the immediate protection of one-day-old progeny provided by maternally derived antibodies vertically transmitted to the young birds. A further example of a breeder vaccination program includes the vaccination of the breeders at one day of age with a live vaccine, and again at 6 weeks of age with a live attenuated vaccine, followed by a vaccination between 14-18 weeks of age with an inactivated vaccine. Alternatively, the live vaccination may be followed by two vaccinations with inactivated vaccines on 10-12 weeks and 16-18 weeks of age.
[0066] The invention also includes combination vaccines including, in addition to an isolated reovirus that comprises an S1 protein that comprises the amino acid sequence set forth in SEQ ID NO: 2, one or more vaccine components of other pathogens infectious to poultry. Typically, the vaccine components in the combination vaccine are the live attenuated or inactivated forms of the pathogens infectious to poultry. For example, a combination vaccine of the present invention can comprise one or more (inactivated) vaccine strains of reovirus, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), fowl adenovirus (FAV), EDS virus and turkey rhinotracheitis virus (TRTV).
[0067] In another aspect, the present invention provides methods of eliciting an immune response in a chicken (Gallus gallus). The methods of this aspect of the invention each comprise the step of introducing into a chicken a composition that comprises an isolated reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. By way of example, the isolated reovirus can be reovirus strain 40973. The compositions typically comprises a pharmaceutically acceptable carrier, and may optionally comprise an adjuvant and/or diluent. The elicited immune response may, or may not, provide the chicken with immunity to further infection by an isolated reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The compositions of the present invention (that comprise an isolated reovirus, wherein the isolated reovirus comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30) can be used in the practice of the methods of the present invention.
[0068] The composition (that comprises an isolated reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30) can be introduced into the chicken by any useful route; for example by intramuscular, intradermal or subcutaneous injection (e.g., by injection into the muscles of the breast, wing, leg, tail, head, or neck), or via intranasal or oral administration (e.g., administration on feed or in drinking water, gels, or sprays). The composition can be administered to a chicken, for example, by a syringe or a needleless apparatus (e.g., Pigjet or Biojector (Bioject, Oregon, USA)).
[0069] In another aspect, the present invention provides methods for measuring the amount of a reovirus in a chicken, wherein the reovirus comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The methods of this aspect of the invention comprise the steps of (a) contacting a chicken sample with an antibody that selectively binds to a reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, and (b) measuring the amount of binding of the antibody to a component of the chicken sample. The antibody can, for example, selectively bind to a portion of the S1 protein that comprises an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The chicken sample can be any biological material obtained from a chicken, such as tissue, bone, blood, urine or faeces. The methods of this aspect of the invention are useful, for example, for determining whether chickens are infected with a reovirus that comprises an S1 protein that comprises an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. Infected chickens can be killed in order to prevent spread of the reovirus to other chickens.
[0070] By way of example, the amount of a reovirus in a chicken can be measured using an Enzyme Linked Immunoassay (abbreviated as ELISA). In the practice of a sandwich-style ELISA, the standards and unknowns are incubated in microtiter wells pre-coated with antibody that captures the reovirus. The samples and standards are diluted in a buffer containing detergent for 1 hour at room temperature prior to loading the microtiter wells. The captured reovirus particles are then detected with tracer-linker antibody (e.g., biotinylated second antibody) that binds to the reovirus which is then detected, for example by using a streptavidin-peroxidase conjugate and colored substrate (e.g., TMB). If TMB is used as the colored substrate then color development is measured at a wavelength of 450 nanometers. The concentration of the reovirus particles in experimental samples is determined using a standard curve prepared from known amounts of the reovirus particles. ELISA techniques are well known to those of ordinary skill in the art and are described, for example, in ELISA and Other Solid Phase Immunoassays: Theoretical and Practical Aspects, Kemeny, D. M., and S. J. Challacombe, eds., Wiley, 1989.
[0071] Antibodies useful in the practice of the present invention include monoclonal antibodies, polyclonal antibodies, single chain antibodies, humanized antibodies, or fragment thereof, also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein (Nature 256:495, 1975), and Galfre, (Meth. Enzymol. 73:3, 1981), which, in brief, involves the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies, or fragments thereof, can be obtained by using methods which are described, for example, in Harlow and Lane, Antibodies, A Laboratory Manual, CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the binding efficiency of the phage antibodies (Schier, Human Antibodies Hybridomas 7:97-105, 1996; Malmborg, J. Immunol. Methods 183:7-13, 1995). The production of chimeric antibodies is described, for example, in WO89/09622. Methods for the production of humanized antibodies are described in, for example, EP-A1 0 239 400 and WO90/07861. A further source of antibodies that can be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies, such as human antibodies in mice, is described in, for example, WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. Useful antibodies can exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab)2, as well as in single chains (see e.g., WO88/09344).
[0072] Useful antibodies, or their corresponding immunoglobulin chain(s), can be further modified using conventional techniques known in the art, for example by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art (see, e.g., Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.).
EXAMPLES
[0073] The foregoing discussion may be better understood in connection with the following representative examples which are presented for purposes of illustrating the principle methods and compositions of the invention and not by way of limitation. Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.
General Materials and Methods
[0074] All parts are by weight (e.g., % w/w), and temperatures are in degrees centigrade (° C.), unless otherwise indicated.
Example 1
[0075] This Example describes the isolation of avian reovirus strain 40973 and related viruses.
Viruses
[0076] Reovirus field isolates, chicken/GA 40973/2005, chicken/AL 40963/2005, chicken/GA 41560/2005, and chicken/GA 41565/2005 were isolated at the Poultry Diagnostic and Research Center at the University of Georgia, from the intestines or feces of chickens exhibiting enteritis. Field isolate chicken/GA 46118/2005 was isolated from a proventricular plaque from an experimentally infected bird. Table 1 provides a summary of the viruses characterized or referred to in all of the studies cited in the Examples. All viruses were isolated and propagated in primary chicken embryo liver cells prepared from 9-11 day of embryonation specific pathogen free (SPF) embryos. All isolates were passaged a total of four times. Upon the development of 70-80% cytopathic effect, observed as syncytial formation, the cell cultures were frozen and stored at -80° C. Each cell culture was frozen and thawed three times before each virus passage in cultured cells.
TABLE-US-00001 TABLE 1 Table of Viruses State of Year of Tissue Species isolation Identification # isolation submitted Chicken GA 40973 2005 Intestines Chicken AL 40963 2005 Proventriculus Chicken GA 41560 2005 Intestines Chicken GA 41565 2005 Intestines Chicken GA 46118 2005 Proventriculus Chicken AR 55957 2006 Intestines Chicken AR 55958 2006 Intestines Chicken AR 55959 2006 Intestines Chicken GA 58554 2007 Intestines Chicken GA 58555 2007 Intestines Chicken GA 60403 2007 Intestines Chicken KY 54647 2006 Tendon, spleen Chicken GA 55255 2006 Spleen Chicken GA 55259 2006 Spleen Chicken MS 42563-1 2005 Intestines Chicken MS 42563-4 2005 Intestines
[0077] Table 2 identifies the nucleotide and amino acid or protein sequences used in the Examples or referred to in this application.
TABLE-US-00002 TABLE 2 Table of Sequences SEQ ID SEQ ID NO NO Name S1 gene Sigma C Description (Isolate) (nt) (aa) (nt, aa) 40973 1 2 GenBank DQ872797, GenBank ABJ09658.1 40963 3 4 GenBank DQ872796, GenBank ABJ09657.1 41560 5 6 GenBank DQ872798, GenBank ABJ09659.1 41565 7 8 GenBank DQ872799, GenBank ABJ09660.1 42563-1 9 10 GenBank DQ872800, GenBank ABJ09661.1 42563-4 11 12 GenBank DQ872801, GenBank ABJ09662.1 55957 13 14 55958 15 16 55959 17 18 58554 19 20 58555 21 22 60403 23 24 54647 25 26 55255 27 28 55259 29 30 Primer P1 31 5'-agt att tgt gag tac gat tg-3' Primer P4 32 5'-agt att tgt gag tac gat tg-3'
RNA Extraction
[0078] Total viral RNA was extracted from primary chicken embryo liver cell passages using the RNeasy kit (Qiagen, Valencia, Calif.) according to the manufacturer's recommendations. Briefly, 48 hours post infection, cell culture media was decanted and the monolayer was lysed with lysing buffer.
RT-PCR
[0079] A cDNA corresponding to the S1 gene was produced by reverse transcription and PCR using SuperScript® III RNase H.sup.- reverse transcriptase and Platinum®Taq DNA polymerase (Invitrogen, Carlsbad, Calif.) using the following primers (Kant, A., et al., Vet. Res. 34:203-212, 2003):
TABLE-US-00003 (SEQ ID NO: 31) P1 (5'- agt att tgt gag tac gat tg -3') and (SEQ ID NO: 32) P4 (5'- ggc gcc aca cct tag gt -3').
[0080] The 1.1 kb amplified products were separated on a 1.0% agarose gel, stained with ethidium bromide and visualized with an ultraviolet transilluminator. The fragment was excised, purified with the QIAEX II gel extraction kit (Qiagen Inc., Valencia, Calif.), and eluted in diethylpyrocarbonate (DEPC)-treated water.
Cloning of Reovirus S1 Gene
[0081] The gel-extracted DNA from the amplification reaction was cloned into plasmid pCR 2.1 (Invitrogen) according to the manufacturer's instructions. Several clones containing plasmids with the 1.1 kb insert were identified by PCR. The clones were expanded and the plasmid DNA purified using Qiagen plasmid miniprep kit according to methods provided by the manufacturer.
Direct Nucleotide Sequencing of Amplified Products
[0082] Purified plasmids and gel-purified PCR products were sequenced with universal sequencing primers or Reovirus S1-specific oligonucleotide primers, respectively, using double-stranded DNA sequencing methods using fluorescently-labeled dideoxynucleotides and Taq polymerase on an ABI 9700 automated sequencer (Applied Biosystems Inc., Foster City, Calif.). A 2× or greater sequence coverage of the S1 gene was obtained for each isolate, with regions of ambiguity resolved by re-sequencing across the desired region.
Sequence Analysis
[0083] Nucleotide sequence editing, predicted amino acid sequence analysis, and multiple alignments of the S1 gene and sigma C protein were performed using CLUSTAL V (Lasergene, v. 5.0, DNASTAR, Madison, Wis.).
Example 2
[0084] This example describes the in vivo evaluation of reovirus isolate 40973, and shows that strain 40973 adversely affects the growth of chickens.
Virus Isolation
[0085] A reovirus field isolate was isolated in primary chicken embryo liver cells (CeLic) from the intestines of 13-day-old commercial broilers experiencing a severe clinical presentation of Runting Stunting Syndrome. Briefly, intestines were homogenized in virus transport medium containing antibiotics, and filtered through a 0.45 micron sterile filter. Complete monolayers of primary CeLic were inoculated with 0.2 ml of the intestinal homogenate. Cell cultures were examined daily. The cell cultures were frozen and stored at -80° C. when 70-80% cytopathic effect, noted as syncytial formation, was obtained. Cell cultures were frozen and thawed three times before subsequent cell culture passages or titration.
Virus Titration
[0086] Titration of virus strain 40973 was performed in primary CeLic in a 96 well microtiter format. Briefly, virus was diluted 1:10 in the first row of the microtiter plate and serially diluted out to the 10th well. A virus and cell control were also included in the plate. Primary cells were added to each well and incubated at 37° C. with 5% CO2. At 96 hours post-inoculation, the cell culture media was decanted, and the cells fixed with methanol and stained with crystal violet. The titer was obtained from the reciprocal of the last dilution where 50% cytopathic effect was observed in the well. The final titer of virus strain 40973 was 105 tissue culture infective dose (TCID)50/ml.
Chickens
[0087] Forty, one-day-old, non-vaccinated commercial broilers were obtained from a local hatchery. Four treatment groups of 10 birds each were placed in forced air positive-pressure Horsfal-Bauer isolation units. Birds were given food and water ad libitum. One group, designated as the negative control birds, was sham-challenged with 0.1 ml sterile phosphate buffered saline (PBS) via the footpad (FP). A second group of negative control birds was sham-challenged with 0.1 ml sterile PBS via the intratracheal (IT) route. A third treatment group was challenged via the FP with 103 TCID50/ml. The fourth treatment group was challenged via IT with 103 TCID50/ml. At two weeks of age, the body weight and length of each bird was recorded and they were humanely sacrificed.
Statistics
[0088] Statistical analysis of the data by the Holm-Sidak method of one way analysis of variance (ANOVA) was performed using SigmaStat software.
TABLE-US-00004 TABLE 3 Average Body Weights and Lengths From Each Treatment Group Mean Body Std. Mean Body Lengths Std. Treatment group Weights (grams) Dev. (cm) Dev Negative control 146.92a 10.1 28.85a 1.07 Sham IT Challenge Negative control 151.45a 9.17 28.78a 1.18 Sham FP Challenge 40973 132.1b 11.5 27.13b 0.68 IT Challenge 40973 116.51b 9.12 26.37c 0.96 FP Challenge Groups with different letters (a, b, or c) were significantly different at p ≦ 0.001.
Example 3
Virus Isolation
[0089] Reovirus field isolates, chicken/GA 40973/2005, chicken/GA 41560/2005, chicken/GA 41565/2005, were isolated at the Poultry Diagnostic and Research Center at the University of Georgia, from the intestines or feces of chickens exhibiting RSS/enteritis. Field isolate chicken/GA 46118/2005 and chicken/AL 40963/2005, were isolated from the proventriculi of RSS-affected experimental birds. Organs (proventriculi or intestines) from clinically-affected chickens were individually homogenized in virus transport media with antibiotics. The homogenized tissues were filtered through a 0.45 micron syringe filter. The filtrates were incubated with Reovirus S1133 antisera (Charles River, SPAFAS, Wilmington, Mass.) at 37° C. for 1 hour. Following neutralization, 0.2 ml of homogenate was inoculated in primary chicken embryo liver cells and cultured for a total of four passages. Upon the development of 70-80% cytopathic effect, observed as syncytial formation, the cell cultures were frozen and stored at -80° C. Cell cultures were frozen and thawed three times prior to subsequent cell culture passage. Viruses designated chicken/MS 42563-1/2005, chicken/MS 42563-2/2005, chicken/MS 42563-3/2005, and chicken/MS 42563-4/2005 were isolated from broilers and submitted to the laboratory for molecular characterization.
RNA Extraction
[0090] Total viral RNA was extracted from primary chicken embryo liver cell passages using the RNeasy kit (Qiagen, Valencia, Calif.) according to the manufacturer's recommendations. Briefly, 48 hours post infection, cell culture media was decanted, the monolayer overlaid with 300 μl RLT buffer containing 0.143 M β-mercaptoethanol and scraped with a cell scraper as recommended by the manufacturer.
RT-PCR
[0091] A cDNA corresponding to the S1 gene was produced by reverse transcription and PCR using SuperScript® III RNase H.sup.- reverse transcriptase and Platinum®Taq DNA polymerase (Invitrogen, Carlsbad, Calif.) with previously published P1 and P4 primers, SEQ ID NOS: 31 and 32, respectively (Kant, A., et al., Vet. Res. 34:203-212, 2003). The 1.1 kb amplified products were separated on a 1.0% agarose gel, stained with ethidium bromide and visualized with a UV transilluminator. The fragment was excised, purified with the QIAEX II gel extraction kit (Qiagen Inc., Valencia, Calif.), eluted in diethylpyrocarbonate (DEPC)-treated water, and stored at -80° C.
