Attenuated avian reovirus strains 94826 C140 and 96139 C140

Abstract

Attenuated isolates of avian reoviruses associated with viral arthritis / tenosynovitis in poultry are provided, including the avian reovirus 94826 C140 strain and its progeny or derivatives, deposited with the ATCC under patent designation PTA-126077, and the avian reovirus 96139 C140 strain and its progeny or derivatives, deposited with the ATCC under patent designation PTA-126078. Compositions and methods for administering the isolates or compositions as vaccines to control reovirus-induced viral arthritis / tenosynovitis in birds of the order Galliformes are also provided.

Description

Technical Field

[0001] Continued Application Data This application claims priority to U.S. Provisional Patent Application No. 62 / 942,426, filed December 2, 2019, which is incorporated herein by reference.

Background Art

[0002] Trileo virus is associated with several poultry diseases, including malabsorption syndrome and runt and stunted syndrome (RSS), but its role as a primary pathogen in these clinical syndromes is not clear. In contrast, the association between trileo virus and clinical cases of viral arthritis / tendosynovitis is fairly clear, as reovirus has been isolated from affected birds. Control of reovirus-induced viral arthritis / tendosynovitis can be achieved by vaccinating broiler breeder hens with a combination of live and / or inactivated vaccines, and maternal immunity is passed on to progeny for initial protection from field challenge. In broilers, live attenuated vaccines are available for use on the day of hatching and for in ovo use. Current commercially available vaccine strains (e.g., S1133, 1733, 2408, and 2177), which were isolated in the 1960s and 1970s, do not provide protection against currently prevalent reovirus isolates obtained from confirmed cases of viral arthritis / tendosynovitis. Therefore, there is a need for the isolation and characterization of currently prevalent trileo viruses and the development of effective vaccines.

Summary of the Invention

[0003] The present invention includes an isolated trileo virus, wherein the isolated trileo virus is the reovirus 94826 C140 strain deposited with the ATCC under Patent Designation PTA-126077, or a progeny or derivative thereof, wherein the progeny or derivative has essentially the same biological and serological characteristics as the reovirus 94826 C140 strain deposited with the ATCC under Patent Designation PTA-126077.

[0004] The present invention includes an isolated reovirus, where the isolated reovirus is the reovirus 96139 C140 strain deposited with the ATCC under patent designation PTA-126078, or a progeny or derivative thereof, where the progeny or derivative thereof has biological and serological characteristics that are essentially the same as those of the reovirus 96139 C140 strain deposited with the ATCC under patent designation PTA-126078.

[0005] In some embodiments, the present invention includes a master seed virus preparation of a reovirus as described herein.

[0006] In some embodiments, the isolated reovirus is a lyophilized, freeze-dried, frozen, or effervescent tablet.

[0007] In some embodiments, the present invention includes a composition comprising an isolated reovirus as described herein. In some embodiments, the composition includes an adjuvant. In some embodiments, the composition includes a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. In some embodiments, the composition is formulated for spraying or aerolizing.

[0008] In some embodiments, the present invention includes a vaccine comprising an isolated reovirus as described herein or a composition as described herein. In some embodiments, the vaccine reduces the susceptibility of birds of the order Galliformes to reovirus-induced viral arthritis / tendosynovitis. In some embodiments, the reovirus-induced viral arthritis / tendosynovitis is variant group 1 / genotype 5-induced viral arthritis / tendosynovitis. In some embodiments, the reovirus-induced viral arthritis / tendosynovitis is variant group 2 / genotype 1-induced viral arthritis / tendosynovitis.

[0009] In some embodiments, the invention includes a vaccine for galliformes birds comprising a sufficient amount of turkey reovirus strain 94826 C140, deposited with the ATCC under Patent Designation PTA-126077, or a progeny or derivative thereof, to protect birds from reovirus-induced viral arthritis / tendon synovitis, and a pharmaceutically acceptable carrier. In some aspects, the reovirus-induced viral arthritis / tendon synovitis is variant group 1 / genotype 5-induced viral arthritis / tendon synovitis.

[0010] In some embodiments, the invention includes a vaccine for galliformes birds comprising a sufficient amount of turkey reovirus strain 96139 C140, deposited with the ATCC under Patent Designation PTA-126078, or a progeny or derivative thereof, to protect birds from reovirus-induced viral arthritis / tendon synovitis, and a pharmaceutically acceptable carrier. In some aspects, the reovirus-induced viral arthritis / tendon synovitis is variant group 2 / genotype 1-induced viral arthritis / tendon synovitis.

[0011] In some embodiments, the invention includes effervescent tablets comprising a turkey reovirus, composition, or vaccine as described herein.

[0012] The invention includes a method for reducing the susceptibility of galliformes birds to reovirus-induced viral arthritis / tendon synovitis, the method comprising administering to the bird a turkey reovirus, composition, or vaccine as described herein.

[0013] The invention includes a method for protecting galliformes birds from reovirus-induced viral arthritis / tendon synovitis, the method comprising administering to the bird a turkey reovirus, composition, or vaccine as described herein.

[0014] In some embodiments of the methods described herein, reovirus-induced viral arthritis / tendosynovitis is variant group 1 / genotype 5-induced viral arthritis / tendosynovitis. In some embodiments, reovirus-induced viral arthritis / tendosynovitis is variant group 2 / genotype 1-induced viral arthritis / tendosynovitis. In some embodiments of the methods described herein, administration is intranasal, intraocular, oral, mucosal, intramuscular, or subcutaneous. In some embodiments of the methods described herein, administration includes in ovo administration. In some embodiments of the methods described herein, the avian reovirus, composition, or vaccine is administered by aerosol. In some embodiments of the methods described herein, the avian reovirus, composition, or vaccine is administered in drinking water. In some embodiments, the bird is a chicken or a turkey. In some embodiments of the methods described herein, administration is to breeder hens or roosters.

