Avian Reovirus Vaccine

Abstract

The present invention relates to novel strains of avian reovirus, which have been isolated from clinical cases of viral arthritis / tenosynovitis in chickens in the southeastern United States. The present invention relates to these novel group 1 and group 2 avian reoviruses, diagnostic assays using nucleotide-specific or amino acid-specific components of such viruses, such as the S1 gene encoding the σC protein, and vaccines for protecting chickens from diseases caused by such viruses.

Description

[0001] Continue to apply for data

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 61 / 932,995, filed January 29, 2014, which is incorporated herein by reference.

[0003] background

[0004] Avian reovirus is associated with several diseases in poultry, including malabsorption syndrome and growth failure and nutrient malabsorption syndrome (RSS), although the role of these avian reoviruses as primary pathogens in these clinical syndromes is unclear. In contrast, the association of avian reovirus with clinical cases of viral arthritis / tenosynovitis is quite clear, as reovirus has been isolated from the tendons of affected birds. Control of reovirus-induced viral arthritis / tenosynovitis can be achieved by inoculating broiler breeders with live and / or inactivated vaccines in combination with maternal immunity that is passed to the offspring for early protection against field stimulation. In broiler chickens, live attenuated vaccines are available for use on the day of hatching and in eggs. Currently commercial vaccine strains (S1133, 1733, 2408, and 2177, to name a few) have been used for decades to control diseases associated with reovirus. However, these commercial vaccine strains were isolated in the 1960s and 1970s and do not provide protection against currently circulating reovirus strains that cause established cases of viral arthritis / tenosynovitis. Therefore, there is a need to isolate and characterize currently circulating avian reoviruses and to develop effective vaccines. SUMMARY OF THE INVENTION

[0006] The present invention includes genetically and serologically distinct group 1 and group 2 avian reoviruses isolated from clinical cases of viral arthritis (VA) / tenosynovitis in chickens. The present invention includes isolated group 1 avian reoviruses, wherein the group 1 avian reovirus comprises a σC protein having an amino acid sequence having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 2 and / or SEQ ID NO: 4. In some aspects, the group 1 avian reovirus comprises a σC protein having an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 2. In some aspects, the group 1 avian reovirus comprises a σC protein having an amino acid sequence with at least about 99% sequence identity to SEQ ID NO: 4. In some aspects, the group 1 avian reovirus comprises a σC protein having an amino acid sequence of SEQ ID NO: 2. In some aspects, the group 1 avian reovirus comprises a σC protein having an amino acid sequence of SEQ ID NO: 4. In some aspects, the group 1 avian reovirus is attenuated, inactivated, or killed. In some aspects, the group 1 avian reovirus is attenuated by at least 25 passages in eggs or a host cell line.

[0007] The present invention includes isolated group 1 avian reoviruses, wherein the group 1 avian reovirus comprises an S1 gene having a nucleotide sequence having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 1 and / or SEQ ID NO: 3. In some aspects, the group 1 avian reovirus comprises an S1 gene having a nucleotide sequence having at least about 99% sequence identity to SEQ ID NO: 1. In some aspects, the group 1 avian reovirus comprises an S1 gene having a nucleotide sequence having at least about 99% sequence identity to SEQ ID NO: 3. In some aspects, the group 1 avian reovirus comprises an S1 gene having a nucleotide sequence having at least about 99% sequence identity to SEQ ID NO: 3. In some aspects, the group 1 avian reovirus comprises an S1 gene having the nucleotide sequence of SEQ ID NO: 3. In some aspects, the group 1 avian reovirus comprises a σC protein having an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 4. In some aspects, the group 1 avian reovirus is attenuated, inactivated, or killed. In some aspects, the group 1 avian reovirus is attenuated by at least 25 passages in eggs or a host cell line.

[0008] The present invention includes isolated group 2 avian reoviruses, wherein the group 2 avian reovirus comprises a σC protein having an amino acid sequence with at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 6. In some aspects, the group 2 avian reovirus comprises a σC protein having an amino acid sequence with at least about 99% sequence identity to SEQ ID NO: 6. In some aspects, the group 2 avian reovirus comprises a σC protein having an amino acid sequence with at least about 99% sequence identity to SEQ ID NO: 6. In some aspects, the group 2 avian reovirus is attenuated, inactivated, or killed. In some aspects, the group 2 avian reovirus is attenuated by at least 25 passages in eggs or a host cell line.