Nucleotide Sequencing of Amplified Products and S1 Clones
[0092] Gel-purified PCR products were sequenced directly using double-stranded DNA sequencing with fluorescently-labeled dideoxynucleotides and Taq polymerase and performed on an ABI 9700 automated sequencer (Applied Biosystems Inc., Foster City, Calif.) (16). PCR primers P1 (SEQ ID NO: 31) and P4 (SEQ ID NO: 32), along with conserved internal S1 gene primers located in conserved regions, were used as needed to complete sequencing. The nucleotide sequences of all primers used for sequencing are available upon request. In addition, gel purified products were cloned into plasmid pCR2TOPO (Invitrogen) and transformed into competent E. coli cells according to protocols provided by the manufacturer. Several clones from each isolate containing plasmids with the 1.1 kb insert were identified by PCR using commercially-available M13 forward and reverse primers. The clones were expanded and the plasmid-purified using a plasmid mini-prep kit (Qiagen, Valencia, Calif.) according to methods of the manufacturer. M13 universal forward and reverse primers along with internal S1 primers were used as needed to complete sequencing. At least three clones or PCR products from three amplifications containing the S1 gene were sequenced in both directions and used to obtain the consensus sequence.
Sequence Analysis
[0093] Nucleotide, predicted amino acid sequence analysis and multiple alignments of the S1 gene and sigma C protein were performed using CLUSTAL W (Lasergene, v. 5.0, DNASTAR, Madison, Wis.). Sequences for the S1 genes of previously published avian reoviruses were obtained from GenBank and had the following accession numbers: DQ872796, chicken/AL 40963/2005; DQ872797, chicken/GA 40973/2005; DQ872798, chicken/GA 41560/2005; and DQ872799, chicken/GA 41565/2005. FIG. 1 illustrates a multiple sequence alignment of avian reovirus sigma C (S1) amino acid sequences of the invention.
[0094] Aligned sequences were compared and a phylogram was generated using maximum parsimony analysis with Neighbor-Joining clustering (Gojobori, T. et al., J. Mol. Evol. 18: 414-423, 1982; Saitou, N. et al., Mol. Biol. Evol. 4: 406-425, 1987) and 1000 bootstrap replicates (confidence levels listed in parentheses) in a heuristic search using the Phylogenetic Analysis Using Parsimony v 4.10b software (PAUP) (Swofford, D. Sinauer and Associates, Inc. Sunderland, Mass., 2001). Predicted antigenic regions within sigma C were obtained using the software program designed by Kolaskar (bio.dfci.harvard.edu/tools/antigenic.html; Kolaskar A. S, and Tongaonkar, P C., FEBS letter 176:172-174, 1990). FIG. 2 illustrates the phylogenetic relationships among avian reoviruses based on the sigma C (S1) amino acid sequences determined by sequencing the S1 genes obtained from avian reoviruses of the invention.
GenBank Accession Numbers
[0095] Sequences obtained for several chicken isolates described here have been submitted to GenBank and assigned the following accession numbers: DQ872796-DQ872801.
Monoclonal Antibody Profiles
[0096] Several reovirus field isolates were sent to Intervet, Inc. (Boxmeer, The Netherlands) for reovirus monoclonal antibody profiling using a plaque reduction assay based on growth in primary chicken embryo liver cell cultures (van Loon, A. A., et al., Vet. Quart. 23(3):129-33, 2001) (Table 4).
TABLE-US-00005 TABLE 4 Monoclonal antibody reactivity pattern Monoclonal antibodiesA Isolates 154 13-6 15-1 14-67 14-11 S1133* + + + + + 2408* + + + + + 1733* + + - + + CO8* + + + + - ERS-1* + - - + - ERS-2* + - - + - 40963 + - - + - 46118 + - - + - 41560 + - - + - 41565 + - - + - 42563-1 + - - + - 42563-2 + - - + - *van Loon, A. A., et al., Vet. Quart. 23(3): 129-33, 2001.
[0097] These results demonstrate that many of the recent southeast United States reovirus isolates have the same reactivity pattern as several European reovirus isolates (ERS).
[0098] While the preferred embodiments of the invention have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. All references, patents, or applications cited herein are incorporated by reference in their entirety, as if written herein.
Sequence CWU
1
321981DNAAvian orthoreovirusCDS(1)..(981) 1atg gaa ggc tta act cag tca cag
cga aga gag gtc gtg ggg ctg ata 48Met Glu Gly Leu Thr Gln Ser Gln
Arg Arg Glu Val Val Gly Leu Ile1 5 10
15ctc tca ttg act tcg agc gtg act ata aat cct ggc gat ttg
acg gaa 96Leu Ser Leu Thr Ser Ser Val Thr Ile Asn Pro Gly Asp Leu
Thr Glu 20 25 30ctg cgt gag
cgc gtc tcg gcg tta gac tcg gcc aat gcg tcg ttg acc 144Leu Arg Glu
Arg Val Ser Ala Leu Asp Ser Ala Asn Ala Ser Leu Thr 35
40 45gaa acc gta aaa ggc gtg tta gat cag ttg gta
gat ttg gta cag aag 192Glu Thr Val Lys Gly Val Leu Asp Gln Leu Val
Asp Leu Val Gln Lys 50 55 60ttg ggc
aat gcg gcg ggt gct ata gtt gac cta cga gga gag cta aac 240Leu Gly
Asn Ala Ala Gly Ala Ile Val Asp Leu Arg Gly Glu Leu Asn65
70 75 80tca tta act acc aga gtc caa
act atc caa tcc tct ttg gga tca ctc 288Ser Leu Thr Thr Arg Val Gln
Thr Ile Gln Ser Ser Leu Gly Ser Leu 85 90
95acg gac agt ata tcg gat ctt tct ggc caa gtg act act
aac acc tct 336Thr Asp Ser Ile Ser Asp Leu Ser Gly Gln Val Thr Thr
Asn Thr Ser 100 105 110tcg ctc
acg aat ctg ggg agt atg atg gct ggt ctc aca gtc gat gtg 384Ser Leu
Thr Asn Leu Gly Ser Met Met Ala Gly Leu Thr Val Asp Val 115
120 125act aat ctt aaa cgc gac gta tcg aat cag
ggt ctt caa att acg agt 432Thr Asn Leu Lys Arg Asp Val Ser Asn Gln
Gly Leu Gln Ile Thr Ser 130 135 140atc
gag cag cgt gta act agt ttg gaa tct ggt gct gga tct att ccc 480Ile
Glu Gln Arg Val Thr Ser Leu Glu Ser Gly Ala Gly Ser Ile Pro145
150 155 160aca ttt gct gct ccc ctt
aaa tta gat agc ggg att gta tca ctc gac 528Thr Phe Ala Ala Pro Leu
Lys Leu Asp Ser Gly Ile Val Ser Leu Asp 165
170 175ctg gat cct tac ttt tgt tct gtg aac cat aat ctc
acg tcg tat tcc 576Leu Asp Pro Tyr Phe Cys Ser Val Asn His Asn Leu
Thr Ser Tyr Ser 180 185 190gca
agc gct ctg cta atg aat ttt cag tgg ctt gtt cga ggt gag gga 624Ala
Ser Ala Leu Leu Met Asn Phe Gln Trp Leu Val Arg Gly Glu Gly 195
200 205gga tcg tct gat tca ttc gat atg aat
gtg aca gct cat agt cac ggc 672Gly Ser Ser Asp Ser Phe Asp Met Asn
Val Thr Ala His Ser His Gly 210 215
220cag agg aca gat ttt atg atg tct act act cag tcg tta act gtt act
720Gln Arg Thr Asp Phe Met Met Ser Thr Thr Gln Ser Leu Thr Val Thr225
230 235 240gga aat tct gtc
act cta gtc ttt gat ctt aac gcg ctt att tct ccc 768Gly Asn Ser Val
Thr Leu Val Phe Asp Leu Asn Ala Leu Ile Ser Pro 245
250 255ccc tct gac tat tct cgt ttg ata cca tgt
cat ggt ttt caa caa gcg 816Pro Ser Asp Tyr Ser Arg Leu Ile Pro Cys
His Gly Phe Gln Gln Ala 260 265
270acg ttc cca gtg gac ctt tcg ttt aag cga gac gac gtc atg cat tca
864Thr Phe Pro Val Asp Leu Ser Phe Lys Arg Asp Asp Val Met His Ser
275 280 285tat cag gtg tat ggt tct tac
aca act ccc cgc att ttc aag ata aca 912Tyr Gln Val Tyr Gly Ser Tyr
Thr Thr Pro Arg Ile Phe Lys Ile Thr 290 295
300ttc tcc cct ggc aat cca gtg cct gcg gtc ata cgt ttc ata acc gtg
960Phe Ser Pro Gly Asn Pro Val Pro Ala Val Ile Arg Phe Ile Thr Val305
310 315 320cgt acg ggc atc
gat act taa 981Arg Thr Gly Ile
Asp Thr 3252326PRTAvian orthoreovirus 2Met Glu Gly Leu Thr
Gln Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15Leu Ser Leu Thr Ser Ser Val Thr Ile Asn Pro
Gly Asp Leu Thr Glu 20 25
30Leu Arg Glu Arg Val Ser Ala Leu Asp Ser Ala Asn Ala Ser Leu Thr
35 40 45Glu Thr Val Lys Gly Val Leu Asp
Gln Leu Val Asp Leu Val Gln Lys 50 55
60Leu Gly Asn Ala Ala Gly Ala Ile Val Asp Leu Arg Gly Glu Leu Asn65
70 75 80Ser Leu Thr Thr Arg
Val Gln Thr Ile Gln Ser Ser Leu Gly Ser Leu 85
90 95Thr Asp Ser Ile Ser Asp Leu Ser Gly Gln Val
Thr Thr Asn Thr Ser 100 105
110Ser Leu Thr Asn Leu Gly Ser Met Met Ala Gly Leu Thr Val Asp Val
115 120 125Thr Asn Leu Lys Arg Asp Val
Ser Asn Gln Gly Leu Gln Ile Thr Ser 130 135
140Ile Glu Gln Arg Val Thr Ser Leu Glu Ser Gly Ala Gly Ser Ile
Pro145 150 155 160Thr Phe
Ala Ala Pro Leu Lys Leu Asp Ser Gly Ile Val Ser Leu Asp
165 170 175Leu Asp Pro Tyr Phe Cys Ser
Val Asn His Asn Leu Thr Ser Tyr Ser 180 185
190Ala Ser Ala Leu Leu Met Asn Phe Gln Trp Leu Val Arg Gly
Glu Gly 195 200 205Gly Ser Ser Asp
Ser Phe Asp Met Asn Val Thr Ala His Ser His Gly 210
215 220Gln Arg Thr Asp Phe Met Met Ser Thr Thr Gln Ser
Leu Thr Val Thr225 230 235
240Gly Asn Ser Val Thr Leu Val Phe Asp Leu Asn Ala Leu Ile Ser Pro
245 250 255Pro Ser Asp Tyr Ser
Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala 260
265 270Thr Phe Pro Val Asp Leu Ser Phe Lys Arg Asp Asp
Val Met His Ser 275 280 285Tyr Gln
Val Tyr Gly Ser Tyr Thr Thr Pro Arg Ile Phe Lys Ile Thr 290
295 300Phe Ser Pro Gly Asn Pro Val Pro Ala Val Ile
Arg Phe Ile Thr Val305 310 315
320Arg Thr Gly Ile Asp Thr 3253978DNAAvian
orthoreovirusCDS(1)..(978)misc_feature(978)..(978)N = a, c, g or t 3atg
gcc gga ctc act cca tca cag cga aga gaa gtc gtc ggg cta ata 48Met
Ala Gly Leu Thr Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1
5 10 15ctc tca ttg act tcg aac acg
agt ata ggt cct gga gat ttg aca ccg 96Leu Ser Leu Thr Ser Asn Thr
Ser Ile Gly Pro Gly Asp Leu Thr Pro 20 25
30atc tac gat cgt ctc tcg gcc gtt gag aca gct tgc gcc act
tta agt 144Ile Tyr Asp Arg Leu Ser Ala Val Glu Thr Ala Cys Ala Thr
Leu Ser 35 40 45gac tct gtg ggc
caa tta gct tca acg aca tct gct tta tcc act cgt 192Asp Ser Val Gly
Gln Leu Ala Ser Thr Thr Ser Ala Leu Ser Thr Arg 50 55
60ttg gat aac ttg gtg agt gtg tca caa gat atg gct aat
gac ctg agg 240Leu Asp Asn Leu Val Ser Val Ser Gln Asp Met Ala Asn
Asp Leu Arg65 70 75
80gat gtt caa agt cgc gtg tct gca ctt caa aca tcg ata gat agt atg
288Asp Val Gln Ser Arg Val Ser Ala Leu Gln Thr Ser Ile Asp Ser Met
85 90 95tcc agt gac att tcc acc
tta tca caa tcg ctg tct aat cac gat tcc 336Ser Ser Asp Ile Ser Thr
Leu Ser Gln Ser Leu Ser Asn His Asp Ser 100
105 110cag att tcc acg ctt tcc tct tct gtc agc act gta
gca act aac gtg 384Gln Ile Ser Thr Leu Ser Ser Ser Val Ser Thr Val
Ala Thr Asn Val 115 120 125tcc aac
ctt caa cgt gac gtg gct gca tct gca ctc aac att tct gat 432Ser Asn
Leu Gln Arg Asp Val Ala Ala Ser Ala Leu Asn Ile Ser Asp 130
135 140ctt cag cga cgt gtt gct gct ctg gaa tcg agt
ccc ggt tca tcc ctt 480Leu Gln Arg Arg Val Ala Ala Leu Glu Ser Ser
Pro Gly Ser Ser Leu145 150 155
160aca ttt ctt gct ccc ctg cgg gct gat ggt ggc tcc gtc tca tta gat