[0015] The present invention includes anti-reovirus antibody production in poultry and the method includes administering to a bird an isolated avian reovirus, composition, or vaccine as described herein. In some embodiments, the bird is a chicken or a turkey.

[0016] The present invention includes a diagnostic kit comprising an isolated avian reovirus as described herein.

[0017] The present invention includes a method of detecting exposure to avian reovirus in a bird and the method includes determining that an antiserum sample obtained from the bird specifically binds to an avian reovirus as described herein. In some embodiments, the bird is a chicken or a turkey.

[0018] The present invention includes a hyperimmune serum against an avian reovirus as described herein.

[0019] The present invention includes antibodies that bind to a trivalent virus as described herein and do not bind to trivalent virus strains S1133, 1733, 2408, and / or 2177. In some embodiments, the antibody is a monoclonal antibody.

[0020] The above summary of the invention is not intended to describe every disclosed embodiment or every implementation of the invention. The following description describes more specifically exemplary embodiments. Throughout the application, references are provided through a list of examples, which can be used in various combinations. In each case, the listed list serves only as a typical group and should not be construed as an exclusive list.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides two attenuated trivalent viruses, trivalent virus strain 94826 C140 and trivalent virus strain 96139 C140, and their progeny and derivatives. These attenuated reovirus strains are safe and effective when administered as a live formulation to prevent reovirus infection and reduce the incidence and severity of reovirus-induced viral arthritis and / or tenosynovitis.

[0022] In one aspect, the present invention includes the reovirus 94826 C140 strain and its progeny and derivatives. The reovirus 94826 C140 strain is an attenuated strain of the reovirus 94826 strain obtained by passaging the reovirus 94826 strain 140 times in chicken embryos. The reovirus 94816 strain is a reovirus of group 1 / genotype 5 associated with viral arthritis and tenosynovitis in poultry. The reovirus 94816 strain is described in detail by International Application No. PCT / US2015 / 013449 (International Publication No. WO 2015 / 116778 Pamphlet) and U.S. Patent Application No. 15 / 223,623, the entire contents of which are incorporated herein by reference. The attenuated reovirus 94826 C140 strain is also a reovirus of group 1 / genotype 5. The attenuated reovirus 94826 C140 strain was deposited on August 13, 2019, under Patent Deposit Designation PTA-126077, with the American Type Culture Collection (ATCC®), University Boulevard 10801, Manassas, Virginia 20110-2209, USA. This deposit was made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The attenuated reovirus 94826 C140 is also archived as PDRC ref: 121116 in the Virus Repository of the Poultry Diagnostic and Research Center (PDRC) of the University of Georgia, located at 953 College Station Road, Athens, Georgia 20602. The safety and efficacy of the reovirus 94826 C140 strain as a live, attenuated reovirus vaccine are described in more detail in Example 3.

[0023] In another aspect, the present invention includes the turkey rhinotracheitis virus 96139 C140 strain and its progeny and derivatives. The turkey rhinotracheitis virus 96139 C140 strain is an attenuated strain of the turkey rhinotracheitis virus 96139 strain obtained by serial passage of the turkey rhinotracheitis virus 96139 strain 140 times in chicken embryos. The turkey rhinotracheitis virus 96139 strain is a group 2 / genotype 1 reovirus associated with viral arthritis and tenosynovitis in poultry. The turkey rhinotracheitis virus 96139 strain is described in detail by International Application No. PCT / US2015 / 013449 (International Publication No. WO 2015 / 116778 Pamphlet) and U.S. Patent Application No. 15 / 223,623, the entire contents of which are incorporated herein by reference. The turkey rhinotracheitis virus 96139 strain was deposited under Patent Deposit Designation PTA-125688 on March 7, 2019, with the American Type Culture Collection (ATCC®). The attenuated turkey rhinotracheitis virus 96139 C140 is also a group 2 / genotype 1 turkey rhinotracheitis virus. The attenuated turkey rhinotracheitis virus 96139 C140 was deposited on August 13, 2019, under Patent Deposit Designation PTA-126078, with the American Type Culture Collection (ATCC®), University Boulevard 10801U, Manassas, Virginia 20110-2209, USA. This deposit was made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The attenuated turkey rhinotracheitis virus 9619 C140 is also archived as PDRC ref: 119812 in the Virus Repository of the Poultry Diagnostic and Research Center (PDRC), University of Georgia, located at 953 College Station Road, Athens, Georgia 20602. The safety and efficacy of the turkey rhinotracheitis virus 96139 C140 strain as a live, attenuated reovirus vaccine are described in more detail in Example 3.

[0024] As more fully discussed in International Application No. PCT / US2015 / 013449 (published as International Publication No. WO 2015 / 116778) and U.S. Patent Application No. 15 / 223,623, strains 94826 and 96139 of avian reovirus correspond to two genetically and serologically distinct groups of avian reoviruses isolated from clinical cases of tenosynovitis. Genetic analysis of their avian reovirus sigma C proteins reveals novel genotypes unrelated to current avian reovirus vaccine strains.