[0009] The present invention includes isolated group 1 avian reoviruses, wherein the group 1 avian reovirus comprises an S1 gene comprising a nucleotide sequence having at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 5. In some aspects, the group 2 avian reovirus comprises an S1 gene having a nucleotide sequence having at least about 99% sequence identity to SEQ ID NO: 5. In some aspects, the group 2 avian reovirus comprises an S1 gene having a nucleotide sequence having the nucleotide sequence SEQ ID NO: 5. In some aspects, the group 2 avian reovirus is attenuated, inactivated, or killed. In some aspects, the group 2 avian reovirus is attenuated by at least 25 passages in eggs or a host cell line.

[0010] In some aspects, the present invention includes group 1 avian reoviruses, wherein group 1 avian reoviruses include avian reovirus strain 94594 and its derivatives and progeny. In some aspects, avian reovirus strain 94595 is killed, inactivated, or attenuated by at least 25 passages in eggs or host cell lines.

[0011] In some aspects, the present invention includes group 1 avian reoviruses, wherein group 1 avian reoviruses include avian reovirus strain 94826 and its derivatives and progeny. In some aspects, strain 948264 is killed, inactivated, or attenuated by at least 25 passages in eggs or host cell lines.

[0012] In some aspects, the present invention includes group 2 avian reoviruses, wherein the avian reovirus includes avian reovirus strain 96139 and its derivatives and progeny. In some aspects, avian reovirus strain 96139 is killed, inactivated, or attenuated by at least 25 passages in eggs or host cell lines.

[0013] The present invention includes compositions comprising one or more of the avian reoviruses described herein. Such compositions may further comprise an adjuvant and / or an antigenic determinant derived from one or more additional pathogens that infect poultry.

[0014] The present invention includes vaccines comprising one or more of the avian reoviruses of the present invention as described herein. Such vaccines may further comprise an adjuvant and / or antigenic determinants from one or more additional pathogens that infect poultry.

[0015] The present invention includes immunological compositions for raising antibodies in poultry, the immunological compositions comprising one or more of the avian reoviruses described above and herein. Such compositions may further comprise an adjuvant and / or antigenic determinants derived from one or more additional pathogens that infect poultry.

[0016] The present invention includes diagnostic kits comprising one or more of the avian reoviruses described above and herein.

[0017] The invention includes a method of producing anti-reovirus antibodies in poultry, the method comprising administering to the bird an isolated avian reovirus, composition, or vaccine of the invention.

[0018] The invention includes a method of protecting a bird of the order Galliformes against pathology or disease induced by an avian reovirus, the method comprising administering to the bird an isolated avian reovirus, composition, or vaccine of the invention.

[0019] The invention includes a method of reducing the susceptibility of a bird of the order Galliformes to a pathology or disease induced by an avian reovirus, the method comprising administering to the bird an isolated avian reovirus, composition, or vaccine of the invention.

[0020] The invention includes a method of reducing viral arthritis and / or tenosynovitis in a bird of the order Galliformes, the method comprising administering to the bird an isolated avian reovirus, composition, or vaccine of the invention.

[0021] The invention includes a method of preventing viral arthritis and / or tenosynovitis in birds of the order Galliformes, the method comprising administering to the bird an isolated avian reovirus, composition, or vaccine of the invention.

[0022] In some aspects of the methods of the invention, the bird comprises a chicken or a turkey.

[0023] In some aspects of the methods of the invention, administering comprises pre-hatch or post-hatch administration.

[0024] In some aspects of the methods of the invention, administering comprises administering in ovo. In some aspects, administering in ovo comprises administering at about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or any range thereof.

[0025] In some aspects of the methods of the invention, administering comprises administering to breeder hens.

[0026] The invention includes antibodies that bind to a Group 1 or Group 2 avian reovirus as described herein and do not bind to avian reovirus strains S1133, 1733, 2408, and / or 2177. In some aspects, the antibody is a monoclonal antibody.

[0027] The invention includes a method for detecting exposure to an avian reovirus in a bird, the method comprising determining specific binding of an antiserum sample obtained from the bird to an avian reovirus of the invention or a component thereof.