528Thr Phe Leu Ala Pro Leu Arg Ala Asp Gly Gly Ser Val Ser Leu Asp
165 170 175atg gat ccg tat ttt
tgc tca gaa agg gcg aac tta acg tcc tac tcg 576Met Asp Pro Tyr Phe
Cys Ser Glu Arg Ala Asn Leu Thr Ser Tyr Ser 180
185 190gcg agt gcg cag ctg ctg cag ttt cag tgg ttc gtc
agg agt gag gga 624Ala Ser Ala Gln Leu Leu Gln Phe Gln Trp Phe Val
Arg Ser Glu Gly 195 200 205ggt tcc
tca gac tcg att aac atg agc gtg gtg gcc cat tgc cac ggt 672Gly Ser
Ser Asp Ser Ile Asn Met Ser Val Val Ala His Cys His Gly 210
215 220cgt cgt acg gac tat ctg atg tcc tct cat gac
tca ctc acc gtc act 720Arg Arg Thr Asp Tyr Leu Met Ser Ser His Asp
Ser Leu Thr Val Thr225 230 235
240ggc aac tca gtg tct tta gtg ttc aac ttg gat tac att act acg tct
768Gly Asn Ser Val Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Thr Ser
245 250 255ggt gtg gat tat gct
cgt ctg att ccg tgt cat ggt ttt cag caa gcc 816Gly Val Asp Tyr Ala
Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala 260
265 270act ttt cct gtt gac atc tct ttc acg aag aat gat
act aca cac acg 864Thr Phe Pro Val Asp Ile Ser Phe Thr Lys Asn Asp
Thr Thr His Thr 275 280 285tac caa
gtc tat ggc gca ttt aat ggc cct cgc gtc ttt aag gtg acc 912Tyr Gln
Val Tyr Gly Ala Phe Asn Gly Pro Arg Val Phe Lys Val Thr 290
295 300ttc tct ccg ggt ggg act agc acg aca aac ata
cgc ttt ctg acc gtg 960Phe Ser Pro Gly Gly Thr Ser Thr Thr Asn Ile
Arg Phe Leu Thr Val305 310 315
320cgt acg ggc atc gac acn
978Arg Thr Gly Ile Asp Thr 3254326PRTAvian
orthoreovirus 4Met Ala Gly Leu Thr Pro Ser Gln Arg Arg Glu Val Val Gly
Leu Ile1 5 10 15Leu Ser
Leu Thr Ser Asn Thr Ser Ile Gly Pro Gly Asp Leu Thr Pro 20
25 30Ile Tyr Asp Arg Leu Ser Ala Val Glu
Thr Ala Cys Ala Thr Leu Ser 35 40
45Asp Ser Val Gly Gln Leu Ala Ser Thr Thr Ser Ala Leu Ser Thr Arg 50
55 60Leu Asp Asn Leu Val Ser Val Ser Gln
Asp Met Ala Asn Asp Leu Arg65 70 75
80Asp Val Gln Ser Arg Val Ser Ala Leu Gln Thr Ser Ile Asp
Ser Met 85 90 95Ser Ser
Asp Ile Ser Thr Leu Ser Gln Ser Leu Ser Asn His Asp Ser 100
105 110Gln Ile Ser Thr Leu Ser Ser Ser Val
Ser Thr Val Ala Thr Asn Val 115 120
125Ser Asn Leu Gln Arg Asp Val Ala Ala Ser Ala Leu Asn Ile Ser Asp
130 135 140Leu Gln Arg Arg Val Ala Ala
Leu Glu Ser Ser Pro Gly Ser Ser Leu145 150
155 160Thr Phe Leu Ala Pro Leu Arg Ala Asp Gly Gly Ser
Val Ser Leu Asp 165 170
175Met Asp Pro Tyr Phe Cys Ser Glu Arg Ala Asn Leu Thr Ser Tyr Ser
180 185 190Ala Ser Ala Gln Leu Leu
Gln Phe Gln Trp Phe Val Arg Ser Glu Gly 195 200
205Gly Ser Ser Asp Ser Ile Asn Met Ser Val Val Ala His Cys
His Gly 210 215 220Arg Arg Thr Asp Tyr
Leu Met Ser Ser His Asp Ser Leu Thr Val Thr225 230
235 240Gly Asn Ser Val Ser Leu Val Phe Asn Leu
Asp Tyr Ile Thr Thr Ser 245 250
255Gly Val Asp Tyr Ala Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala
260 265 270Thr Phe Pro Val Asp
Ile Ser Phe Thr Lys Asn Asp Thr Thr His Thr 275
280 285Tyr Gln Val Tyr Gly Ala Phe Asn Gly Pro Arg Val
Phe Lys Val Thr 290 295 300Phe Ser Pro
Gly Gly Thr Ser Thr Thr Asn Ile Arg Phe Leu Thr Val305
310 315 320Arg Thr Gly Ile Asp Thr
3255978DNAAvian orthoreovirusCDS(1)..(978) 5atg gcc gga tta act
ccc tca cag cga aga gaa gtc gtg gga ttg ata 48Met Ala Gly Leu Thr
Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15ctc tcc ttg act tcg agc gcg aat aca aat tgt
gga gat ttg acg ccc 96Leu Ser Leu Thr Ser Ser Ala Asn Thr Asn Cys
Gly Asp Leu Thr Pro 20 25
30gta tat gac cgt ttg ctt agt ctg gaa tcc act att acg tca ttg aat
144Val Tyr Asp Arg Leu Leu Ser Leu Glu Ser Thr Ile Thr Ser Leu Asn
35 40 45ggc tct gtt agc gac ttg tcg cgg
aag gta tcg gat ctg gaa tct gat 192Gly Ser Val Ser Asp Leu Ser Arg
Lys Val Ser Asp Leu Glu Ser Asp 50 55
60ttg caa gac gtc att tca tct tta ggt cag gct aat tca acg ttg acc
240Leu Gln Asp Val Ile Ser Ser Leu Gly Gln Ala Asn Ser Thr Leu Thr65
70 75 80gag tta tct aaa gaa
cta cgt cag tta agt ggc tct gtt gac act ctc 288Glu Leu Ser Lys Glu
Leu Arg Gln Leu Ser Gly Ser Val Asp Thr Leu 85
90 95gtc acg tct gtt tcg gat cta tcg acc act gta
tcc ggg cat cag aac 336Val Thr Ser Val Ser Asp Leu Ser Thr Thr Val
Ser Gly His Gln Asn 100 105
110gcg ata gcc gct ata caa act tcc gtt cac gct aat acg act gac att
384Ala Ile Ala Ala Ile Gln Thr Ser Val His Ala Asn Thr Thr Asp Ile
115 120 125agt aac ctg aaa agt agc ata
tcg act atc ggc tta aat atc act gac 432Ser Asn Leu Lys Ser Ser Ile
Ser Thr Ile Gly Leu Asn Ile Thr Asp 130 135
140cta gag caa cgt gtg gga aac atc gaa tca ggg tcg ggt cct act ttg
480Leu Glu Gln Arg Val Gly Asn Ile Glu Ser Gly Ser Gly Pro Thr Leu145
150 155 160aag ttt atg tca
cct ctg agc ttg tcc caa ggc gta gcg tca ctg att 528Lys Phe Met Ser
Pro Leu Ser Leu Ser Gln Gly Val Ala Ser Leu Ile 165
170 175atg gat cct tac ttc tgt tca gac aat aag
gcg cta acc tca tac tct 576Met Asp Pro Tyr Phe Cys Ser Asp Asn Lys
Ala Leu Thr Ser Tyr Ser 180 185
190aca gac gct cag ctg atg cag ttc cag tgg ctg gct cga ggt gag gat
624Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu Ala Arg Gly Glu Asp
195 200 205ggt tca tcg agc tct gtt gag
atg ctt gtt aat gcg cac tgt cat ggg 672Gly Ser Ser Ser Ser Val Glu
Met Leu Val Asn Ala His Cys His Gly 210 215
220cgt cgc act gac tat atg atg tcc act aca gag aat ttc act gtc acg
720Arg Arg Thr Asp Tyr Met Met Ser Thr Thr Glu Asn Phe Thr Val Thr225
230 235 240ggc aac tcc gta
tcg ttg gtt ttc agt ctg gac tat att act aaa ccg 768Gly Asn Ser Val
Ser Leu Val Phe Ser Leu Asp Tyr Ile Thr Lys Pro 245
250 255cct tct gac ata tca cgt tta gta cca cgt
gtc gga ttc cag gct gct 816Pro Ser Asp Ile Ser Arg Leu Val Pro Arg
Val Gly Phe Gln Ala Ala 260 265
270tcc ttt cct gtg gac gtc tct ttc act cgt gac gca act ccg cat gct
864Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Ala Thr Pro His Ala
275 280 285tac cag gta tac ggc gct ttt
tct agc cct cgc gtc ttc aag atc acg 912Tyr Gln Val Tyr Gly Ala Phe
Ser Ser Pro Arg Val Phe Lys Ile Thr 290 295
300ttc ctc aca ggt ggc act ggg act gcg aat ctc cgt ttc ttg acc gta
960Phe Leu Thr Gly Gly Thr Gly Thr Ala Asn Leu Arg Phe Leu Thr Val305
310 315 320cgt acg ggc atc
gac acc 978Arg Thr Gly Ile
Asp Thr 3256326PRTAvian orthoreovirus 6Met Ala Gly Leu Thr
Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15Leu Ser Leu Thr Ser Ser Ala Asn Thr Asn Cys
Gly Asp Leu Thr Pro 20 25
30Val Tyr Asp Arg Leu Leu Ser Leu Glu Ser Thr Ile Thr Ser Leu Asn
35 40 45Gly Ser Val Ser Asp Leu Ser Arg
Lys Val Ser Asp Leu Glu Ser Asp 50 55
60Leu Gln Asp Val Ile Ser Ser Leu Gly Gln Ala Asn Ser Thr Leu Thr65
70 75 80Glu Leu Ser Lys Glu
Leu Arg Gln Leu Ser Gly Ser Val Asp Thr Leu 85
90 95Val Thr Ser Val Ser Asp Leu Ser Thr Thr Val
Ser Gly His Gln Asn 100 105
110Ala Ile Ala Ala Ile Gln Thr Ser Val His Ala Asn Thr Thr Asp Ile
115 120 125Ser Asn Leu Lys Ser Ser Ile
Ser Thr Ile Gly Leu Asn Ile Thr Asp 130 135
140Leu Glu Gln Arg Val Gly Asn Ile Glu Ser Gly Ser Gly Pro Thr
Leu145 150 155 160Lys Phe
Met Ser Pro Leu Ser Leu Ser Gln Gly Val Ala Ser Leu Ile
165 170 175Met Asp Pro Tyr Phe Cys Ser
Asp Asn Lys Ala Leu Thr Ser Tyr Ser 180 185
190Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu Ala Arg Gly
Glu Asp 195 200 205Gly Ser Ser Ser
Ser Val Glu Met Leu Val Asn Ala His Cys His Gly 210
215 220Arg Arg Thr Asp Tyr Met Met Ser Thr Thr Glu Asn
Phe Thr Val Thr225 230 235
240Gly Asn Ser Val Ser Leu Val Phe Ser Leu Asp Tyr Ile Thr Lys Pro
245 250 255Pro Ser Asp Ile Ser
Arg Leu Val Pro Arg Val Gly Phe Gln Ala Ala 260
265 270Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Ala
Thr Pro His Ala 275 280 285Tyr Gln
Val Tyr Gly Ala Phe Ser Ser Pro Arg Val Phe Lys Ile Thr 290
295 300Phe Leu Thr Gly Gly Thr Gly Thr Ala Asn Leu
Arg Phe Leu Thr Val305 310 315
320Arg Thr Gly Ile Asp Thr 3257978DNAAvian
orthoreovirusCDS(1)..(978) 7atg gcc gga ctc act cca tca cag cga aga gaa
gtc gtc ggg ctg ata 48Met Ala Gly Leu Thr Pro Ser Gln Arg Arg Glu
Val Val Gly Leu Ile1 5 10
15ctc tca ttg act tcg aac acg agt ata agt cct gga gat ttg aca ccg
96Leu Ser Leu Thr Ser Asn Thr Ser Ile Ser Pro Gly Asp Leu Thr Pro
20 25 30atc tac gat cgt ctc tcg gcc
gtt gag aca gct tat tct act tta agc 144Ile Tyr Asp Arg Leu Ser Ala
Val Glu Thr Ala Tyr Ser Thr Leu Ser 35 40
45gac tct gtg ggt caa ttg gct tca acg aca tct gct tta tct act
cgt 192Asp Ser Val Gly Gln Leu Ala Ser Thr Thr Ser Ala Leu Ser Thr
Arg 50 55 60ctg gat gac ttg gtg agt
atg tca caa gat ata gta aat gac ttg agg 240Leu Asp Asp Leu Val Ser
Met Ser Gln Asp Ile Val Asn Asp Leu Arg65 70
75 80gat att caa agt cgt gta tct gca ttt caa ata
tcg ata gat agt ctg 288Asp Ile Gln Ser Arg Val Ser Ala Phe Gln Ile
Ser Ile Asp Ser Leu 85 90
95tcc agt gac att tcc acc cta tca cag tcg ctg tct aat cat gat tcc
336Ser Ser Asp Ile Ser Thr Leu Ser Gln Ser Leu Ser Asn His Asp Ser
100 105 110cag att tcc acg ctt tcc
tct tct gtc ggc acg cta tcc act gac atg 384Gln Ile Ser Thr Leu Ser
Ser Ser Val Gly Thr Leu Ser Thr Asp Met 115 120
125tcc aac ctc caa cgc gac gta act gca tct gca ctc aac att
tct gat 432Ser Asn Leu Gln Arg Asp Val Thr Ala Ser Ala Leu Asn Ile
Ser Asp 130 135 140ctt cag cga cgt gtt
gct gct ctg gaa tcg agt tcc ggt tca ccc ctt 480Leu Gln Arg Arg Val
Ala Ala Leu Glu Ser Ser Ser Gly Ser Pro Leu145 150
155 160aca ttt ctt gct ccc ctg cga gct gat ggt
ggc tcc gtc tcg tta gat 528Thr Phe Leu Ala Pro Leu Arg Ala Asp Gly
Gly Ser Val Ser Leu Asp 165 170
175atg gat ccg tac ttt tgc tcg gaa agg gcg aac tta acg tct tac tca
576Met Asp Pro Tyr Phe Cys Ser Glu Arg Ala Asn Leu Thr Ser Tyr Ser
180 185 190gcg agt gcg cag ttg ctg
cag ttt cag tgg ttt gtc agg agt gag gga 624Ala Ser Ala Gln Leu Leu
Gln Phe Gln Trp Phe Val Arg Ser Glu Gly 195 200
205ggt tcc tca gac tca atc gac atg agc gtg gtg gct cac tgc
cac ggc 672Gly Ser Ser Asp Ser Ile Asp Met Ser Val Val Ala His Cys
His Gly 210 215 220cgt cgc acg gat tat
ctg atg tct tcc cat gac tca ctc acc gtc act 720Arg Arg Thr Asp Tyr
Leu Met Ser Ser His Asp Ser Leu Thr Val Thr225 230
235 240ggc aac tca gtg tct cta gtg ttc aac ctg
gat cac atc acc acg tct 768Gly Asn Ser Val Ser Leu Val Phe Asn Leu
Asp His Ile Thr Thr Ser 245 250
255ggt gtg gat tat gct cgt ctg att ccg tgt cat ggt ttt caa caa gcc
816Gly Val Asp Tyr Ala Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala
260 265 270act ttt cct gtt gac atc
tct ttc acg aaa aat gac act aca cac acg 864Thr Phe Pro Val Asp Ile
Ser Phe Thr Lys Asn Asp Thr Thr His Thr 275 280
285tac caa gtc tat ggc gcg ttt gat ggc cct cgc gtc ttt aag
gtg acc 912Tyr Gln Val Tyr Gly Ala Phe Asp Gly Pro Arg Val Phe Lys
Val Thr 290 295 300ttc tct ccg ggt ggg