[0025] Tritheoreovirus, together with mammalian orthoreovirus, constitutes the genus Orthoreovirus within the family Reoviridae. These viruses contain 10 double-stranded RNA genomic segments encapsulated within a non-enveloped icosahedral double capsid approximately 80 nm in diameter. The genomic segments can be divided into three large (L1, L2, L3), three medium (M1, M2, M3), and four small (S1, S2, S3, S4) fragments, which encode proteins λ1, λ2, λ3, μ1, μ2, μNS, σ3, σ1, σ2, σNS, respectively, based on their electrophoretic mobility. The σ2 protein is an outer capsid protein with type-specific neutralizing epitopes. It also binds double-stranded RNA and has been identified as a zinc metalloprotein. Sigma C protein, a minor outer capsid protein encoded by the σ1 fragment, is a target for determining the molecular characteristics of tritheoreovirus and is involved in cell attachment and induction of type-specific neutralizing antibodies. The tritheoreovirus virion contains a capsid, a core, and a nucleoprotein complex. The virus capsid is not covered by an envelope. The capsid / nucleocapsid is isometric with icosahedral symmetry and has a diameter of approximately 80 - 82 nm. The capsid shell of the virion is composed of two layers. All shells are usually present, or the outer shell is often lost during preparation. The capsid appears round. The surface structure of the capsid shows a regular pattern with prominent features. The capsomere arrangement is clearly visible. Surface projections are absent. The inner capsid core has a diameter of approximately 60 nm. Virus preparations contain single particle components. The core is spherical and consists of a double-stranded RNA genome approximately 49 nm in diameter. The fiber ends protrude almost to the capsid surface.

[0026] The present invention includes a master seed virus preparation prepared from the reovirus 94826 C140 strain (deposited with the ATCC® under Patent Deposit Designation PTA-126077 on August 13, 2019), or the reovirus 96139 C140 strain (deposited with the ATCC® under Patent Deposit Designation PTA-126078 on August 13, 2019). The master seed virus preparation of the present invention may be prepared and tested in accordance with Title 9 of the Code of Federal Regulations (CFR) §113.332 (tendon synovitis vaccine).

[0027] Isolated progeny and isolated derivatives having equivalent or similar biological, serological, and / or genetic characteristics to the reovirus 94826 C140 strain (deposited with the ATCC® under Patent Deposit Designation PTA-126077 on August 13, 2019) and the reovirus 96139 C140 strain (deposited with the ATCC® under Patent Deposit Designation PTA-126078 on August 13, 2019) are also encompassed by the present invention. As used herein, serological, biological, and genetic characteristics may include one or more of the characteristics described in the data of the examples included herein. More specifically, progeny or derived strains of PTA-126077 or PTA-1260789495 may retain particularly preferred protective properties belonging to the present invention, as described in more detail by the examples included herein.

[0028] The reovirus according to the present invention can be propagated by conventional methods including, but not limited to, those described in any of the example sections included herein. Briefly, the substrate capable of supporting the replication of the reovirus is inoculated with the reovirus of the present invention and grown until the virus replicates to the desired infectious titer or antigen mass content. Next, the material containing the reovirus is collected. Suitable substrates include embryonated chicken eggs; primary (avian) cell cultures such as, for example, chicken embryo liver cells, chicken embryo fibroblasts, or chicken kidney cells; mammalian cell lines such as, for example, the VERO cell line or the BGM-70 cell line; or avian cell lines such as, for example, QT-35, QM-7, or LMH.

[0029] The present invention includes compositions and vaccines comprising one or more of the isolated viruses described herein. The viral preparations of the present invention may be prepared and tested in accordance with Title 9 of the Code of Federal Regulations (CFR) § 113.332 (tendon synovitis vaccine).

[0030] In some embodiments, the virus is live. In some embodiments, the virus is inactivated or killed. In some embodiments, the virus or its composition or vaccine may be lyophilized. In some embodiments, the virus or its composition or vaccine may be frozen. In some embodiments, the virus or its composition or vaccine may be freeze-dried.

[0031] In some embodiments, the virus or its composition or vaccine may be formulated as an effervescent tablet. Such effervescent tablets can be packaged, for example, within lightweight aluminum blisters. The tablet is dissolved in water and may be administered, for example, orally, nasally, or by aerosol spray, whereby the droplets penetrate through the mucosa of the bird.

[0032] Such compositions and vaccines may be administered as active ingredients to immunize birds, thereby inducing an immune response against type 1 and / or type 2 avian reoviruses and / or inducing immunity against such avian reoviruses. The immunity may include induction of a higher level of protection in the bird population after vaccination as compared to an unvaccinated group. The immune response may or may not confer protective immunity. The immune response may include, for example, one or more of a cell-mediated immune response involving lymphocyte production in response to antigen exposure and / or a humoral immune response involving plasma lymphocyte (B cell) production in response to antigen exposure followed by antibody production. Immunization may result in a decrease, inhibition, or prevention of one or more of the symptoms of avian reovirus-associated viral arthritis (VA) / tendon synovitis. Such symptoms include, but are not limited to, weight suppression, decreased egg production, mortality, macroscopic lesions (such as tendon swelling, tendon synovitis, tendon rupture, and pericardial edema), and histological changes (such as lymphocytic tendon synovitis, lymphocytic epicarditis, and lymphocytic myocarditis).

[0033] The compositions or vaccines of the present invention may also include one or more compounds having adjuvant activity. Suitable compounds or compositions for this purpose include aluminum hydroxide, aluminum phosphate, aluminum oxide, vegetable oils, animal oils, oil-in-water or water-in-oil emulsions based on mineral oils such as, for example, Bayol F™ or Marcol 52®, complete Freund's adjuvant, incomplete Freund's adjuvant, or vegetable oils such as vitamin E acetate, and saponins.

[0034] The composition or vaccine of the present invention may contain one or more suitable pharmaceutically acceptable carriers or diluents. The immunogenic composition or vaccine of the present invention may also contain one or more stabilizers. Carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, or glucose; proteins such as albumin or casein; and any suitable stabilizers including buffers such as alkaline metal phosphates can be used. Stabilizers are particularly advantageous when a dry vaccine formulation is prepared by lyophilization.