[0028] The invention includes a method for detecting exposure to an avian reovirus in a bird, the method comprising measuring specific binding of an antiserum sample obtained from the bird to the σC protein of a Group 1 avian reovirus or a Group 2 avian reovirus of the invention.

[0029] The present invention includes a method for detecting an avian reovirus infectious agent in a sample, the method comprising detecting hybridization of a polynucleotide comprising SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or a fragment thereof to the sample.

[0030] The present invention includes a method for detecting avian reovirus in a sample, the method comprising generating a polymerase chain reaction (PCR) amplification product using at least one primer derived from SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 . S1 nucleotide sequence encoding σC protein (base pairs 1-931) from cluster 1 2012VA variant wild-type isolate 94594 (SEQ ID NO: 1).

[0033] Figure 2 . S1 amino acid sequence encoding σC (amino acids 1-310) from cluster 1 2012VA variant wild-type isolate 94594 (SEQ ID NO: 2).

[0034] Figure 3 . S1 nucleotide sequence encoding σC protein (base pairs 1-931) from cluster 1 2012VA variant wild-type isolate 94826 (SEQ ID NO: 3).

[0035] Figure 4 . S1 amino acid sequence encoding σC (amino acids 1-310) from cluster 1 2012VA variant wild-type isolate 94826 (SEQ ID NO: 4).

[0036] Figure 5 . S1 nucleotide sequence encoding σC protein (base pairs 1-931) from cluster 2 2012VA variant wild-type isolate 96139 (SEQ ID NO: 5).

[0037] Figure 6 . S1 amino acid sequence encoding σC protein (amino acids 1-310) from cluster 2 2012VA variant wild-type isolate 96139 (SEQ ID NO: 6).

[0038] Figure 7 Multiple alignments of σC amino acid sequences were performed using Clustal W, and a phylogenetic tree with 1000 bootstrap replicates was generated. The bootstrap confidence level is indicated by a number at each tree node.

[0039] Figure 8 Percent identity and dispersion based on multiple alignments generated using the Clustal W algorithm.

[0040] Figure 9 Mean footpad measurements in millimeters at the days indicated on the X-axis. Significant differences in bird size were observed in the S1133 and 94286 challenged groups compared to the negative control, starting at seven days of age. Standard deviations are indicated by bars for each group.

[0041] Figure 10 Mean body weight at the day of termination (14 days of age). Standard deviation is represented by a bar for each group.

[0042] Figure 11 Measurements were taken above the digital flexor tendons (just below the hock joint) at 10 days of age and again on the day of termination, 14 days of age. Significant swelling was observed in the 94286 challenge group at 14 days of age. Standard deviations are indicated by bars for each group.

[0043] Figure 12 Clinical signs recorded on the day of termination. Six birds were noted to be removed from the S1133 flock at 10 days due to lameness and lack of access to feed or water.

[0044] Figure 13 Mean body weight in grams at the end of the study (23 days of age). Standard deviations are indicated by end bars.

[0045] Figure 14 Average footpad measurements in millimeters were taken at 12, 14, 16, 19, 21, and 23 days of age. The first measurement was taken at 12 days of age, just before challenge.

[0046] Figure 15Mean hock measurements in millimeters taken at 19, 21, and 23 days of age. Hock 1 measurements were taken at the hock joint and hock 2 measurements were taken just below the hock joint, above the digital flexor tendon.

[0047] Figure 16 Clinical signs and lesions on the day of termination (23 days).

[0048] Figure 17 .Serology on the day of incubation.

[0049] Figure 18 . Body weight in birds stimulated by cluster 2.

[0050] Figure 19 For birds challenged with cluster 2, footpad swelling as a percentage of body weight was measured on day 14.

[0051] Figure 20 For birds challenged with Cluster 2, measurement of tendon swelling as a percentage of body weight.

[0052] Figure 21 Genbank accession numbers for cluster 1 and cluster 2 isolates. Entries shaded in gray indicate cluster 2 avian reoviruses. All other entries indicate cluster 1 reoviruses.