acc agc acg acg agc gta cgt ttt ctg acc gtg 960Phe Ser Pro Gly Gly
Thr Ser Thr Thr Ser Val Arg Phe Leu Thr Val305 310
315 320cgt acg ggc atc gac acc
978Arg Thr Gly Ile Asp Thr
3258326PRTAvian orthoreovirus 8Met Ala Gly Leu Thr Pro Ser Gln Arg Arg
Glu Val Val Gly Leu Ile1 5 10
15Leu Ser Leu Thr Ser Asn Thr Ser Ile Ser Pro Gly Asp Leu Thr Pro
20 25 30Ile Tyr Asp Arg Leu Ser
Ala Val Glu Thr Ala Tyr Ser Thr Leu Ser 35 40
45Asp Ser Val Gly Gln Leu Ala Ser Thr Thr Ser Ala Leu Ser
Thr Arg 50 55 60Leu Asp Asp Leu Val
Ser Met Ser Gln Asp Ile Val Asn Asp Leu Arg65 70
75 80Asp Ile Gln Ser Arg Val Ser Ala Phe Gln
Ile Ser Ile Asp Ser Leu 85 90
95Ser Ser Asp Ile Ser Thr Leu Ser Gln Ser Leu Ser Asn His Asp Ser
100 105 110Gln Ile Ser Thr Leu
Ser Ser Ser Val Gly Thr Leu Ser Thr Asp Met 115
120 125Ser Asn Leu Gln Arg Asp Val Thr Ala Ser Ala Leu
Asn Ile Ser Asp 130 135 140Leu Gln Arg
Arg Val Ala Ala Leu Glu Ser Ser Ser Gly Ser Pro Leu145
150 155 160Thr Phe Leu Ala Pro Leu Arg
Ala Asp Gly Gly Ser Val Ser Leu Asp 165
170 175Met Asp Pro Tyr Phe Cys Ser Glu Arg Ala Asn Leu
Thr Ser Tyr Ser 180 185 190Ala
Ser Ala Gln Leu Leu Gln Phe Gln Trp Phe Val Arg Ser Glu Gly 195
200 205Gly Ser Ser Asp Ser Ile Asp Met Ser
Val Val Ala His Cys His Gly 210 215
220Arg Arg Thr Asp Tyr Leu Met Ser Ser His Asp Ser Leu Thr Val Thr225
230 235 240Gly Asn Ser Val
Ser Leu Val Phe Asn Leu Asp His Ile Thr Thr Ser 245
250 255Gly Val Asp Tyr Ala Arg Leu Ile Pro Cys
His Gly Phe Gln Gln Ala 260 265
270Thr Phe Pro Val Asp Ile Ser Phe Thr Lys Asn Asp Thr Thr His Thr
275 280 285Tyr Gln Val Tyr Gly Ala Phe
Asp Gly Pro Arg Val Phe Lys Val Thr 290 295
300Phe Ser Pro Gly Gly Thr Ser Thr Thr Ser Val Arg Phe Leu Thr
Val305 310 315 320Arg Thr
Gly Ile Asp Thr 3259981DNAAvian
orthoreovirusCDS(1)..(981)misc_feature(831)..(831)y = c or t 9atg gcc gga
tta act ccc tcc cag cga aga gaa gtc gtg gga ttg ata 48Met Ala Gly
Leu Thr Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15ctc tcc ttg act tcg agc gtg aat aca
aac tgt gga gat ttg acg cct 96Leu Ser Leu Thr Ser Ser Val Asn Thr
Asn Cys Gly Asp Leu Thr Pro 20 25
30gta tat gac cgt ctg ctt agt ctg gaa tcc gct gtt gcg tcg ttg aat
144Val Tyr Asp Arg Leu Leu Ser Leu Glu Ser Ala Val Ala Ser Leu Asn
35 40 45aac tct gta aat ggc ttg ttg
cag aag gta tca gat ttc gag tct gat 192Asn Ser Val Asn Gly Leu Leu
Gln Lys Val Ser Asp Phe Glu Ser Asp 50 55
60ttg caa gac gtc act tca tcc cta ggc cag gcc aat tcg atg tta acc
240Leu Gln Asp Val Thr Ser Ser Leu Gly Gln Ala Asn Ser Met Leu Thr65
70 75 80gag cta tct aag
gaa ctg cgt cag ttg agc agc tct gtt gac aat ctc 288Glu Leu Ser Lys
Glu Leu Arg Gln Leu Ser Ser Ser Val Asp Asn Leu 85
90 95gtc acg tcc gtt tct gat cta tcg acc gct
gta tcc ggg cac cag aac 336Val Thr Ser Val Ser Asp Leu Ser Thr Ala
Val Ser Gly His Gln Asn 100 105
110gcc ata gcc gat ata caa gct tcc gtt cac gcc aat gca act gat att
384Ala Ile Ala Asp Ile Gln Ala Ser Val His Ala Asn Ala Thr Asp Ile
115 120 125aac aac ctg aaa agc agt gtc
tct act att agc tta aat atc act gat 432Asn Asn Leu Lys Ser Ser Val
Ser Thr Ile Ser Leu Asn Ile Thr Asp 130 135
140ctc gag caa cgc gtg gca aac atc gag tca ggg tca gac tct aat tta
480Leu Glu Gln Arg Val Ala Asn Ile Glu Ser Gly Ser Asp Ser Asn Leu145
150 155 160agg ttt gtg tca
cct ctg aac ttg tcc caa ggc gtg gtg tca ttg atc 528Arg Phe Val Ser
Pro Leu Asn Leu Ser Gln Gly Val Val Ser Leu Ile 165
170 175atg gat cct tac ttc tgt tct gac aat aag
gca cta acc tca tat tct 576Met Asp Pro Tyr Phe Cys Ser Asp Asn Lys
Ala Leu Thr Ser Tyr Ser 180 185
190acg gac gcg cag ctg atg cag ttt cag tgg ctg gct aga ggt gaa gat
624Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu Ala Arg Gly Glu Asp
195 200 205ggg tca tcg agc tct gtt gat
atg ctc gtt aac gca cac tgt cat ggg 672Gly Ser Ser Ser Ser Val Asp
Met Leu Val Asn Ala His Cys His Gly 210 215
220cgt cgc act gac tat atg atg tcc act aca gag agt ttt act gtc acg
720Arg Arg Thr Asp Tyr Met Met Ser Thr Thr Glu Ser Phe Thr Val Thr225
230 235 240ggc aat tct gta
tca ttg gtc ttc aat ctg gat tat atc acc aag ccg 768Gly Asn Ser Val
Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Lys Pro 245
250 255ccc tct gac atg tcg cgg cta gta ccc cgt
gct gga ttc cag gct gct 816Pro Ser Asp Met Ser Arg Leu Val Pro Arg
Ala Gly Phe Gln Ala Ala 260 265
270tcg ttt ccc gtg gay gtc tcc ttc act cgt gac acg aca ccg cat gca
864Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Thr Thr Pro His Ala
275 280 285tac cag gtg tat ggt gct ttc
tct agc cct cgc att ttt aag atc acg 912Tyr Gln Val Tyr Gly Ala Phe
Ser Ser Pro Arg Ile Phe Lys Ile Thr 290 295
300ttc ctc aca ggt ggt act gga act gcg aat ctc cgt ttc ttg acc gtg
960Phe Leu Thr Gly Gly Thr Gly Thr Ala Asn Leu Arg Phe Leu Thr Val305
310 315 320cgt acg ggc atc
gac act taa 981Arg Thr Gly Ile
Asp Thr 32510326PRTAvian orthoreovirus 10Met Ala Gly Leu
Thr Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15Leu Ser Leu Thr Ser Ser Val Asn Thr Asn
Cys Gly Asp Leu Thr Pro 20 25
30Val Tyr Asp Arg Leu Leu Ser Leu Glu Ser Ala Val Ala Ser Leu Asn
35 40 45Asn Ser Val Asn Gly Leu Leu Gln
Lys Val Ser Asp Phe Glu Ser Asp 50 55
60Leu Gln Asp Val Thr Ser Ser Leu Gly Gln Ala Asn Ser Met Leu Thr65
70 75 80Glu Leu Ser Lys Glu
Leu Arg Gln Leu Ser Ser Ser Val Asp Asn Leu 85
90 95Val Thr Ser Val Ser Asp Leu Ser Thr Ala Val
Ser Gly His Gln Asn 100 105
110Ala Ile Ala Asp Ile Gln Ala Ser Val His Ala Asn Ala Thr Asp Ile
115 120 125Asn Asn Leu Lys Ser Ser Val
Ser Thr Ile Ser Leu Asn Ile Thr Asp 130 135
140Leu Glu Gln Arg Val Ala Asn Ile Glu Ser Gly Ser Asp Ser Asn
Leu145 150 155 160Arg Phe
Val Ser Pro Leu Asn Leu Ser Gln Gly Val Val Ser Leu Ile
165 170 175Met Asp Pro Tyr Phe Cys Ser
Asp Asn Lys Ala Leu Thr Ser Tyr Ser 180 185
190Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu Ala Arg Gly
Glu Asp 195 200 205Gly Ser Ser Ser
Ser Val Asp Met Leu Val Asn Ala His Cys His Gly 210
215 220Arg Arg Thr Asp Tyr Met Met Ser Thr Thr Glu Ser
Phe Thr Val Thr225 230 235
240Gly Asn Ser Val Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Lys Pro
245 250 255Pro Ser Asp Met Ser
Arg Leu Val Pro Arg Ala Gly Phe Gln Ala Ala 260
265 270Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Thr
Thr Pro His Ala 275 280 285Tyr Gln
Val Tyr Gly Ala Phe Ser Ser Pro Arg Ile Phe Lys Ile Thr 290
295 300Phe Leu Thr Gly Gly Thr Gly Thr Ala Asn Leu
Arg Phe Leu Thr Val305 310 315
320Arg Thr Gly Ile Asp Thr 32511981DNAAvian
orthoreovirusCDS(1)..(981) 11atg gcc gga ctc act cca tta cag cga aga gaa
gtc gtc gga ctg ata 48Met Ala Gly Leu Thr Pro Leu Gln Arg Arg Glu
Val Val Gly Leu Ile1 5 10
15ctc tca ttg act tcg agc acg agt ata agt cct gga gat ttg aca cca
96Leu Ser Leu Thr Ser Ser Thr Ser Ile Ser Pro Gly Asp Leu Thr Pro
20 25 30ata tat gat cgg ctg tcc gcc
gtt gag acg atc tgc gac act cta agt 144Ile Tyr Asp Arg Leu Ser Ala
Val Glu Thr Ile Cys Asp Thr Leu Ser 35 40
45ggc tcc gtg ggc cag ttg acg act atg atg tct aat tta tct gct
cgt 192Gly Ser Val Gly Gln Leu Thr Thr Met Met Ser Asn Leu Ser Ala
Arg 50 55 60ctg gat gat tta acg att
gtc tcc cga gat atg gcc act gat ttg aga 240Leu Asp Asp Leu Thr Ile
Val Ser Arg Asp Met Ala Thr Asp Leu Arg65 70
75 80aac gtc caa ggt agt gtg tct gcg ctt cga acg
tca tta gat cat ctg 288Asn Val Gln Gly Ser Val Ser Ala Leu Arg Thr
Ser Leu Asp His Leu 85 90
95tcc ggt gat gtt tcc gct tta tct caa tca tta tcc gcc cat gat tca
336Ser Gly Asp Val Ser Ala Leu Ser Gln Ser Leu Ser Ala His Asp Ser
100 105 110cag ctc tca aca ctc tcc
gtc tct gtt agc gcc ctt tcg act gac gtg 384Gln Leu Ser Thr Leu Ser
Val Ser Val Ser Ala Leu Ser Thr Asp Val 115 120
125tct aac ctc aaa cgc gat gtg gca tca tct gcg ctc aat atc
tct gac 432Ser Asn Leu Lys Arg Asp Val Ala Ser Ser Ala Leu Asn Ile
Ser Asp 130 135 140att cag cgg cgt gtc
gcc gct ttg gaa tct ggt tct ggc gcg gtc cta 480Ile Gln Arg Arg Val
Ala Ala Leu Glu Ser Gly Ser Gly Ala Val Leu145 150
155 160aca ttt cgt gct ccc ctt cgg ctc gat ggc
gac tcc gtc tca tta gat 528Thr Phe Arg Ala Pro Leu Arg Leu Asp Gly
Asp Ser Val Ser Leu Asp 165 170
175atg gat cct tac ttt tgc tcg gaa agg gcg aac tta acg tca tac tcg
576Met Asp Pro Tyr Phe Cys Ser Glu Arg Ala Asn Leu Thr Ser Tyr Ser
180 185 190gcg agc gcg cag ctg ttg
cag ttt cag tgg ttt gtc agg agt gag gat 624Ala Ser Ala Gln Leu Leu
Gln Phe Gln Trp Phe Val Arg Ser Glu Asp 195 200
205ggt tct tcc gat tcg att gac atg agt gtc gtt gcc cat tgc
cac ggt 672Gly Ser Ser Asp Ser Ile Asp Met Ser Val Val Ala His Cys
His Gly 210 215 220cgt cgt acg gat tac
tta atg tcc tct cat gat tca ctc act gtc act 720Arg Arg Thr Asp Tyr
Leu Met Ser Ser His Asp Ser Leu Thr Val Thr225 230
235 240ggt aac tcg gtc tct ctc gtg ttc aac tta
gat tac atc aca tcg cct 768Gly Asn Ser Val Ser Leu Val Phe Asn Leu
Asp Tyr Ile Thr Ser Pro 245 250
255ggc gtg gat tat gct cgt ctg att cca tgt cat gga ttt caa caa gca
816Gly Val Asp Tyr Ala Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala
260 265 270acc ttc ccc gtt gac att
tcc ttc aca aaa agc gac gtt aca cac acg 864Thr Phe Pro Val Asp Ile
Ser Phe Thr Lys Ser Asp Val Thr His Thr 275 280
285tat cag gtc tat ggc gca ttc gat ggg cct cgt gtc ttc aaa
gta acg 912Tyr Gln Val Tyr Gly Ala Phe Asp Gly Pro Arg Val Phe Lys
Val Thr 290 295 300ttc tcg ccg ggt gaa
act ggt gcg acg aat ata cga ttc ttg acc gtg 960Phe Ser Pro Gly Glu
Thr Gly Ala Thr Asn Ile Arg Phe Leu Thr Val305 310
315 320cgt acg ggc atc gac acc taa
981Arg Thr Gly Ile Asp Thr
32512326PRTAvian orthoreovirus 12Met Ala Gly Leu Thr Pro Leu Gln Arg Arg
Glu Val Val Gly Leu Ile1 5 10
15Leu Ser Leu Thr Ser Ser Thr Ser Ile Ser Pro Gly Asp Leu Thr Pro
20 25 30Ile Tyr Asp Arg Leu Ser
Ala Val Glu Thr Ile Cys Asp Thr Leu Ser 35 40
45Gly Ser Val Gly Gln Leu Thr Thr Met Met Ser Asn Leu Ser
Ala Arg 50 55 60Leu Asp Asp Leu Thr
Ile Val Ser Arg Asp Met Ala Thr Asp Leu Arg65 70
75 80Asn Val Gln Gly Ser Val Ser Ala Leu Arg
Thr Ser Leu Asp His Leu 85 90
95Ser Gly Asp Val Ser Ala Leu Ser Gln Ser Leu Ser Ala His Asp Ser
100 105 110Gln Leu Ser Thr Leu
Ser Val Ser Val Ser Ala Leu Ser Thr Asp Val 115
120 125Ser Asn Leu Lys Arg Asp Val Ala Ser Ser Ala Leu
Asn Ile Ser Asp 130 135 140Ile Gln Arg
Arg Val Ala Ala Leu Glu Ser Gly Ser Gly Ala Val Leu145
150 155 160Thr Phe Arg Ala Pro Leu Arg
Leu Asp Gly Asp Ser Val Ser Leu Asp 165
170 175Met Asp Pro Tyr Phe Cys Ser Glu Arg Ala Asn Leu
Thr Ser Tyr Ser 180 185 190Ala
Ser Ala Gln Leu Leu Gln Phe Gln Trp Phe Val Arg Ser Glu Asp 195
200 205Gly Ser Ser Asp Ser Ile Asp Met Ser