[0035] The composition or vaccine of the present invention may further contain one or more immunogens derived from other pathogens that are infectious to poultry. Such immunogens may be derived from, for example, Marek's disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), egg drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV), poxvirus, reovirus, avian parvovirus, and avian nephritis virus (including but not limited to ANV-1 and ANV-2).

[0036] The composition or vaccine of the present invention may be administered by any suitable known method of poultry vaccination, including but not limited to intranasal, ophthalmic, injection, in drinking water, in feed, exposure, in ovo, maternally, respiratory inhalation, etc. The immunogenic composition or vaccine may be administered by mass administration techniques such as putting the vaccine in drinking water or spraying or vaporizing it. When administered by injection, the immunogenic composition or vaccine may be administered parenterally. Examples of parenteral administration include administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.

[0037] In some embodiments, the live vaccine may be administered at a dose of 10 2 antibody titer units or more per bird (the antibody titer unit is defined in 9 CFR 113.332), and the vaccine may be 10 4 ~10 10 TCID 50It may contain antigen equivalents (TCID is an abbreviation for tissue culture infectious dose).

[0038] In some embodiments, the live vaccine may be administered at a dose of about 10 1.5 ~ about 10 7 of TCID or EID (embryo infectious dose (does)).

[0039] In some aspects, the vaccine may contain about 10 1.5 of TCID 50 , about 10 2 of TCID 50 , about 10 3 of TCID 50 , about 10 4 of TCID 50 , about 10 5 of TCID 50 , about 10 6 of TCID 50 , about 10 7 of TCID 50 , about 10 8 of TCID 50 , about 10 9 of TCID 50 , about 10 10 of TCID 50 , or may contain antigen equivalents in any range thereof.

[0040] The compositions and vaccines of the present invention may be substantially pure. As used herein, "substantially pure" means a material that essentially does not contain any similar macromolecules or other biological entities commonly found in nature.

[0041] The compositions and vaccines of the present invention may be administered to any bird susceptible to infectious diseases caused by avian reoviruses, including but not limited to poultry, birds of the order Galliformes, and exotic birds. Examples of birds of the order Galliformes include, but are not limited to, chickens, turkeys, pheasants, quails, and partridges. As used herein, poultry includes domesticated birds raised for the purpose of collecting their eggs or slaughtering them for meat and / or feathers. These are most typically members of the superorder Galloanserae (birds), particularly the order Galliformes (including, for example, chickens, turkeys, pheasants, and quails), and the family Anatidae (order Anseriformes), commonly known as "waterfowl" (including, for example, ducks, geese, and swans). Poultry may also include other birds slaughtered for their meat, such as pigeons or doves, or birds considered game animals, such as partridges. Examples of chickens include, but are not limited to, hens, roosters, broilers, roasters, laying hens, breeding hens, descendants of breeding hens, and pullets. As used herein, the term "susceptible" means the likelihood or actuality of a detrimental reaction to the microorganism in question, and / or one or more pathological conditions that are indicators of avian reovirus infection, compared to individuals or populations that are less susceptible.

[0042] The vaccine of the present invention may be administered to poultry before or after hatching. Poultry may be vaccinated at various ages. For example, broilers may be vaccinated in ovo, at 1 day of age, or at 2 - 3 weeks of age. Laying or breeding lines may be vaccinated, for example, at about 6 - 12 weeks of age and boostered at about 16 - 20 weeks of age. Such laying or breeding lines may be vaccinated at about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks of age. Also, in some embodiments, such laying or breeding lines may be vaccinated within about 2 weeks after birth. Such laying or breeding lines may be boostered at about 16, about 17, about 18, about 19, or about 20 weeks of age. The progeny of such laying or breeding lines may exhibit antibody titers against the polypeptides as described herein, which may prevent or reduce the symptoms of avian reovirus infection in the progeny. Vaccination in ovo may be performed, for example, on about day 13, about day 14, about day 15, about day 16, about day 17, about day 18, about day 19, about day 20, or any range thereof.

[0043] Chickens may be vaccinated at any suitable age, usually about 1 - 3 days of age before the first vaccination. Chickens may be vaccinated only once. Alternatively, if two vaccine administrations are used, the first dose is administered, for example, when the chicken is between 3 days and 1 week of age, followed by an additional dose 1 - 10 weeks later.

[0044] Multiple administrations of the composition may be administered throughout the life of the chicken. Since maternal immunity is the primary source of protection for broiler progeny, breeder chickens are typically vaccinated, but if desired, broiler chickens may also be vaccinated.

[0045] The present invention includes a method of generating an anti-mutant group 1 and / or mutant group 2 avian reovirus immune response in poultry, the method comprising administering an isolated virus, composition or vaccine as described herein. In some embodiments, the immunity includes humoral and / or cellular immunity. In some embodiments, the immunity includes mucosal immunity.

[0046] The present invention includes a method of preventing avian reovirus infection in poultry, the method comprising administering an isolated virus, composition or vaccine as described herein.

[0047] In some embodiments of the methods of the present invention, the administration includes injection, spraying, oral administration, or respiratory administration. In some embodiments of the methods of the present invention, the administration induces mucosal immunity. In some embodiments of the methods of the present invention, the administration includes in ovo administration. In some embodiments, the in ovo administration includes administration on about day 13, about day 14, about day 15, about day 16, about day 17, about day 18, about day 19, about day 20, or any range thereof.

