CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application Ser. No. 60/862,958, filed Oct. 25, 2006. 
     
    
     FIELD OF THE INVENTION 
       [0002]    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 
       [0003]    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, 4 th  ed., vol. 2. Knipe, D. M., and Howley, P. M., eds. Lippincott Williams &amp; Wilkins, Philadelphia, Pa., p. 3087, 2001). 
         [0004]    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, 11 th  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. 
         [0005]    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. 
         [0006]    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. 
         [0007]    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. 
         [0008]    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&#39;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&#39;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. 
         [0009]    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 
       [0010]    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. 
         [0011]    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. 
         [0012]    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. 
         [0013]    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 
         [0014]    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: 
           [0015]      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. 
           [0016]      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 
       [0017]    The following is a list of terms and their definitions used throughout the specification and the claims: 
         [0018]    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%). 
         [0019]    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. 
         [0020]    The term “Galliformes” refers to an order of birds containing turkeys, grouse, chickens, quails, and pheasants. About 256 species are found worldwide. 
         [0021]    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 
       [0022]    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. 
         [0023]    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. 
         [0024]    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 10 2  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. 
         [0025]    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 10 2  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. 
         [0026]    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. 
         [0027]    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. 
         [0028]    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. 
         [0029]    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. 
         [0030]    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. 
         [0031]    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). 
         [0032]    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. 
         [0033]    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. 
         [0034]    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. 
         [0035]    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. 
         [0036]    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. 
         [0037]    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. 
         [0038]    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. 
         [0039]    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. 
         [0040]    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. 
         [0041]    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. 
         [0042]    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. 
         [0043]    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. 
         [0044]    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. 
         [0045]    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. 
         [0046]    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. 
         [0047]    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. 
         [0048]    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). 
         [0049]    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. 
         [0050]    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. 
         [0051]    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. 
         [0052]    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. 
         [0053]    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). 
         [0054]    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. 
         [0055]    Typically, the live vaccine according to the invention can be administered in a dose of not less than 10 2  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 10 4 -10 10  TCID 50  per bird (wherein TCID is an abbreviation for Tissue Culture Infective Dose). 
         [0056]    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. 
         [0057]    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. 
         [0058]    The following, exemplary, vaccination regime can be applied to breeders, grandparents, and to great-grandparents:
       Day 1: Mild live tissue culture attenuated (subcutaneous).   Weeks 3-4: Attenuated live (oral in the drinking water).   Weeks 6-7: Attenuated live (oral in the drinking water).   Week 12: Killed vaccine (subcutaneous or intramuscular).   Week 18: Killed vaccine (subcutaneous or intramuscular).       
 
         [0064]    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. 
         [0065]    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). 
         [0066]    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. 
         [0067]    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)). 
         [0068]    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. 
         [0069]    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. 
         [0070]    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 Galfré, ( 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). 
         [0071]    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 
       [0072]    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 
       [0073]    All parts are by weight (e.g., % w/w), and temperatures are in degrees centigrade (° C.), unless otherwise indicated. 
       Example 1 
       [0074]    This Example describes the isolation of avian reovirus strain 40973 and related viruses. 
       Viruses 
       [0075]    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. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Table of Viruses 
               
             
          
           
               
                   
                 State of 
                 Identification 
                 Year of 
                 Tissue 
               
               
                 Species 
                 isolation 
                 # 
                 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 
               
               
                   
               
             
          
         
       
     
         [0076]    Table 2 identifies the nucleotide and amino acid or protein sequences used in the Examples or referred to in this application. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Table of Sequences 
               
             
          
           
               
                   
                 SEQ ID 
                 SEQ ID 
                   
               
               
                   
                 NO 
                 NO 
               
               
                 Name 
                 S1 gene 
                 Sigma 
                 Description 
               