Val Val Ala His Cys His Gly 210 215
220Arg Arg Thr Asp Tyr Leu Met Ser Ser His Asp Ser Leu Thr Val Thr225
230 235 240Gly Asn Ser Val
Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Ser Pro 245
250 255Gly Val Asp Tyr Ala Arg Leu Ile Pro Cys
His Gly Phe Gln Gln Ala 260 265
270Thr Phe Pro Val Asp Ile Ser Phe Thr Lys Ser Asp Val Thr His Thr
275 280 285Tyr Gln Val Tyr Gly Ala Phe
Asp Gly Pro Arg Val Phe Lys Val Thr 290 295
300Phe Ser Pro Gly Glu Thr Gly Ala Thr Asn Ile Arg Phe Leu Thr
Val305 310 315 320Arg Thr
Gly Ile Asp Thr 32513978DNAAvian
orthoreovirusCDS(1)..(978) 13atg gaa ggt ctc aat cag tca cag cga aga gag
gtc gtg ggg ctg ata 48Met Glu Gly Leu Asn Gln Ser Gln Arg Arg Glu
Val Val Gly Leu Ile1 5 10
15ctc tca ttg act tcg agc gtg act ata agt cct ggc gat ttg acg caa
96Leu Ser Leu Thr Ser Ser Val Thr Ile Ser Pro Gly Asp Leu Thr Gln
20 25 30cta cgt gag cgt atc tcg gca
tta gaa tct gct aac gcg tcg tta aat 144Leu Arg Glu Arg Ile Ser Ala
Leu Glu Ser Ala Asn Ala Ser Leu Asn 35 40
45gag gct att gaa ggc gtg tta act cag ttg gtg gat ttg tca cag
aag 192Glu Ala Ile Glu Gly Val Leu Thr Gln Leu Val Asp Leu Ser Gln
Lys 50 55 60ctg agc aat gca gcg gac
gct atg gtt gaa tta cga gga gag tta aat 240Leu Ser Asn Ala Ala Asp
Ala Met Val Glu Leu Arg Gly Glu Leu Asn65 70
75 80tca ttg act gct agc gtt cag att atc cag tcc
tct ttg aaa tca ctt 288Ser Leu Thr Ala Ser Val Gln Ile Ile Gln Ser
Ser Leu Lys Ser Leu 85 90
95aca gac agt atg tcg gat ctt tct gac cga acg act gct aat acc tcg
336Thr Asp Ser Met Ser Asp Leu Ser Asp Arg Thr Thr Ala Asn Thr Ser
100 105 110tcg atc acg aat ctg gca
agt aag gtg gat ggt ctt acg gtc gat gtg 384Ser Ile Thr Asn Leu Ala
Ser Lys Val Asp Gly Leu Thr Val Asp Val 115 120
125act aac ctt aag cgg gac gtg tca aat cag ggt ctt aaa gtt
act agt 432Thr Asn Leu Lys Arg Asp Val Ser Asn Gln Gly Leu Lys Val
Thr Ser 130 135 140ctc gaa cag cgt gta
tct agt tta gag tct ggc gtt ggt tct gtc cct 480Leu Glu Gln Arg Val
Ser Ser Leu Glu Ser Gly Val Gly Ser Val Pro145 150
155 160aca ttt gct gct ccc ctc aaa ttg gaa agc
ggg act gtc tct ctc gat 528Thr Phe Ala Ala Pro Leu Lys Leu Glu Ser
Gly Thr Val Ser Leu Asp 165 170
175tta gat cct tat ttt tgt tct gtg gac cat aat ctt acg tcg tat tcc
576Leu Asp Pro Tyr Phe Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser
180 185 190gca agt gcg ata ttg atg
aat ttt cag tgg ctt gtc cgc agt gag gga 624Ala Ser Ala Ile Leu Met
Asn Phe Gln Trp Leu Val Arg Ser Glu Gly 195 200
205gga tcg tct gac tcg ttt gat atg aat gtg aca gct cat agc
cat ggc 672Gly Ser Ser Asp Ser Phe Asp Met Asn Val Thr Ala His Ser
His Gly 210 215 220cag agg aca gat tat
atg atg tct act act cag tcg ttg act gtt act 720Gln Arg Thr Asp Tyr
Met Met Ser Thr Thr Gln Ser Leu Thr Val Thr225 230
235 240ggg aat cct gtc acc cta gtc ttt gat ctt
aac gct ctt att tcc ccg 768Gly Asn Pro Val Thr Leu Val Phe Asp Leu
Asn Ala Leu Ile Ser Pro 245 250
255ccc tcc gat tat tct cgc tta ata cca tgt cat ggc ttc caa caa gca
816Pro Ser Asp Tyr Ser Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala
260 265 270acg ttc ccg gtg gat ctt
tcc ttc aag cgc gat gat gtc acg cac tca 864Thr Phe Pro Val Asp Leu
Ser Phe Lys Arg Asp Asp Val Thr His Ser 275 280
285tat cag gtg tat ggt tcg tat aca acc cct cgt gtt ttc aag
ata acc 912Tyr Gln Val Tyr Gly Ser Tyr Thr Thr Pro Arg Val Phe Lys
Ile Thr 290 295 300ttc tct cct ggt aac
cca gtg cct gcg gtc ata cgc ttc ata acc gtg 960Phe Ser Pro Gly Asn
Pro Val Pro Ala Val Ile Arg Phe Ile Thr Val305 310
315 320cgt acg ggc atc gat acc
978Arg Thr Gly Ile Asp Thr
32514326PRTAvian orthoreovirus 14Met Glu Gly Leu Asn Gln Ser Gln Arg Arg
Glu Val Val Gly Leu Ile1 5 10
15Leu Ser Leu Thr Ser Ser Val Thr Ile Ser Pro Gly Asp Leu Thr Gln
20 25 30Leu Arg Glu Arg Ile Ser
Ala Leu Glu Ser Ala Asn Ala Ser Leu Asn 35 40
45Glu Ala Ile Glu Gly Val Leu Thr Gln Leu Val Asp Leu Ser
Gln Lys 50 55 60Leu Ser Asn Ala Ala
Asp Ala Met Val Glu Leu Arg Gly Glu Leu Asn65 70
75 80Ser Leu Thr Ala Ser Val Gln Ile Ile Gln
Ser Ser Leu Lys Ser Leu 85 90
95Thr Asp Ser Met Ser Asp Leu Ser Asp Arg Thr Thr Ala Asn Thr Ser
100 105 110Ser Ile Thr Asn Leu
Ala Ser Lys Val Asp Gly Leu Thr Val Asp Val 115
120 125Thr Asn Leu Lys Arg Asp Val Ser Asn Gln Gly Leu
Lys Val Thr Ser 130 135 140Leu Glu Gln
Arg Val Ser Ser Leu Glu Ser Gly Val Gly Ser Val Pro145
150 155 160Thr Phe Ala Ala Pro Leu Lys
Leu Glu Ser Gly Thr Val Ser Leu Asp 165
170 175Leu Asp Pro Tyr Phe Cys Ser Val Asp His Asn Leu
Thr Ser Tyr Ser 180 185 190Ala
Ser Ala Ile Leu Met Asn Phe Gln Trp Leu Val Arg Ser Glu Gly 195
200 205Gly Ser Ser Asp Ser Phe Asp Met Asn
Val Thr Ala His Ser His Gly 210 215
220Gln Arg Thr Asp Tyr Met Met Ser Thr Thr Gln Ser Leu Thr Val Thr225
230 235 240Gly Asn Pro Val
Thr Leu Val Phe Asp Leu Asn Ala Leu Ile Ser Pro 245
250 255Pro Ser Asp Tyr Ser Arg Leu Ile Pro Cys
His Gly Phe Gln Gln Ala 260 265
270Thr Phe Pro Val Asp Leu Ser Phe Lys Arg Asp Asp Val Thr His Ser
275 280 285Tyr Gln Val Tyr Gly Ser Tyr
Thr Thr Pro Arg Val Phe Lys Ile Thr 290 295
300Phe Ser Pro Gly Asn Pro Val Pro Ala Val Ile Arg Phe Ile Thr
Val305 310 315 320Arg Thr
Gly Ile Asp Thr 32515969DNAAvian
orthoreovirusCDS(1)..(969)misc_feature(968)..(969)n = a, c, g or t 15atg
gaa ggg tta act cag tca caa cga aga gaa gtc gtg ggg ctg ata 48Met
Glu Gly Leu Thr Gln Ser Gln Arg Arg Glu Val Val Gly Leu Ile1
5 10 15ctc tct ttg act tcg agc gcg
aat ata agc act gga gat tta gct cag 96Leu Ser Leu Thr Ser Ser Ala
Asn Ile Ser Thr Gly Asp Leu Ala Gln 20 25
30att cgt agc cgc cta tcg gcg ttg gaa tca tcc aat gcg tca
ttg agt 144Ile Arg Ser Arg Leu Ser Ala Leu Glu Ser Ser Asn Ala Ser
Leu Ser 35 40 45gag act gta aat
ggt gcg gtg agt cag tta gta gtc ttg tct tcg cga 192Glu Thr Val Asn
Gly Ala Val Ser Gln Leu Val Val Leu Ser Ser Arg 50 55
60atc gac aat cta gcc gct act gtg gct gat gga cag ctg
gaa ttg cgc 240Ile Asp Asn Leu Ala Ala Thr Val Ala Asp Gly Gln Leu
Glu Leu Arg65 70 75
80tct ttg gtc atg gat gtt aag aat atc cga tcg tta tta gat gat gtc
288Ser Leu Val Met Asp Val Lys Asn Ile Arg Ser Leu Leu Asp Asp Val
85 90 95tct acg acc gtg gta tct
tta tcc gct tca gtt cgc gaa cat gac tca 336Ser Thr Thr Val Val Ser
Leu Ser Ala Ser Val Arg Glu His Asp Ser 100
105 110tca att att gat tta agg cgt cag ttc ggt ttg ctt
tcc acc gat aca 384Ser Ile Ile Asp Leu Arg Arg Gln Phe Gly Leu Leu
Ser Thr Asp Thr 115 120 125gcc aat
ctt aaa gct gat gtt gcc gct cag tcc ctc ata gtc act agt 432Ala Asn
Leu Lys Ala Asp Val Ala Ala Gln Ser Leu Ile Val Thr Ser 130
135 140ctt gag cag cgt gtt act gcc cta gaa tct agt
acc ggc tcc ctc cct 480Leu Glu Gln Arg Val Thr Ala Leu Glu Ser Ser
Thr Gly Ser Leu Pro145 150 155
160tcg ttc tcc gcc cca ctc aaa cta gat gat ggg acc gtt tca tta gac
528Ser Phe Ser Ala Pro Leu Lys Leu Asp Asp Gly Thr Val Ser Leu Asp
165 170 175tta gat cct tac ttc
tgy tcg gtg gat cat aat cta aca tct tat tct 576Leu Asp Pro Tyr Phe
Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser 180
185 190gcc agt gcg cag tta atg caa ttc caa tgg ttc gtg
aga ggt gag gga 624Ala Ser Ala Gln Leu Met Gln Phe Gln Trp Phe Val
Arg Gly Glu Gly 195 200 205ggt tca
tct gat tcc ata gat atg agc gtc act gct cat tgc cac ggc 672Gly Ser
Ser Asp Ser Ile Asp Met Ser Val Thr Ala His Cys His Gly 210
215 220cgg cgg act gat tac atg atg tcc acc act caa
tcc cta act gtt act 720Arg Arg Thr Asp Tyr Met Met Ser Thr Thr Gln
Ser Leu Thr Val Thr225 230 235
240ggt acc tcc gtg tcc ctt gtc ttt gat tta aat aca ctc gtt act cca
768Gly Thr Ser Val Ser Leu Val Phe Asp Leu Asn Thr Leu Val Thr Pro
245 250 255cct tct gac tac tcg
cgc ctt att ccg tgt cat ggg ttc cag cag gca 816Pro Ser Asp Tyr Ser
Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala 260
265 270aca ttt cca gtt gat ctt tcg ttc aaa agg gat gaa
gag aca cac tct 864Thr Phe Pro Val Asp Leu Ser Phe Lys Arg Asp Glu
Glu Thr His Ser 275 280 285tat cag
gta tat ggt tca tac tcg acg ccc gcg tgt ctt aag gtg act 912Tyr Gln
Val Tyr Gly Ser Tyr Ser Thr Pro Ala Cys Leu Lys Val Thr 290
295 300ttc tcc ccc tgg ggc ttc cag tta ccg gca gtc
att cga ttc cta acc 960Phe Ser Pro Trp Gly Phe Gln Leu Pro Ala Val
Ile Arg Phe Leu Thr305 310 315
320ctg cga cnn
969Leu Arg Xaa16323PRTAvian orthoreovirusmisc_feature(323)..(323)The
'Xaa' at location 323 stands for Gln, His, Arg, Pro, or Leu. 16Met
Glu Gly Leu Thr Gln Ser Gln Arg Arg Glu Val Val Gly Leu Ile1
5 10 15Leu Ser Leu Thr Ser Ser Ala
Asn Ile Ser Thr Gly Asp Leu Ala Gln 20 25
30Ile Arg Ser Arg Leu Ser Ala Leu Glu Ser Ser Asn Ala Ser
Leu Ser 35 40 45Glu Thr Val Asn
Gly Ala Val Ser Gln Leu Val Val Leu Ser Ser Arg 50 55
60Ile Asp Asn Leu Ala Ala Thr Val Ala Asp Gly Gln Leu
Glu Leu Arg65 70 75
80Ser Leu Val Met Asp Val Lys Asn Ile Arg Ser Leu Leu Asp Asp Val
85 90 95Ser Thr Thr Val Val Ser
Leu Ser Ala Ser Val Arg Glu His Asp Ser 100
105 110Ser Ile Ile Asp Leu Arg Arg Gln Phe Gly Leu Leu
Ser Thr Asp Thr 115 120 125Ala Asn
Leu Lys Ala Asp Val Ala Ala Gln Ser Leu Ile Val Thr Ser 130
135 140Leu Glu Gln Arg Val Thr Ala Leu Glu Ser Ser
Thr Gly Ser Leu Pro145 150 155
160Ser Phe Ser Ala Pro Leu Lys Leu Asp Asp Gly Thr Val Ser Leu Asp
165 170 175Leu Asp Pro Tyr
Phe Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser 180
185 190Ala Ser Ala Gln Leu Met Gln Phe Gln Trp Phe
Val Arg Gly Glu Gly 195 200 205Gly
Ser Ser Asp Ser Ile Asp Met Ser Val Thr Ala His Cys His Gly 210
215 220Arg Arg Thr Asp Tyr Met Met Ser Thr Thr
Gln Ser Leu Thr Val Thr225 230 235
240Gly Thr Ser Val Ser Leu Val Phe Asp Leu Asn Thr Leu Val Thr
Pro 245 250 255Pro Ser Asp
Tyr Ser Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala 260
265 270Thr Phe Pro Val Asp Leu Ser Phe Lys Arg
Asp Glu Glu Thr His Ser 275 280
285Tyr Gln Val Tyr Gly Ser Tyr Ser Thr Pro Ala Cys Leu Lys Val Thr 290
295 300Phe Ser Pro Trp Gly Phe Gln Leu
Pro Ala Val Ile Arg Phe Leu Thr305 310
315 320Leu Arg Xaa17966DNAAvian
orthoreovirusCDS(1)..(966) 17atg gca gga tta act ccc tta cag cga aga gaa
gtc gtg ggc ttg ata 48Met Ala Gly Leu Thr Pro Leu Gln Arg Arg Glu
Val Val Gly Leu Ile1 5 10
15ctc tcc ttg act tcg agc gcg aat aca aac agt gga gat ttg acg ccc
96Leu Ser Leu Thr Ser Ser Ala Asn Thr Asn Ser Gly Asp Leu Thr Pro
20 25 30ata tat gac cgt ctg ctt agt
atg gag tcc gcc gtt gcg tca ttg agc 144Ile Tyr Asp Arg Leu Leu Ser
Met Glu Ser Ala Val Ala Ser Leu Ser 35 40
45gct tcc gtt agc ggc tta ttg cag aaa gta tca gat ttg gac tct
gat 192Ala Ser Val Ser Gly Leu Leu Gln Lys Val Ser Asp Leu Asp Ser
Asp 50 55 60ttg cag aat gtc act tca