[0048] The present invention includes a method of detecting exposure to mutant group 1 and / or mutant group 2 avian reovirus in birds, the method comprising determining whether an antiserum sample obtained from a bird specifically binds to the virus of the present invention. For example, the present invention includes the use of one or more avian reoviruses of the present invention in a method of detecting exposure to avian reovirus in birds, the method comprising determining that an antiserum sample obtained from a bird specifically binds to the avian reovirus of the present invention.

[0049] The present invention also includes a diagnostic kit comprising one or more of the triviruses of the present invention. The kit may include one or more containers filled with the triviruses of the present invention. In some embodiments, the virus may be, for example, lyophilized, freeze-dried, frozen, or formulated as a foaming tablet. The kit may include additional separate containers of other strains of triviruses or other pathogens of poultry. Such a kit may include additional components such as, for example, a positive control virus, a negative control virus, a secondary antibody, and / or a detectable marker. Further, the kit may include other reagents such as a buffer solution and also includes solutions necessary to carry out the present invention. Optionally, such containers may be accompanied by relevant notifications or printed instructions. The kit of the present invention may include a "packaging material". As used herein, the term "packaging material" refers to one or more physical structures used to contain the contents of the kit. The packaging material is constructed by well-known methods and preferably provides a sterile and contaminant-free environment. The packaging material may be a solid matrix or a material such as glass, plastic, paper, foil, etc.

[0050] The present invention provides a method for detecting and / or measuring the amount of type 1 or type 2 trivirus in a sample obtained from a bird. Such a method may include contacting the sample with an antibody that selectively binds to a trivirus as described herein to measure the amount of antibody binding to the virus or protein in the sample. The sample may be any biological material such as tissue, bone, blood, urine, or feces. The method of this aspect of the present invention is useful, for example, in determining whether a poultry is infected with the triviruses of the present invention. Such identified infected animals may then be isolated or culled to prevent the spread of reovirus to other animals.

[0051] As used herein, "isolated" refers to a substance that has been removed from its original environment (e.g., its natural environment if it occurs naturally) and thus has been modified "by the hand of man" from its natural state.

[0052] The term "and / or" means one or all of the recited elements, or any combination of two or more of the recited elements.

[0053] The terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain advantages in certain circumstances. However, in the same or other circumstances, other embodiments may also be suitable. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and it is not intended to exclude other embodiments from the scope of the invention.

[0054] The term "comprising" and variations thereof do not have a limiting meaning when these terms appear in the description and claims.

[0055] Unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably and mean one or more than one.

[0056] Also herein, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0057] In any method disclosed herein that includes discrete steps, the steps may be performed in any executable order. And, if desired, any combination of two or more steps may be performed simultaneously.

[0058] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0059] The numerical ranges and parameters set forth in this invention are approximations, but the numerical values set forth in the specific examples are reported as precisely as possible. However, all numerical values inherently contain a range necessarily resulting from the standard deviation found in their respective test measurements.

[0060] Throughout the application, indices are provided in several places through the list of examples, and the examples can be used in various combinations. In each case, the recited list serves only as a typical group and should not be construed as an exclusive list. Specific examples, substances, amounts, and procedures are to be understood as being construed broadly in accordance with the scope and spirit of the invention described herein.

[0061] All headings are for the convenience of the reader and should not be used to limit the meaning of the text following the heading unless so specified.

[0062] The present invention is defined in the claims. However, a non-exhaustive list of non-limiting embodiments is provided below. Any one or more of the features of these embodiments may be combined with any one or more of the features of any other example, embodiment, or aspect described herein.

[0063] 1. An isolated orthoreovirus, wherein the isolated orthoreovirus is the orthoreovirus strain 94826 C140 deposited with the ATCC under Patent Designation PTA - 126077, or a progeny or derivative thereof, wherein the progeny or derivative has biological and serological characteristics essentially the same as those of the orthoreovirus strain 94826 C140 deposited with the ATCC under Patent Designation PTA - 126077.

[0064] 2. An isolated orthoreovirus, wherein the isolated orthoreovirus is the orthoreovirus strain 96139 C140 deposited with the ATCC under Patent Designation PTA - 126078, or a progeny or derivative thereof, wherein the progeny or derivative has biological and serological characteristics essentially the same as those of the orthoreovirus strain 96139 C140 deposited with the ATCC under Patent Designation PTA - 126078.

[0065] 3. The isolated orthoreovirus according to embodiment 1 or 2, wherein the isolated orthoreovirus comprises a master seed virus.

[0066] 4. The isolated orthoreovirus according to any one of embodiments 1 to 3, wherein the isolated orthoreovirus is in the form of lyophilization, freeze - drying, freezing, or effervescent tablets.

[0067] 5. A composition comprising the isolated orthoreovirus according to any one of embodiments 1 to 4.

[0068] 6. The composition according to embodiment 5, comprising an adjuvant.

[0069] 7. The composition according to embodiment 5 or 6, comprising a pharmaceutically acceptable carrier.

[0070] 8. The composition according to any one of embodiments 5 to 7, formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or intra - egg administration.

[0071] 9. The composition according to any one of embodiments 5-8, formulated for spraying or aerolizing.

[0072] 10. A vaccine comprising the isolated trivalent virus according to any one of embodiments 1-4, or the composition according to any one of embodiments 5-9.

[0073] 11. The vaccine according to embodiment 10, wherein the vaccine reduces the susceptibility of birds of the order Galliformes to reovirus-induced viral arthritis / tendosynovitis.

[0074] 12. A vaccine for birds of the order Galliformes comprising an amount sufficient to protect birds from reovirus-induced viral arthritis / tendosynovitis of the trivalent virus strain 94826 C140 deposited with the ATCC under Patent Designation PTA-126077, or a progeny or derivative thereof, and a pharmaceutically acceptable carrier.