               
                 (Isolate) 
                 (nt) 
                 C (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 
       [0077]    Total viral RNA was extracted from primary chicken embryo liver cell passages using the RNeasy kit (Qiagen, Valencia, Calif.) according to the manufacturer&#39;s recommendations. Briefly, 48 hours post infection, cell culture media was decanted and the monolayer was lysed with lysing buffer. 
       RT-PCR 
       [0078]    A cDNA corresponding to the S1 gene was produced by reverse transcription and PCR using SuperScript™ III RNase H −  reverse transcriptase and Platinum®Taq DNA polymerase (Invitrogen, Carlsbad, Calif.) using the following primers (Kant, A., et al.,  Vet. Res.  34:203-212, 2003): 
         [0000]    
       
         
               
               
             
               
               
             
               
               
             
               
               
             
           
               
                   
                 (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′). 
               
             
          
         
       
     
         [0079]    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 
       [0080]    The gel-extracted DNA from the amplification reaction was cloned into plasmid pCR 2.1 (Invitrogen) according to the manufacturer&#39;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 
       [0081]    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 
       [0082]    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 
       [0083]    This example describes the in vivo evaluation of reovirus isolate 40973, and shows that strain 40973 adversely affects the growth of chickens. 
       Virus Isolation 
       [0084]    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 
       [0085]    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 10 th  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% CO 2 . 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 10 5  tissue culture infective dose (TCID) 50 /ml. 
       Chickens 
       [0086]    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 10 3  TCID 50 /ml. The fourth treatment group was challenged via IT with 10 3  TCID 50 /ml. At two weeks of age, the body weight and length of each bird was recorded and they were humanely sacrificed. 
       Statistics 
       [0087]    Statistical analysis of the data by the Holm-Sidak method of one way analysis of variance (ANOVA) was performed using SigmaStat software. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Average Body Weights and Lengths From Each Treatment Group 
               
             
          
           
               
                   
                 Mean Body 
                   
                 Mean Body 
                   
               
               
                   
                 Weights 
                 Std. 
                 Lengths 
                 Std. 
               
               
                 Treatment group 
                 (grams) 
                 Dev. 
                 (cm) 
                 Dev 
               
               
                   
               
             
          
           
               
                 Negative control 
                 146.92 a   
                 10.1 
                 28.85 a   
                 1.07 
               
               
                 Sham IT 
               
               
                 Challenge 
               
               
                 Negative control 
                 151.45 a   
                 9.17 
                 28.78 a   
                 1.18 
               
               
                 Sham FP 
               
               
                 Challenge 
               
               
                 40973 
                 132.1 b   
                 11.5 
                 27.13 b   
                 0.68 
               
               
                 IT Challenge 
               
               
                 40973 
                 116.51 b   
                 9.12 
                 26.37 c   
                 0.96 
               
               
                 FP Challenge 
               
               
                   
               
               
                 Groups with different letters ( a ,  b , or  c ) were significantly different at p ≦ 0.001. 
               
             
          
         
       
     
       Example 3 
     Virus Isolation 
       [0088]    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 
       [0089]    Total viral RNA was extracted from primary chicken embryo liver cell passages using the RNeasy kit (Qiagen, Valencia, Calif.) according to the manufacturer&#39;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 
       [0090]    A cDNA corresponding to the S1 gene was produced by reverse transcription and PCR using SuperScript™ III RNase H −  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 
       [0091]    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 
       [0092]    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. 
         [0093]    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 
       [0094]    Sequences obtained for several chicken isolates described here have been submitted to GenBank and assigned the following accession numbers: DQ872796-DQ872801. 
       Monoclonal Antibody Profiles 
       [0095]    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). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Monoclonal antibody reactivity pattern 
               
             
          
           
               
                   
                 Monoclonal antibodies A   
               
             
          
           
               
                   
                 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. 
               
             
          
         
       
     
         [0096]    These results demonstrate that many of the recent southeast United States reovirus isolates have the same reactivity pattern as several European reovirus isolates (ERS). 
         [0097]    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.
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 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 SI 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%.