tcc tta ggc cag gct gat tcg gcg ttg acc 240Leu Gln Asn Val Thr Ser
Ser Leu Gly Gln Ala Asp Ser Ala Leu Thr65 70
75 80gca ttg tcc aag gaa ctg cac cag ttg agc agc
tct att gaa aat gtc 288Ala Leu Ser Lys Glu Leu His Gln Leu Ser Ser
Ser Ile Glu Asn Val 85 90
95tca acg tct gtt tca gat gtg tcg act act gta tca ggg cat cag gct
336Ser Thr Ser Val Ser Asp Val Ser Thr Thr Val Ser Gly His Gln Ala
100 105 110gcg ata gcc gcc gta caa
act tcc gtt cac gct aat acg act gac att 384Ala Ile Ala Ala Val Gln
Thr Ser Val His Ala Asn Thr Thr Asp Ile 115 120
125agc aat ctg aaa agc agt gta tct act atc agc tta aat ctc
gct gat 432Ser Asn Leu Lys Ser Ser Val Ser Thr Ile Ser Leu Asn Leu
Ala Asp 130 135 140ctt gaa cga cgt gta
gac gac att gga tca ggt tca ggt tcc aac ctg 480Leu Glu Arg Arg Val
Asp Asp Ile Gly Ser Gly Ser Gly Ser Asn Leu145 150
155 160agg ttt tcg tcc ccg ctg agt ttg tcc cag
ggt gtg gtg tca ctg atc 528Arg Phe Ser Ser Pro Leu Ser Leu Ser Gln
Gly Val Val Ser Leu Ile 165 170
175atg gat cct tac ttc tgc tcg gac aat cag gcg tta acc tcg tat tcc
576Met Asp Pro Tyr Phe Cys Ser Asp Asn Gln Ala Leu Thr Ser Tyr Ser
180 185 190aca gac gcg cag ttg atg
caa ttc cag tgg cta gct aga ggt gag ggt 624Thr Asp Ala Gln Leu Met
Gln Phe Gln Trp Leu Ala Arg Gly Glu Gly 195 200
205ggc tca tcg agt tcc gtc gaa atg ctc gtc aat gca cat tgt
cat gga 672Gly Ser Ser Ser Ser Val Glu Met Leu Val Asn Ala His Cys
His Gly 210 215 220cgc cgt acc gat tat
atg atg tcc act aca gaa aac ttc acc gtt aca 720Arg Arg Thr Asp Tyr
Met Met Ser Thr Thr Glu Asn Phe Thr Val Thr225 230
235 240gat aac tct gtg tca ctg gtt ttc aat cta
gac tat att act aaa cca 768Asp Asn Ser Val Ser Leu Val Phe Asn Leu
Asp Tyr Ile Thr Lys Pro 245 250
255cct tct gat atg tcg cgc ttg ata cca cgc gct ggg ttc cag gcc gct
816Pro Ser Asp Met Ser Arg Leu Ile Pro Arg Ala Gly Phe Gln Ala Ala
260 265 270tct ttt ccc gtg gac gtc
tcc ttc acc cgt gac acg aca acg cac gct 864Ser Phe Pro Val Asp Val
Ser Phe Thr Arg Asp Thr Thr Thr His Ala 275 280
285tac cag gtg tat ggt act ttt tct agt cct cgc att ttt aag
atc aca 912Tyr Gln Val Tyr Gly Thr Phe Ser Ser Pro Arg Ile Phe Lys
Ile Thr 290 295 300ttt ctt aca ggt ggt
act gga acc tgc aaa cat tcg ttt ctt gag ccg 960Phe Leu Thr Gly Gly
Thr Gly Thr Cys Lys His Ser Phe Leu Glu Pro305 310
315 320gcg acg
966Ala Thr18322PRTAvian orthoreovirus 18Met
Ala Gly Leu Thr Pro Leu Gln Arg Arg Glu Val Val Gly Leu Ile1
5 10 15Leu Ser Leu Thr Ser Ser Ala
Asn Thr Asn Ser Gly Asp Leu Thr Pro 20 25
30Ile Tyr Asp Arg Leu Leu Ser Met Glu Ser Ala Val Ala Ser
Leu Ser 35 40 45Ala Ser Val Ser
Gly Leu Leu Gln Lys Val Ser Asp Leu Asp Ser Asp 50 55
60Leu Gln Asn Val Thr Ser Ser Leu Gly Gln Ala Asp Ser
Ala Leu Thr65 70 75
80Ala Leu Ser Lys Glu Leu His Gln Leu Ser Ser Ser Ile Glu Asn Val
85 90 95Ser Thr Ser Val Ser Asp
Val Ser Thr Thr Val Ser Gly His Gln Ala 100
105 110Ala Ile Ala Ala Val Gln Thr Ser Val His Ala Asn
Thr Thr Asp Ile 115 120 125Ser Asn
Leu Lys Ser Ser Val Ser Thr Ile Ser Leu Asn Leu Ala Asp 130
135 140Leu Glu Arg Arg Val Asp Asp Ile Gly Ser Gly
Ser Gly Ser Asn Leu145 150 155
160Arg Phe Ser Ser Pro Leu Ser Leu Ser Gln Gly Val Val Ser Leu Ile
165 170 175Met Asp Pro Tyr
Phe Cys Ser Asp Asn Gln Ala Leu Thr Ser Tyr Ser 180
185 190Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu
Ala Arg Gly Glu Gly 195 200 205Gly
Ser Ser Ser Ser Val Glu Met Leu Val Asn Ala His Cys His Gly 210
215 220Arg Arg Thr Asp Tyr Met Met Ser Thr Thr
Glu Asn Phe Thr Val Thr225 230 235
240Asp Asn Ser Val Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Lys
Pro 245 250 255Pro Ser Asp
Met Ser Arg Leu Ile Pro Arg Ala Gly Phe Gln Ala Ala 260
265 270Ser Phe Pro Val Asp Val Ser Phe Thr Arg
Asp Thr Thr Thr His Ala 275 280
285Tyr Gln Val Tyr Gly Thr Phe Ser Ser Pro Arg Ile Phe Lys Ile Thr 290
295 300Phe Leu Thr Gly Gly Thr Gly Thr
Cys Lys His Ser Phe Leu Glu Pro305 310
315 320Ala Thr19960DNAAvian
orthoreovirusCDS(1)..(960)misc_feature(960)..(960)n = a, c, g or t 19atg
gcc gga ctc act cca tca cag cga aga gaa gtc gtc ggg atg ata 48Met
Ala Gly Leu Thr Pro Ser Gln Arg Arg Glu Val Val Gly Met Ile1
5 10 15ctc tca ttg act tcg agc acg
agt ata agt cct gga gat ttg gca cca 96Leu Ser Leu Thr Ser Ser Thr
Ser Ile Ser Pro Gly Asp Leu Ala Pro 20 25
30ata tac gat cgc ctc tct gcc gtt gag acg att tgc gcc acc
tta agt 144Ile Tyr Asp Arg Leu Ser Ala Val Glu Thr Ile Cys Ala Thr
Leu Ser 35 40 45gac tct gtg agt
cag ctg att tca aag acg tct gat cta tct aat cgt 192Asp Ser Val Ser
Gln Leu Ile Ser Lys Thr Ser Asp Leu Ser Asn Arg 50 55
60tta aat ggc ttg gtg aat gtg tcc caa gat ata gct aac
gac tta aga 240Leu Asn Gly Leu Val Asn Val Ser Gln Asp Ile Ala Asn
Asp Leu Arg65 70 75
80gat att caa agt cgt gtg tct gca ctt caa aca ttg gta gat ggt gtg
288Asp Ile Gln Ser Arg Val Ser Ala Leu Gln Thr Leu Val Asp Gly Val
85 90 95tcc agt gat gtt tct ctc
cta tca cag tcg ttg tct gct cac gat tca 336Ser Ser Asp Val Ser Leu
Leu Ser Gln Ser Leu Ser Ala His Asp Ser 100
105 110cag ctc tcc gag ctt tcc tct tcg gtt agc act ctg
tct act gac atg 384Gln Leu Ser Glu Leu Ser Ser Ser Val Ser Thr Leu
Ser Thr Asp Met 115 120 125tca aac
ctt caa cgt gat gtg act gca tct gca ctc aat att tct gat 432Ser Asn
Leu Gln Arg Asp Val Thr Ala Ser Ala Leu Asn Ile Ser Asp 130
135 140ctt cag caa cgt gtc gcc ggt ttg gaa tcg agc
tct ggg tca tct ctc 480Leu Gln Gln Arg Val Ala Gly Leu Glu Ser Ser
Ser Gly Ser Ser Leu145 150 155
160acg ttc ctt gct cca ctt cgg gtt gat gga ggt tcc gtc tcg ttg gac
528Thr Phe Leu Ala Pro Leu Arg Val Asp Gly Gly Ser Val Ser Leu Asp
165 170 175atg gat ccg tac ttt
tgc tcc gaa agg tcg aac tta aca tcc tac tcg 576Met Asp Pro Tyr Phe
Cys Ser Glu Arg Ser Asn Leu Thr Ser Tyr Ser 180
185 190gcg agc gca cag ttg ttg cag ttt cag tgg tac gtt
agg agt gag ggt 624Ala Ser Ala Gln Leu Leu Gln Phe Gln Trp Tyr Val
Arg Ser Glu Gly 195 200 205ggc tct
tca gat tcg att gac atg agc gtg gtg gct cat tgc cac ggt 672Gly Ser
Ser Asp Ser Ile Asp Met Ser Val Val Ala His Cys His Gly 210
215 220cgt cgc acg gat tat ttg atg tcc act cat gat
tcg ctc acc gtc act 720Arg Arg Thr Asp Tyr Leu Met Ser Thr His Asp
Ser Leu Thr Val Thr225 230 235
240ggc aac tca gtg tcc tta gta ttc aat cta gac tac atc acc acg tct
768Gly Asn Ser Val Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Thr Ser
245 250 255ggt gtg gag tac gct
cgt ctg atc ccg agt cat ggt ttc caa cag gct 816Gly Val Glu Tyr Ala
Arg Leu Ile Pro Ser His Gly Phe Gln Gln Ala 260
265 270act ttt ccc gtt gat atc tct ttc acg aga aat gac
acc aca cat acg 864Thr Phe Pro Val Asp Ile Ser Phe Thr Arg Asn Asp
Thr Thr His Thr 275 280 285tac caa
gtt tat ggt gca ttt gat ggc cct cgc gtg ttt aaa gta aca 912Tyr Gln
Val Tyr Gly Ala Phe Asp Gly Pro Arg Val Phe Lys Val Thr 290
295 300ttc tcg ccg ggc gag aac cag cac gac caa atg
tgc gat tca acc gan 960Phe Ser Pro Gly Glu Asn Gln His Asp Gln Met
Cys Asp Ser Thr Xaa305 310 315
32020320PRTAvian orthoreovirusmisc_feature(320)..(320)The 'Xaa' at
location 320 stands for Glu, or Asp. 20Met Ala Gly Leu Thr Pro Ser
Gln Arg Arg Glu Val Val Gly Met Ile1 5 10
15Leu Ser Leu Thr Ser Ser Thr Ser Ile Ser Pro Gly Asp
Leu Ala Pro 20 25 30Ile Tyr
Asp Arg Leu Ser Ala Val Glu Thr Ile Cys Ala Thr Leu Ser 35
40 45Asp Ser Val Ser Gln Leu Ile Ser Lys Thr
Ser Asp Leu Ser Asn Arg 50 55 60Leu
Asn Gly Leu Val Asn Val Ser Gln Asp Ile Ala Asn Asp Leu Arg65
70 75 80Asp Ile Gln Ser Arg Val
Ser Ala Leu Gln Thr Leu Val Asp Gly Val 85
90 95Ser Ser Asp Val Ser Leu Leu Ser Gln Ser Leu Ser
Ala His Asp Ser 100 105 110Gln
Leu Ser Glu Leu Ser Ser Ser Val Ser Thr Leu Ser Thr Asp Met 115
120 125Ser Asn Leu Gln Arg Asp Val Thr Ala
Ser Ala Leu Asn Ile Ser Asp 130 135
140Leu Gln Gln Arg Val Ala Gly Leu Glu Ser Ser Ser Gly Ser Ser Leu145
150 155 160Thr Phe Leu Ala
Pro Leu Arg Val Asp Gly Gly Ser Val Ser Leu Asp 165
170 175Met Asp Pro Tyr Phe Cys Ser Glu Arg Ser
Asn Leu Thr Ser Tyr Ser 180 185
190Ala Ser Ala Gln Leu Leu Gln Phe Gln Trp Tyr Val Arg Ser Glu Gly
195 200 205Gly Ser Ser Asp Ser Ile Asp
Met Ser Val Val Ala His Cys His Gly 210 215
220Arg Arg Thr Asp Tyr Leu Met Ser Thr His Asp Ser Leu Thr Val
Thr225 230 235 240Gly Asn
Ser Val Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Thr Ser
245 250 255Gly Val Glu Tyr Ala Arg Leu
Ile Pro Ser His Gly Phe Gln Gln Ala 260 265
270Thr Phe Pro Val Asp Ile Ser Phe Thr Arg Asn Asp Thr Thr
His Thr 275 280 285Tyr Gln Val Tyr
Gly Ala Phe Asp Gly Pro Arg Val Phe Lys Val Thr 290
295 300Phe Ser Pro Gly Glu Asn Gln His Asp Gln Met Cys
Asp Ser Thr Xaa305 310 315
32021963DNAAvian orthoreovirusCDS(1)..(963) 21atg gaa ggc tta act cag
tca cag cga aga gag gtc gtg ggg ctg ata 48Met Glu Gly Leu Thr Gln
Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15ctc tca ttg act tcg agc gtg act aca agt cct ggc
gat ttg acg gaa 96Leu Ser Leu Thr Ser Ser Val Thr Thr Ser Pro Gly
Asp Leu Thr Glu 20 25 30ctg
cgt gaa cgc gtc tca gcg tta gaa tct gcc act gcg tcc ttg aat 144Leu
Arg Glu Arg Val Ser Ala Leu Glu Ser Ala Thr Ala Ser Leu Asn 35
40 45gag act ata aaa ggc gtg ttg ggc caa
ttg gta gat ttg gca cag aag 192Glu Thr Ile Lys Gly Val Leu Gly Gln
Leu Val Asp Leu Ala Gln Lys 50 55
60ttg agt aat gcg gca gac gct ata gtc ggg ttg cga gga gag ttg aac
240Leu Ser Asn Ala Ala Asp Ala Ile Val Gly Leu Arg Gly Glu Leu Asn65
70 75 80tca tta gct gct agc
gtc caa act att caa tct tct ttg aga tca ctt 288Ser Leu Ala Ala Ser
Val Gln Thr Ile Gln Ser Ser Leu Arg Ser Leu 85
90 95aca gac agt gtg tcg gat ctt tct ggc caa gtg
act act aat gcg tcg 336Thr Asp Ser Val Ser Asp Leu Ser Gly Gln Val
Thr Thr Asn Ala Ser 100 105
110tcg atc acg aat gtg aag ggt atg ttg gat ggt ctt acg gtc gac gtg
384Ser Ile Thr Asn Val Lys Gly Met Leu Asp Gly Leu Thr Val Asp Val
115 120 125gct aat ctt aag cgt gac gca
tca aat cag ggt ctt aaa att act gat 432Ala Asn Leu Lys Arg Asp Ala
Ser Asn Gln Gly Leu Lys Ile Thr Asp 130 135
140ctc gag cag cgt gta gct ggt tta gag tct ggt tct gga tct att ccc
480Leu Glu Gln Arg Val Ala Gly Leu Glu Ser Gly Ser Gly Ser Ile Pro145
150 155 160aca ttc gcc gct
cct ctt aag tta gac agc ggg att gta tca ctc gac 528Thr Phe Ala Ala
Pro Leu Lys Leu Asp Ser Gly Ile Val Ser Leu Asp 165
170 175ctg gat cct tac ttt tgt tcc gta gat cat
aat ctt acg tcg tat tcc 576Leu Asp Pro Tyr Phe Cys Ser Val Asp His
Asn Leu Thr Ser Tyr Ser 180 185
190gcc agt gcg tca tta atg aat ttc cag tgg ctt gtt cga ggt gag gga
624Ala Ser Ala Ser Leu Met Asn Phe Gln Trp Leu Val Arg Gly Glu Gly
195 200 205gga tct tct gac tca ttc gat