[0075] 13. The vaccine according to embodiment 11 or 12, wherein the reovirus-induced viral arthritis / tendosynovitis is variant group 1 / genotype 5-induced viral arthritis / tendosynovitis.

[0076] 14. A vaccine for birds of the order Galliformes comprising an amount sufficient to protect birds from reovirus-induced viral arthritis / tendosynovitis of the trivalent virus strain 96139 C140 deposited with the ATCC under Patent Designation PTA-126078, or a progeny or derivative thereof, and a pharmaceutically acceptable carrier.

[0077] 15. The vaccine according to embodiment 13 or 14, wherein the reovirus-induced viral arthritis / tendosynovitis is variant group 2 / genotype 1-induced viral arthritis / tendosynovitis.

[0078] 16. An effervescent tablet comprising the trivalent virus, composition, or vaccine according to any one of embodiments 1-15.

[0079] 17. A method for reducing the susceptibility of Galliformes birds to reovirus-induced viral arthritis / tendon synovitis, comprising administering to the bird a trivalent reovirus, composition, or vaccine according to any one of Embodiments 1 to 16.

[0080] 18. A method for protecting Galliformes birds from reovirus-induced viral arthritis / tendon synovitis, comprising administering to the bird a trivalent reovirus, composition, or vaccine according to any one of Embodiments 1 to 16.

[0081] 19. The method according to Embodiment 17 or 18, wherein the reovirus-induced viral arthritis / tendon synovitis is mutant group 1 / genotype 5-induced viral arthritis / tendon synovitis.

[0082] 20. The method according to Embodiment 17 or 18, wherein the reovirus-induced viral arthritis / tendon synovitis is mutant group 2 / genotype 1-induced viral arthritis / tendon synovitis.

[0083] 21. The method according to any one of Embodiments 17 to 20, wherein the administration is intranasal, intraocular, oral, mucosal, intramuscular, or subcutaneous.

[0084] 22. The method according to any one of Embodiments 17 to 20, wherein the administration includes in ovo administration.

[0085] 23. The method according to any one of Embodiments 17 to 20, wherein the trivalent reovirus, composition, or vaccine is administered by aerosol.

[0086] 24. The method according to any one of Embodiments 17 to 20, wherein the trivalent reovirus, composition, or vaccine is administered by drinking water.

[0087] 25. The method according to any one of Embodiments 17 to 24, wherein the administration includes administration to breeder hens or roosters.

[0088] 26. A method for producing anti-reovirus antibodies in poultry, comprising administering to a bird an isolated reovirus, composition, or vaccine according to any one of Embodiments 1 to 16.

[0089] 27. The method according to any one of Embodiments 17 to 26, wherein the bird is a chicken or a turkey.

[0090] 28. A diagnostic kit comprising the isolated reovirus according to any one of Embodiments 1 to 4.

[0091] 29. A method for detecting exposure to reovirus in a bird, comprising determining that an antiserum sample obtained from the bird specifically binds to the reovirus according to any one of Embodiments 1 to 3.

[0092] 30. A hyperimmune serum against the reovirus according to any one of Embodiments 1 to 3.

[0093] 31. An antibody that binds to the reovirus according to any one of Embodiments 1 to 3 and does not bind to the S1133, 1733, 2408, and / or 2177 strains of reovirus.

[0094] 32. The antibody according to Embodiment 31, wherein the antibody is a monoclonal antibody.

[0095] The present invention is illustrated by the following examples. It is understood that specific examples, substances, amounts, and procedures are to be construed broadly in accordance with the scope and spirit of the invention described herein.

Examples

[0096] Example 1 94826 C140-mutant group 1 / genotype 5 reovirus The attenuated turkey rhinotracheitis virus 94826 C140 was obtained by passaging the 94826 turkey rhinotracheitis virus 140 times in chicken embryos. The attenuated turkey rhinotracheitis virus 94826 C140 was deposited with the ATCC® under Patent Deposit Designation PTA-126077 on August 13, 2019. The attenuated turkey rhinotracheitis virus 94826 C140 is also archived as PDRC ref:121116 in the virus repository of the Poultry Diagnostic and Research Center (PDRC) of the University of Georgia, located at 953 College Station Road, Athens, GA 20602. The safety and efficacy of the turkey rhinotracheitis 94826 C140 as a live, attenuated rhinotracheitis virus vaccine are described in more detail in Example 3.

[0097] The parental 94826 turkey rhinotracheitis virus is a Group 1 / Genotype 5 turkey rhinotracheitis virus and is described in more detail in International Application No. PCT / US2015 / 013449 (published as WO 2015 / 116778) and U.S. Patent Application No. 15 / 223,623, both of which are incorporated herein by reference.

[0098] Example 2 96139 C140 - Mutant Group 2 / Genotype 1 turkey rhinotracheitis virus The attenuated turkey rhinotracheitis virus 96139 C140 was obtained by passaging the 96139 turkey rhinotracheitis virus 140 times in chicken embryos. The attenuated turkey rhinotracheitis virus 96139 C140 was deposited with the ATCC® under Patent Deposit Designation PTA-126078 on August 13, 2019. The attenuated turkey rhinotracheitis virus 9619 C140 is also archived as PDRC ref:119812 in the virus repository of the Poultry Diagnostic and Research Center (PDRC) of the University of Georgia, located at 953 College Station Road, Athens, GA 20602. The safety and efficacy of the turkey rhinotracheitis 96139 C140 as a live, attenuated rhinotracheitis virus vaccine are described in more detail in Example 3.

[0099] The parental 96139 triple reassortant virus is a group 2 / genotype 1 triple reassortant virus, both of which are described in detail in International Application No. PCT / US2015 / 013449 (International Publication No. WO 2015 / 116778 Pamphlet) and U.S. Patent Application No. 15 / 223,623, which are incorporated herein by reference, and was deposited with the ATCC® under Patent Deposit Designation PTA-125688 on March 7, 2019.

[0100] Example 3 Live attenuated reovirus vaccine candidates 94826 C140 and 96139 C140 Isolate The 94826 C140 triple reassortant virus, which is a mutant group 1 / genotype 5 triple reassortant virus, is as described in Example 1.

[0101] The 96139 C140 triple reassortant virus, which is a mutant group 2 / genotype 1 triple reassortant virus, is as described in Example 2. 96139 C140

[0102] Embryo passage Both virus isolates were serially passaged in specific pathogen free (SPF) embryos via the chorioallantoic membrane (CAM) inoculation route. Specifically, the CAM from the original isolate was diluted 1:100 in sterile minimum essential medium (MEM), and 0.1 ml was inoculated into the CAM of 9 - 11 day old SPF embryos. At 48 hours post-inoculation, the embryos were refrigerated at 4°C for 4 hours, the CAM was collected from the embryos and pooled, homogenized in virus transport medium (VTM) using an Omni Bead Mill, clarified at 1500 × g, and stored at -80 until the next embryo passage day.

[0103] C140 strain titer The titers of both the 94826 C140 strain and the 96139 C140 strain are shown in Table 1 below.

[0104] [Table 1]

[0105] Identity test Identity tests were performed every 10 - 20 embryo passages. Briefly, aliquots of CAM passages were subjected to reovirus sigma C RT - PCR for sequencing, and nucleotide and amino acid sequences were compared to the original and previous passages to confirm identity.

[0106] Purity test Purity tests were performed on the C140 strain according to the guidelines specified in Title 9 of the Code of Federal Regulations (9 CFR) Part 113.332 (9 CFR §113.332), and by culture and RT - PCR / PCR methods.

[0107] Cultures for bacteria, fungi, and mold showed that both the 94826 C140 strain and the 96139 C140 strain were negative for bacteria, fungi, and mold by culture.

[0108] PCR showed that both the 94826 C140 strain and the 96139 C140 strain were negative for Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS).

[0109] PCR / RT - PCR for adventitious viruses showed that both the 94826 C140 strain and the 96139 C140 strain were negative for avian adventitious viruses.

[0110] The results of the lymphoid leukosis contamination test of the stock virus according to the guidelines specified in 9 CFR §113.332(b)(2)(3) are shown in Table 2 below, indicating that both the 94826 C140 strain and the 96139 C140 strain were free of leukosis contamination.

[0111]

Table 2

[0112] Safety test The CAM passage 140 (C140) of the isolates from both parties was subjected to safety tests in accordance with the guidelines outlined in Title 9 of the Code of Federal Regulations (9 CFR) Part 113.332.

[0113] Experiment Summary ● Avian source ● Parent birds (broiler breed) from Aviagen ● Breeder hens vaccinated with commercially available inactivated S1133 ● Broiler chicks negative for reovirus 94826 antibody and 96139 antibody ● Safety tests in accordance with Title 9 of the Code of Federal Regulations ● Evaluation parameters in accordance with Title 9 of the Code of Federal Regulations ● EID50 / 0.2 ml of 94826 C140 (VG1 / GT5)10 4 [29 chicks] ● EID50 / 0.2 ml of 94826 C140 (VG1 / GT5)10 5 [30 chicks] ● EID50 / 0.2 ml of 96139 C140 (VG2 / GT1)10 4 [30 chicks] ● EID50 / 0.2 ml of 96139 C140 (VG2 / GT1)10 5 [30 chicks] ● Monitored daily for 21 days ● Evaluation of lameness, swelling of tendon / hock ● Mortality

[0114] The results of 10 evaluations in 1-day-old commercial broilers negative for antibodies against 94826 and 96139 are shown in Table 3 below. The results indicated that no clinical symptoms and gross lesions were observed during the 21-day evaluation period in broilers subcutaneously administered with the EID50 of 10 4 or the EID50 amount of 10 5 of 94826 or 96139. The vaccine is considered safe for use in 1-day-old broilers at these doses.

[0115]

Table 3

[0116] The results of 10 evaluations in 28-day-old commercial broilers negative for antibodies against 94826 and 96139 are shown in Table 4 below. The results show that in broilers subcutaneously administered with the EID50 of 10 of 94826 or 96139 or the EID50 amount of 10, no clinical symptoms and gross lesions were observed during the 21-day evaluation period after inoculation. The vaccine is considered safe even when used in 28-day-old broilers at these doses. 4 of EID50 or 10 5 of the EID50 amount.

[0117]

Table 4

[0118] Efficacy test In accordance with 9 CFR 113.332(4)(c)(1, 2, 3), the C140 strain was evaluated for efficacy.

[0119] Efficacy test in 1-day-old broilers negative for reovirus antibodies against 94826 and 96139 ●Vaccination / Subcutaneous injection ●94826 C140 (VG1 / GT5) 10 2.5 of EID50 / 0.2 ml [31 chicks] ●94826 C140 (VG1 / GT5) 10 3 of EID50 / 0.2 ml [31 chicks] ●96139 C140 (VG2 / GT1) 10 2.5 of EID50 / 0.2 ml [30 chicks] ●96139 C140 (VG2 / GT1) 10 3 of EID50 / 0.2 ml [30 chicks] ●Unvaccinated control ●For the unvaccinated / challenged control, 20 chicks placed in an isolation unit at 1 day of age ●21 days of age (vaccinated / subcutaneous and unvaccinated) ●104 All groups challenged via plantar injection of 94826 Li7 or 96139 Li5 (parent virus) at an EID50 / 0.05 ml ● Observed daily for 14 days ● Mortality ● Depression ● Lameness / dragging gait ● Swollen plantar and / or tendon

[0120] The results of the efficacy test in 1-day-old broilers are shown in Table 5 below. The results showed that 1-day-old chicks vaccinated at an EID50 of 10 2.5 or 10 3 were protected after challenge with the homologous parent virus.