atg aat gta aca gct cat agc cat ggc 672Gly Ser Ser Asp Ser Phe Asp
Met Asn Val Thr Ala His Ser His Gly 210 215
220cag agg acg gac tat atg atg tct act act caa tca ttg act gtt act
720Gln Arg Thr Asp Tyr Met Met Ser Thr Thr Gln Ser Leu Thr Val Thr225
230 235 240gga aat tca gtc
acc ctg gtc ttt gac ctt aac gct ctt atc tct ccg 768Gly Asn Ser Val
Thr Leu Val Phe Asp Leu Asn Ala Leu Ile Ser Pro 245
250 255cca tct gat tat tct cgc ttg ata cca tgt
cat ggt ttc caa caa gca 816Pro Ser Asp Tyr Ser Arg Leu Ile Pro Cys
His Gly Phe Gln Gln Ala 260 265
270acg ttt cca gta gac ctt tca ttt aag cga gat gac gtc aca cac tca
864Thr Phe Pro Val Asp Leu Ser Phe Lys Arg Asp Asp Val Thr His Ser
275 280 285tat cag gtg tat ggt tcg tac
acg act cct cgc gtt ttc aag ata acc 912Tyr Gln Val Tyr Gly Ser Tyr
Thr Thr Pro Arg Val Phe Lys Ile Thr 290 295
300ttc tct cct ggc aat cca gtg cct gca gtc ata cgc ttc ata acc ggc
960Phe Ser Pro Gly Asn Pro Val Pro Ala Val Ile Arg Phe Ile Thr Gly305
310 315 320gac
963Asp22321PRTAvian
orthoreovirus 22Met Glu Gly Leu Thr Gln Ser Gln Arg Arg Glu Val Val Gly
Leu Ile1 5 10 15Leu Ser
Leu Thr Ser Ser Val Thr Thr Ser Pro Gly Asp Leu Thr Glu 20
25 30Leu Arg Glu Arg Val Ser Ala Leu Glu
Ser Ala Thr Ala Ser Leu Asn 35 40
45Glu Thr Ile Lys Gly Val Leu Gly Gln Leu Val Asp Leu Ala Gln Lys 50
55 60Leu Ser Asn Ala Ala Asp Ala Ile Val
Gly Leu Arg Gly Glu Leu Asn65 70 75
80Ser Leu Ala Ala Ser Val Gln Thr Ile Gln Ser Ser Leu Arg
Ser Leu 85 90 95Thr Asp
Ser Val Ser Asp Leu Ser Gly Gln Val Thr Thr Asn Ala Ser 100
105 110Ser Ile Thr Asn Val Lys Gly Met Leu
Asp Gly Leu Thr Val Asp Val 115 120
125Ala Asn Leu Lys Arg Asp Ala Ser Asn Gln Gly Leu Lys Ile Thr Asp
130 135 140Leu Glu Gln Arg Val Ala Gly
Leu Glu Ser Gly Ser Gly Ser Ile Pro145 150
155 160Thr Phe Ala Ala Pro Leu Lys Leu Asp Ser Gly Ile
Val Ser Leu Asp 165 170
175Leu Asp Pro Tyr Phe Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser
180 185 190Ala Ser Ala Ser Leu Met
Asn Phe Gln Trp Leu Val Arg Gly Glu Gly 195 200
205Gly Ser Ser Asp Ser Phe Asp Met Asn Val Thr Ala His Ser
His Gly 210 215 220Gln Arg Thr Asp Tyr
Met Met Ser Thr Thr Gln Ser Leu Thr Val Thr225 230
235 240Gly Asn Ser Val Thr Leu Val Phe Asp Leu
Asn Ala Leu Ile Ser Pro 245 250
255Pro Ser Asp Tyr Ser Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala
260 265 270Thr Phe Pro Val Asp
Leu Ser Phe Lys Arg Asp Asp Val Thr His Ser 275
280 285Tyr Gln Val Tyr Gly Ser Tyr Thr Thr Pro Arg Val
Phe Lys Ile Thr 290 295 300Phe Ser Pro
Gly Asn Pro Val Pro Ala Val Ile Arg Phe Ile Thr Gly305
310 315 320Asp23978DNAAvian
orthoreovirusCDS(1)..(978)misc_feature(161)..(161)k = g or t 23atg gcc
gga tta act ccc tca cag cga aga gaa gtc gtg gga ttg ata 48Met Ala
Gly Leu Thr Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15ctc tcc ttg act tcg agc gcg aat
aca aat tgt gga gat ttg acg ccc 96Leu Ser Leu Thr Ser Ser Ala Asn
Thr Asn Cys Gly Asp Leu Thr Pro 20 25
30gtg tat gat cgt ctg ctc agt ctg gag tcc gct gtt gca tca ctg
aat 144Val Tyr Asp Arg Leu Leu Ser Leu Glu Ser Ala Val Ala Ser Leu
Asn 35 40 45ggc tct gtt agc ggc
tcg tct cag aag gtg tta gat ttg gag tca ggt 192Gly Ser Val Ser Gly
Ser Ser Gln Lys Val Leu Asp Leu Glu Ser Gly 50 55
60tta caa gac gtc gct tca tct tta ggc cag act aat tca acg
ttg act 240Leu Gln Asp Val Ala Ser Ser Leu Gly Gln Thr Asn Ser Thr
Leu Thr65 70 75 80gag
tta tcg aaa gga ctg cgt cag ctg agc agt tcc gtt gac agc ctc 288Glu
Leu Ser Lys Gly Leu Arg Gln Leu Ser Ser Ser Val Asp Ser Leu
85 90 95gtc tcg tct gtt tca gat cta
tcg acc gct gtg tcc ggg cat caa gac 336Val Ser Ser Val Ser Asp Leu
Ser Thr Ala Val Ser Gly His Gln Asp 100 105
110gcc ata gtt act att caa acg tcc gtc cac gct aac tca act
gat atc 384Ala Ile Val Thr Ile Gln Thr Ser Val His Ala Asn Ser Thr
Asp Ile 115 120 125agt aac ctg aga
agc agc gta tct acc att agc ttg aat atc acc gat 432Ser Asn Leu Arg
Ser Ser Val Ser Thr Ile Ser Leu Asn Ile Thr Asp 130
135 140ctc gag caa cgc gtg gaa aag att gaa tca ggc gca
agc tcc agt cta 480Leu Glu Gln Arg Val Glu Lys Ile Glu Ser Gly Ala
Ser Ser Ser Leu145 150 155
160cgg ttt acg tcg cct ctg agt ttg tct caa ggc gtg gtg tca ctg att
528Arg Phe Thr Ser Pro Leu Ser Leu Ser Gln Gly Val Val Ser Leu Ile
165 170 175atg gat cct tac ttc
tgt tca gac aac atg gca cta acc tca tat tct 576Met Asp Pro Tyr Phe
Cys Ser Asp Asn Met Ala Leu Thr Ser Tyr Ser 180
185 190aca gat gct caa ctg atg cag ttt cag tgg ttg gct
aga ggt gag gat 624Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu Ala
Arg Gly Glu Asp 195 200 205ggg tca
tca agc tct gtt gat atg ctc gtt aat gct cac tgt cat ggg 672Gly Ser
Ser Ser Ser Val Asp Met Leu Val Asn Ala His Cys His Gly 210
215 220cgt cgc act gat tat atg atg tcc acc aaa gag
agt ttc act gtt acg 720Arg Arg Thr Asp Tyr Met Met Ser Thr Lys Glu
Ser Phe Thr Val Thr225 230 235
240ggc aat tct gtg tcc ttg gtt ttc aat ctg gac tat att act aaa ccg
768Gly Asn Ser Val Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Lys Pro
245 250 255ccc tct gac atg tcg
cgt tta gta ccc cgc gct gga ttc cgg gct gct 816Pro Ser Asp Met Ser
Arg Leu Val Pro Arg Ala Gly Phe Arg Ala Ala 260
265 270tct ttt cct gta gac gtc tct ttc acc cgt gac aca
aca ccg cat gct 864Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Thr
Thr Pro His Ala 275 280 285tac cag
gtg tac ggt gcc ttc tct agt cct cgc atc ttt aag att aca 912Tyr Gln
Val Tyr Gly Ala Phe Ser Ser Pro Arg Ile Phe Lys Ile Thr 290
295 300ttc ctt aca ggt ggc act ggg act gca aat ctc
cgt ttc ttg acc gtg 960Phe Leu Thr Gly Gly Thr Gly Thr Ala Asn Leu
Arg Phe Leu Thr Val305 310 315
320cgt acg ggc atc gac acc
978Arg Thr Gly Ile Asp Thr 32524326PRTAvian
orthoreovirus 24Met Ala Gly Leu Thr Pro Ser Gln Arg Arg Glu Val Val Gly
Leu Ile1 5 10 15Leu Ser
Leu Thr Ser Ser Ala Asn Thr Asn Cys Gly Asp Leu Thr Pro 20
25 30Val Tyr Asp Arg Leu Leu Ser Leu Glu
Ser Ala Val Ala Ser Leu Asn 35 40
45Gly Ser Val Ser Gly Ser Ser Gln Lys Val Leu Asp Leu Glu Ser Gly 50
55 60Leu Gln Asp Val Ala Ser Ser Leu Gly
Gln Thr Asn Ser Thr Leu Thr65 70 75
80Glu Leu Ser Lys Gly Leu Arg Gln Leu Ser Ser Ser Val Asp
Ser Leu 85 90 95Val Ser
Ser Val Ser Asp Leu Ser Thr Ala Val Ser Gly His Gln Asp 100
105 110Ala Ile Val Thr Ile Gln Thr Ser Val
His Ala Asn Ser Thr Asp Ile 115 120
125Ser Asn Leu Arg Ser Ser Val Ser Thr Ile Ser Leu Asn Ile Thr Asp
130 135 140Leu Glu Gln Arg Val Glu Lys
Ile Glu Ser Gly Ala Ser Ser Ser Leu145 150
155 160Arg Phe Thr Ser Pro Leu Ser Leu Ser Gln Gly Val
Val Ser Leu Ile 165 170
175Met Asp Pro Tyr Phe Cys Ser Asp Asn Met Ala Leu Thr Ser Tyr Ser
180 185 190Thr Asp Ala Gln Leu Met
Gln Phe Gln Trp Leu Ala Arg Gly Glu Asp 195 200
205Gly Ser Ser Ser Ser Val Asp Met Leu Val Asn Ala His Cys
His Gly 210 215 220Arg Arg Thr Asp Tyr
Met Met Ser Thr Lys Glu Ser Phe Thr Val Thr225 230
235 240Gly Asn Ser Val Ser Leu Val Phe Asn Leu
Asp Tyr Ile Thr Lys Pro 245 250
255Pro Ser Asp Met Ser Arg Leu Val Pro Arg Ala Gly Phe Arg Ala Ala
260 265 270Ser Phe Pro Val Asp
Val Ser Phe Thr Arg Asp Thr Thr Pro His Ala 275
280 285Tyr Gln Val Tyr Gly Ala Phe Ser Ser Pro Arg Ile
Phe Lys Ile Thr 290 295 300Phe Leu Thr
Gly Gly Thr Gly Thr Ala Asn Leu Arg Phe Leu Thr Val305
310 315 320Arg Thr Gly Ile Asp Thr
32525951DNAAvian orthoreovirusCDS(1)..(951) 25atg gac gga tta act
ccc tca cag cga aga gaa gtc gtg gga ttg ata 48Met Asp Gly Leu Thr
Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15ctc tcc ttg act tcg agc gtg aat aca aac tgt
gga gat ttg acg cct 96Leu Ser Leu Thr Ser Ser Val Asn Thr Asn Cys
Gly Asp Leu Thr Pro 20 25
30gta tac gac cgt ctg ctt agt ctg gag tcc gcc gtt gcg tcg ctg aac
144Val Tyr Asp Arg Leu Leu Ser Leu Glu Ser Ala Val Ala Ser Leu Asn
35 40 45aac tct gtt aat gac ttg ttg cag
aag gta tca gat ttt gaa tct gac 192Asn Ser Val Asn Asp Leu Leu Gln
Lys Val Ser Asp Phe Glu Ser Asp 50 55
60ttg caa gac gcc acc tca tct cta ggc caa gcc aat ttg atg cta acc
240Leu Gln Asp Ala Thr Ser Ser Leu Gly Gln Ala Asn Leu Met Leu Thr65
70 75 80aag tta tct gag gac
ttg cgt cag ttg agc agc tct gtt gac aac ctc 288Lys Leu Ser Glu Asp
Leu Arg Gln Leu Ser Ser Ser Val Asp Asn Leu 85
90 95gtc acg tcc gtt tct gat cta tcg acc act gta
tcc ggg cat cag gac 336Val Thr Ser Val Ser Asp Leu Ser Thr Thr Val
Ser Gly His Gln Asp 100 105
110gca ata gcc gct ata caa act tcc gtt cat gcc aat aca acc gat att
384Ala Ile Ala Ala Ile Gln Thr Ser Val His Ala Asn Thr Thr Asp Ile
115 120 125agt aac tta aaa agc aac gta
tct act att ggc ttg aat atc aca gat 432Ser Asn Leu Lys Ser Asn Val
Ser Thr Ile Gly Leu Asn Ile Thr Asp 130 135
140ctc gcg caa cgt gtg gca aac att gaa tca agc tcg ggc tct agt tta
480Leu Ala Gln Arg Val Ala Asn Ile Glu Ser Ser Ser Gly Ser Ser Leu145
150 155 160aga ttt gtg tca
cct ctg agc ttg tcc caa ggt gta gtg tcc ttg atc 528Arg Phe Val Ser
Pro Leu Ser Leu Ser Gln Gly Val Val Ser Leu Ile 165
170 175atg gat ccc tac ttc tgt tcg gac aat aag
gca tta acc tcc tat tcc 576Met Asp Pro Tyr Phe Cys Ser Asp Asn Lys
Ala Leu Thr Ser Tyr Ser 180 185
190acg gac gct cag ctg atg cag ttc cag tgg ctg gct aga ggt gag gat
624Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu Ala Arg Gly Glu Asp
195 200 205ggg tca tcg agc tct gtt gat
atg ctt gtt aac gca cac tgt cat ggg 672Gly Ser Ser Ser Ser Val Asp
Met Leu Val Asn Ala His Cys His Gly 210 215
220cgt cgc act gat tat atg atg tcc act aca gag agt ttt act gtc acg
720Arg Arg Thr Asp Tyr Met Met Ser Thr Thr Glu Ser Phe Thr Val Thr225
230 235 240ggc aat tct gta
tca ttg gtc ttc aac ctg gat tat atc acc aaa ccg 768Gly Asn Ser Val
Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Lys Pro 245
250 255ccc tct gac atg tcg cgg cta gta cca cgt
gct ggt ttc cag gct gct 816Pro Ser Asp Met Ser Arg Leu Val Pro Arg
Ala Gly Phe Gln Ala Ala 260 265
270tct ttc cct gtg gac gtc tcc ttc act cgt gac aca aca ccg cac gct
864Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Thr Thr Pro His Ala
275 280 285tac cag gtg tac ggc gct ttc
tct agc cct cgc att ttt aag atc acg 912Tyr Gln Val Tyr Gly Ala Phe
Ser Ser Pro Arg Ile Phe Lys Ile Thr 290 295
300ttc ctc aca ggc ggt act gga act gca aat ctc cgt ttc
951Phe Leu Thr Gly Gly Thr Gly Thr Ala Asn Leu Arg Phe305
310 31526317PRTAvian orthoreovirus 26Met Asp Gly Leu
Thr Pro Ser Gln Arg Arg Glu Val Val Gly Leu Ile1 5
10 15Leu Ser Leu Thr Ser Ser Val Asn Thr Asn
Cys Gly Asp Leu Thr Pro 20 25
30Val Tyr Asp Arg Leu Leu Ser Leu Glu Ser Ala Val Ala Ser Leu Asn
35 40 45Asn Ser Val Asn Asp Leu Leu Gln
Lys Val Ser Asp Phe Glu Ser Asp 50 55