[0121]

Table 5

[0122] The results from unvaccinated / parent virus-challenged broilers are shown in Table 6 below. The results showed that the efficacy test was effective based on the clinical signs observed in unvaccinated / challenged broilers.

[0123]

Table 6

[0124] Results of the reverse titration of the C140 strain for the 1-day-old efficacy study - aliquots of the diluted C140 strain titrated in embryos (10 2.5 and 10 3 EID50 / dose) (5 replicate tests) are shown in Table 7 below.

[0125]

Table 7

[0126] Summary of the efficacy test in 1-day-old broilers 10 2.5 and 10 3 Vaccination at day 1 with 94826 C140 or 96139 C140 of the dosage of

[0127] Unvaccinated / challenged controls were within the acceptable range outlined in 9 CFR 113.332(4)(c)(3) for both parental viruses.

[0128] The tests in 1-day-old broilers were conducted in accordance with the guidelines of 9 CFR.

[0129] The complete disclosures of all patents, patent applications, and literature, and electronically available information (e.g., nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions such as in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq, etc.) cited herein are incorporated by reference. If any conflict exists between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are provided only to clarify understanding. No unnecessary limitations are to be construed therefrom. The invention is not limited to the exact details shown and described, and the variations obvious to one of ordinary skill in the art are included within the invention as defined by the claims.

Claims

**Claim 1** An isolated turkey reovirus, wherein the isolated turkey reovirus is the turkey reovirus strain 94826 C140 deposited with the ATCC under Patent Designation PTA-126077. **Claim 2** An isolated turkey reovirus, wherein the isolated turkey reovirus is the turkey reovirus strain 96139 C140 deposited with the ATCC under Patent Designation PTA-126078. **Claim 3** The isolated turkey reovirus according to claim 1 or 2, wherein the isolated turkey reovirus comprises a master seed virus. **Claim 4** The isolated turkey reovirus according to any one of claims 1 to 3, wherein the isolated turkey reovirus is lyophilized, freeze-dried, or frozen, or is a foaming tablet. **Claim 5** A composition comprising the isolated turkey reovirus according to any one of claims 1 to 4. **Claim 6** The composition according to claim 5, further comprising an adjuvant and / or a pharmaceutically acceptable carrier. **Claim 7** The composition according to claim 5 or 6, formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration, or for spraying or aerosolization. **Claim 8** A vaccine comprising the isolated turkey reovirus according to any one of claims 1 to 4, or the composition according to any one of claims 5 to 7. **Claim 9** The vaccine according to claim 8, wherein the vaccine is for reducing susceptibility to reovirus-induced viral arthritis / tendon synovitis in turkeys of the order Galliformes. **Claim 10** A vaccine for turkeys of the order Galliformes, comprising an amount of the turkey reovirus strain 94826 C140 deposited with the ATCC under Patent Designation PTA-126077 sufficient to protect turkeys from reovirus-induced viral arthritis / tendon synovitis, and a pharmaceutically acceptable carrier. **Claim 11** The vaccine according to claim 10, wherein the reovirus-induced viral arthritis / tendon synovitis is variant group 1 / genotype 5-induced viral arthritis / tendon synovitis. **Claim 12** A vaccine for Galliformes birds comprising an amount sufficient to protect birds from reovirus-induced viral arthritis / tendosynovitis, the turkey reovirus turkey reovirus 96139 C140 strain deposited with the ATCC under patent designation PTA-126078, and a pharmaceutically acceptable carrier.

13. The vaccine according to claim 12, wherein the reovirus-induced viral arthritis / tendosynovitis is variant group 2 / genotype 1-induced viral arthritis / tendosynovitis.

14. A effervescent tablet comprising a turkey reovirus according to any one of claims 1 to 4, a composition according to any one of claims 5 to 7, or a vaccine according to any one of claims 8 to 13.

15. A method for reducing the susceptibility of Galliformes birds to reovirus-induced viral arthritis / tendosynovitis, comprising administering to the birds a turkey reovirus according to any one of claims 1 to 4, a composition according to any one of claims 5 to 7, a vaccine according to any one of claims 8 to 13, or an effervescent tablet according to claim 14.

16. A method for protecting Galliformes birds from reovirus-induced viral arthritis / tendosynovitis, comprising administering to the birds a turkey reovirus according to any one of claims 1 to 4, a composition according to any one of claims 5 to 7, a vaccine according to any one of claims 8 to 13, or an effervescent tablet according to claim 14.

17. The method according to claim 15 or 16, wherein the administration comprises intranasal, intraocular, oral, mucosal, intramuscular, or subcutaneous administration; in ovo administration; aerosol administration; or administration in drinking water.

18. The method according to any one of claims 15 to 17, wherein the administration comprises administration to breeder hens or roosters.

19. A method for generating anti-reovirus antibodies in poultry, comprising the step of administering to the poultry an isolated turkey reovirus according to any one of claims 1 to 4, a composition according to any one of claims 5 to 7, a vaccine according to any one of claims 8 to 13, or an effervescent tablet according to claim 14.

20. The method according to any one of claims 15 to 19, wherein the bird is a chicken or a guinea fowl.

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