60Leu Gln Asp Ala Thr Ser Ser Leu Gly Gln Ala Asn Leu Met Leu Thr65
70 75 80Lys Leu Ser Glu Asp
Leu Arg Gln Leu Ser Ser Ser Val Asp Asn Leu 85
90 95Val Thr Ser Val Ser Asp Leu Ser Thr Thr Val
Ser Gly His Gln Asp 100 105
110Ala Ile Ala Ala Ile Gln Thr Ser Val His Ala Asn Thr Thr Asp Ile
115 120 125Ser Asn Leu Lys Ser Asn Val
Ser Thr Ile Gly Leu Asn Ile Thr Asp 130 135
140Leu Ala Gln Arg Val Ala Asn Ile Glu Ser Ser Ser Gly Ser Ser
Leu145 150 155 160Arg Phe
Val Ser Pro Leu Ser Leu Ser Gln Gly Val Val Ser Leu Ile
165 170 175Met Asp Pro Tyr Phe Cys Ser
Asp Asn Lys Ala Leu Thr Ser Tyr Ser 180 185
190Thr Asp Ala Gln Leu Met Gln Phe Gln Trp Leu Ala Arg Gly
Glu Asp 195 200 205Gly Ser Ser Ser
Ser Val Asp Met Leu Val Asn Ala His Cys His Gly 210
215 220Arg Arg Thr Asp Tyr Met Met Ser Thr Thr Glu Ser
Phe Thr Val Thr225 230 235
240Gly Asn Ser Val Ser Leu Val Phe Asn Leu Asp Tyr Ile Thr Lys Pro
245 250 255Pro Ser Asp Met Ser
Arg Leu Val Pro Arg Ala Gly Phe Gln Ala Ala 260
265 270Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Thr
Thr Pro His Ala 275 280 285Tyr Gln
Val Tyr Gly Ala Phe Ser Ser Pro Arg Ile Phe Lys Ile Thr 290
295 300Phe Leu Thr Gly Gly Thr Gly Thr Ala Asn Leu
Arg Phe305 310 31527966DNAAvian
orthoreovirusCDS(1)..(966) 27atg gaa ggt tta agt cag tta cag cga aga gaa
gtc gtg ggg ctg ata 48Met Glu Gly Leu Ser Gln Leu Gln Arg Arg Glu
Val Val Gly Leu Ile1 5 10
15ctc tcg ttg act tcg aac gtg act ata agt cct ggc gat tta acg gaa
96Leu Ser Leu Thr Ser Asn Val Thr Ile Ser Pro Gly Asp Leu Thr Glu
20 25 30ctg cgt gaa cgc gtc tca gcg
tta gaa tct gct act gcg tcg ttg aat 144Leu Arg Glu Arg Val Ser Ala
Leu Glu Ser Ala Thr Ala Ser Leu Asn 35 40
45gag act gta aag ggc gtg cta ggt cag ctg gtg gat ttg gcg cag
aag 192Glu Thr Val Lys Gly Val Leu Gly Gln Leu Val Asp Leu Ala Gln
Lys 50 55 60ttg agc aac gcg gca gat
gct gtg gtt gag ctg cga gga gat ttg aat 240Leu Ser Asn Ala Ala Asp
Ala Val Val Glu Leu Arg Gly Asp Leu Asn65 70
75 80tca tta acc gtt agc gtc caa act atc caa tcc
tct ttg gga tca ctt 288Ser Leu Thr Val Ser Val Gln Thr Ile Gln Ser
Ser Leu Gly Ser Leu 85 90
95aca gaa agc atg tcg gat ctt tcc ggc cag gtg act act aat gct tcg
336Thr Glu Ser Met Ser Asp Leu Ser Gly Gln Val Thr Thr Asn Ala Ser
100 105 110tcg atc acg aat ttg agg
agt gtg gtg gat ggt ctc aca gtc gac gtg 384Ser Ile Thr Asn Leu Arg
Ser Val Val Asp Gly Leu Thr Val Asp Val 115 120
125gct aat ctt aag cgc gac atg tca aac cag ggt ctt aaa gtc
act ggt 432Ala Asn Leu Lys Arg Asp Met Ser Asn Gln Gly Leu Lys Val
Thr Gly 130 135 140ctc gag cag cgt gta
gct gat ttg gag tct ggc gct gga tct ctt ccc 480Leu Glu Gln Arg Val
Ala Asp Leu Glu Ser Gly Ala Gly Ser Leu Pro145 150
155 160aca ttt gct gct ccc ctt aaa tta gat agc
ggg gtt gta tca ctc gac 528Thr Phe Ala Ala Pro Leu Lys Leu Asp Ser
Gly Val Val Ser Leu Asp 165 170
175ctg gat cct tac ttt tgt tct gtg gac cat aat ctt acg tcg tat tcc
576Leu Asp Pro Tyr Phe Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser
180 185 190gca agt gca tcg tta atg
aat ttt cag tgg ctc gtt cga ggt gag ggc 624Ala Ser Ala Ser Leu Met
Asn Phe Gln Trp Leu Val Arg Gly Glu Gly 195 200
205gga tcg tct gac tct ttc gat atg aat gtg aca gct cat agc
cat ggc 672Gly Ser Ser Asp Ser Phe Asp Met Asn Val Thr Ala His Ser
His Gly 210 215 220cag agg aca gac tat
atg atg tct act act caa tca ttg act gtt act 720Gln Arg Thr Asp Tyr
Met Met Ser Thr Thr Gln Ser Leu Thr Val Thr225 230
235 240gga aat tca gtc acc cta gtc ttt gat ctt
aac gcc ctt att tct cca 768Gly Asn Ser Val Thr Leu Val Phe Asp Leu
Asn Ala Leu Ile Ser Pro 245 250
255ccc tct gat tac tct cgc ttg ata cca tgt cat ggc ttc caa caa gcg
816Pro Ser Asp Tyr Ser Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala
260 265 270acg ttc cca gtg gac ctt
tcg ttt aag cga gat gac gtc ata cac tca 864Thr Phe Pro Val Asp Leu
Ser Phe Lys Arg Asp Asp Val Ile His Ser 275 280
285tat cag gtg tat ggt tcg tac aca act ccc cgc gtt ttc agg
ata aca 912Tyr Gln Val Tyr Gly Ser Tyr Thr Thr Pro Arg Val Phe Arg
Ile Thr 290 295 300ttc tct ccc ggc aat
cca gtg ccc gcg gtc ata cgc ttc ata acc ggc 960Phe Ser Pro Gly Asn
Pro Val Pro Ala Val Ile Arg Phe Ile Thr Gly305 310
315 320gac gag
966Asp Glu28322PRTAvian orthoreovirus 28Met
Glu Gly Leu Ser Gln Leu Gln Arg Arg Glu Val Val Gly Leu Ile1
5 10 15Leu Ser Leu Thr Ser Asn Val
Thr Ile Ser Pro Gly Asp Leu Thr Glu 20 25
30Leu Arg Glu Arg Val Ser Ala Leu Glu Ser Ala Thr Ala Ser
Leu Asn 35 40 45Glu Thr Val Lys
Gly Val Leu Gly Gln Leu Val Asp Leu Ala Gln Lys 50 55
60Leu Ser Asn Ala Ala Asp Ala Val Val Glu Leu Arg Gly
Asp Leu Asn65 70 75
80Ser Leu Thr Val Ser Val Gln Thr Ile Gln Ser Ser Leu Gly Ser Leu
85 90 95Thr Glu Ser Met Ser Asp
Leu Ser Gly Gln Val Thr Thr Asn Ala Ser 100
105 110Ser Ile Thr Asn Leu Arg Ser Val Val Asp Gly Leu
Thr Val Asp Val 115 120 125Ala Asn
Leu Lys Arg Asp Met Ser Asn Gln Gly Leu Lys Val Thr Gly 130
135 140Leu Glu Gln Arg Val Ala Asp Leu Glu Ser Gly
Ala Gly Ser Leu Pro145 150 155
160Thr Phe Ala Ala Pro Leu Lys Leu Asp Ser Gly Val Val Ser Leu Asp
165 170 175Leu Asp Pro Tyr
Phe Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser 180
185 190Ala Ser Ala Ser Leu Met Asn Phe Gln Trp Leu
Val Arg Gly Glu Gly 195 200 205Gly
Ser Ser Asp Ser Phe Asp Met Asn Val Thr Ala His Ser His Gly 210
215 220Gln Arg Thr Asp Tyr Met Met Ser Thr Thr
Gln Ser Leu Thr Val Thr225 230 235
240Gly Asn Ser Val Thr Leu Val Phe Asp Leu Asn Ala Leu Ile Ser
Pro 245 250 255Pro Ser Asp
Tyr Ser Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala 260
265 270Thr Phe Pro Val Asp Leu Ser Phe Lys Arg
Asp Asp Val Ile His Ser 275 280
285Tyr Gln Val Tyr Gly Ser Tyr Thr Thr Pro Arg Val Phe Arg Ile Thr 290
295 300Phe Ser Pro Gly Asn Pro Val Pro
Ala Val Ile Arg Phe Ile Thr Gly305 310
315 320Asp Glu29957DNAAvian orthoreovirusCDS(1)..(957)
29atg gaa ggc tta act cag tca cag cga aga gag gtc gtg ggg ctg ata
48Met Glu Gly Leu Thr Gln Ser Gln Arg Arg Glu Val Val Gly Leu Ile1
5 10 15ctc tca ttg act tcg aac
gtg act ata aat cct ggc gat ttg atg gaa 96Leu Ser Leu Thr Ser Asn
Val Thr Ile Asn Pro Gly Asp Leu Met Glu 20 25
30ctg cgt gag cgc gtc tca gcg tta gaa tcg gtc act gcg
tcg ttg aat 144Leu Arg Glu Arg Val Ser Ala Leu Glu Ser Val Thr Ala
Ser Leu Asn 35 40 45ggg act ata
aaa ggc gtg tta gac cag ctg gtg gat ttg gca cag aag 192Gly Thr Ile
Lys Gly Val Leu Asp Gln Leu Val Asp Leu Ala Gln Lys 50
55 60tta ggt aat gcg gcg ggt gct gta gtt gac cta cga
ggc gag ctg aac 240Leu Gly Asn Ala Ala Gly Ala Val Val Asp Leu Arg
Gly Glu Leu Asn65 70 75
80tca tta act gcc agc gtt caa act gtc caa tcc tct ttg gaa tca ctc
288Ser Leu Thr Ala Ser Val Gln Thr Val Gln Ser Ser Leu Glu Ser Leu
85 90 95acg gac agt atg tcg gat
ctt tct ggc caa gtg act act aac acc tcg 336Thr Asp Ser Met Ser Asp
Leu Ser Gly Gln Val Thr Thr Asn Thr Ser 100
105 110tcg ctc acg aat ctg gag agt acg gtg gct ggt ctt
aca gtc gat gta 384Ser Leu Thr Asn Leu Glu Ser Thr Val Ala Gly Leu
Thr Val Asp Val 115 120 125act aat
ctt aaa cgt gac gta tcg agt cag ggt ctt caa att acg agt 432Thr Asn
Leu Lys Arg Asp Val Ser Ser Gln Gly Leu Gln Ile Thr Ser 130
135 140ctc gag cag cgt gta act agt ttg gaa tct ggc
gct gga tct att ccc 480Leu Glu Gln Arg Val Thr Ser Leu Glu Ser Gly
Ala Gly Ser Ile Pro145 150 155
160acg ttt gct gct ccc ctt aaa tta gat agc ggg att gta tca ctc gac
528Thr Phe Ala Ala Pro Leu Lys Leu Asp Ser Gly Ile Val Ser Leu Asp
165 170 175ttg gat cct tac ttt
tgt tct gta gac cat aac ctc acg tcg tat tcc 576Leu Asp Pro Tyr Phe
Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser 180
185 190gca agc gct ctg cta atg aat ttt cag tgg ctt gtt
cga ggt gag gga 624Ala Ser Ala Leu Leu Met Asn Phe Gln Trp Leu Val
Arg Gly Glu Gly 195 200 205gga tcg
tct gat tca ttc gat atg aat gtg aca gct cat agc cat ggt 672Gly Ser
Ser Asp Ser Phe Asp Met Asn Val Thr Ala His Ser His Gly 210
215 220cag agg aca gac ttt atg atg tct act acc cag
tcg tta act gtt act 720Gln Arg Thr Asp Phe Met Met Ser Thr Thr Gln
Ser Leu Thr Val Thr225 230 235
240gga aat tct gtc act cta gtc ttt gat ctt aac gcg ctt att tct cca
768Gly Asn Ser Val Thr Leu Val Phe Asp Leu Asn Ala Leu Ile Ser Pro
245 250 255ccc cct gac tac tct
cgc ttg ata ccc tgc cat ggt ttt caa caa gcg 816Pro Pro Asp Tyr Ser
Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala 260
265 270acg ttc cca gtg gac ctt tcg ttc aag cga gac gac
gtc acg cac tca 864Thr Phe Pro Val Asp Leu Ser Phe Lys Arg Asp Asp
Val Thr His Ser 275 280 285tat cag
gtg tat ggt tcg tac acg act cct cgc att ttc aag ata acc 912Tyr Gln
Val Tyr Gly Ser Tyr Thr Thr Pro Arg Ile Phe Lys Ile Thr 290
295 300ttc tct cct ggc aat acc agt gcc tgc ggt cat
acg ttt ata agc 957Phe Ser Pro Gly Asn Thr Ser Ala Cys Gly His
Thr Phe Ile Ser305 310 31530319PRTAvian
orthoreovirus 30Met Glu Gly Leu Thr Gln Ser Gln Arg Arg Glu Val Val Gly
Leu Ile1 5 10 15Leu Ser
Leu Thr Ser Asn Val Thr Ile Asn Pro Gly Asp Leu Met Glu 20
25 30Leu Arg Glu Arg Val Ser Ala Leu Glu
Ser Val Thr Ala Ser Leu Asn 35 40
45Gly Thr Ile Lys Gly Val Leu Asp Gln Leu Val Asp Leu Ala Gln Lys 50
55 60Leu Gly Asn Ala Ala Gly Ala Val Val
Asp Leu Arg Gly Glu Leu Asn65 70 75
80Ser Leu Thr Ala Ser Val Gln Thr Val Gln Ser Ser Leu Glu
Ser Leu 85 90 95Thr Asp
Ser Met Ser Asp Leu Ser Gly Gln Val Thr Thr Asn Thr Ser 100
105 110Ser Leu Thr Asn Leu Glu Ser Thr Val
Ala Gly Leu Thr Val Asp Val 115 120
125Thr Asn Leu Lys Arg Asp Val Ser Ser Gln Gly Leu Gln Ile Thr Ser
130 135 140Leu Glu Gln Arg Val Thr Ser
Leu Glu Ser Gly Ala Gly Ser Ile Pro145 150
155 160Thr Phe Ala Ala Pro Leu Lys Leu Asp Ser Gly Ile
Val Ser Leu Asp 165 170
175Leu Asp Pro Tyr Phe Cys Ser Val Asp His Asn Leu Thr Ser Tyr Ser
180 185 190Ala Ser Ala Leu Leu Met
Asn Phe Gln Trp Leu Val Arg Gly Glu Gly 195 200
205Gly Ser Ser Asp Ser Phe Asp Met Asn Val Thr Ala His Ser
His Gly 210 215 220Gln Arg Thr Asp Phe
Met Met Ser Thr Thr Gln Ser Leu Thr Val Thr225 230
235 240Gly Asn Ser Val Thr Leu Val Phe Asp Leu
Asn Ala Leu Ile Ser Pro 245 250
255Pro Pro Asp Tyr Ser Arg Leu Ile Pro Cys His Gly Phe Gln Gln Ala
260 265 270Thr Phe Pro Val Asp
Leu Ser Phe Lys Arg Asp Asp Val Thr His Ser 275
280 285Tyr Gln Val Tyr Gly Ser Tyr Thr Thr Pro Arg Ile
Phe Lys Ile Thr 290 295 300Phe Ser Pro
Gly Asn Thr Ser Ala Cys Gly His Thr Phe Ile Ser305 310
3153120DNAArtificial SequenceSynthetic Primer 31agtatttgtg
agtacgattg
203217DNAArtificial SequenceSynthetic Primer 32ggcgccacac cttaggt
17
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