[0053] Detailed Description of Illustrative Embodiments

[0054] With the present invention, two genetically and serologically distinct groups of avian reovirus have been isolated from clinical cases of viral arthritis (VA) / tenosynovitis from across the United States and Canada. Genetic analysis of the avian reovirus σC protein revealed novel group 1 and group 2 genotypes that are unrelated to current avian reovirus vaccine strains. Serological evaluation of these viruses revealed little or no cross neutralization with known antisera to current commercial reovirus vaccines. In addition, pathogenicity and progeny protection studies confirmed that current commercial vaccines do not provide adequate protection to progeny from vaccinated breeder hens. Current reovirus vaccine strains were isolated in the 1960s and 1970s and do not provide protection against currently prevalent reovirus strains from established cases of viral arthritis / tenosynovitis.

[0055] Avian reoviruses, along with mammalian reoviruses, include the genus Orthoreovirus in the family Reoviridae. These viruses contain 10 dsRNA genomic segments enclosed within a non-enveloped, icosahedral double capsid of approximately 80 nm. The genomic segments can be separated based on electrophoretic mobility into three large (L1, L2, L3), three medium (M1, M2, M3), and four small (S1, S2, S3, S4) segments, each encoding proteins λ1, λ2, λ3, μ1, μ2, μNS, σ3, σ1, σ2, σNS. The σ2 protein is an outer capsid protein that carries group-specific neutralizing epitopes. It also binds double-stranded RNA and has been identified as a zinc metalloprotein. The σC protein, the smaller outer capsid protein encoded by the σ1 segment, is the target of molecular characterization of avian reoviruses and is responsible for cell attachment as well as the induction of type-specific neutralizing antibodies.

[0056] The avian reovirus particle comprises a capsid, a core and a nucleoprotein complex. The viral capsid is non-enveloped. The capsid / nucleocapsid is icosahedrally symmetrical and isometric and has a diameter of about 80-82 nm. The capsid of the virion is composed of two layers. Usually all shells are present, or the outer shell often disappears during preparation. The capsid is round. The capsid surface structure reveals a regular pattern with distinct characteristics. The capsid particle arrangement is clearly visible. Surface protrusions are absent. The inner capsid core has a diameter of about 60 nm. The viral preparation contains a particle component. The core, which has a diameter of about 49 nm, is spherical and consists of the dsRNA genome. The ends of these fibers almost protrude through the capsid surface.

[0057] In the avian reovirus vaccine strain S1133, the s1 mRNA transcript is 1644 nucleotides long and contains three out-of-phase and partially overlapping cistrons. The first cistron (ORF-1, nucleotides 25–319) expresses p10, the second cistron (ORF-2, nucleotides 293–731) expresses p17, and the third cistron (ORF-3, nucleotides 630–1608) expresses cell attachment protein C.

[0058] Analysis of the σC amino acid sequences of reovirus vaccine strains S1133, 1733, 2408, and 2177 revealed >99% similarity among these vaccine strains. Genotypic characterization of wild-type reovirus isolates included comparison of wild-type isolates with vaccine strains, along with all available reovirus sequences in the public domain and PDRC databases. Genotypic similarity to wild-type isolates was reported for either the wild-type isolate or the vaccine strain, with the highest percentage of sequence similarity reported.

[0059] With the present invention, two genetically and serologically distinct groups of avian reoviruses have been isolated from clinical cases of tenosynovitis. Genetic analysis of the avian reovirus σC protein revealed novel genotypes that are unrelated to current avian reovirus vaccine strains. Serological evaluation of these viruses revealed little or no cross-neutralization with known antisera against currently commercial reovirus vaccines. In addition, pathogenicity and progeny protection studies have been performed to confirm that currently commercial vaccines do not provide adequate protection to progeny from vaccinated breeder hens.

[0060] The two genetic / serological groups of the wild-type isolates described herein are referred to as group 1 2012VA variants (also referred to herein as "group 1 avian reoviruses") and group 2 2012VA variants (also referred to herein as "group 2 avian reoviruses"). Reoviruses from the group 1 2012VA variants and / or group 2 2012VA variants can be used in live attenuated vaccines and killed / inactivated vaccines. In some aspects, such vaccines can be autovaccines.

[0061] The group 1 avian reoviruses of the present invention can have a Cσ protein having an amino acid sequence having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 2. In some aspects, the group 1 avian reoviruses of the present invention can have a Cσ protein having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the group 1 avian reoviruses as described herein have a Cσ protein having at least one substitution modification relative to SEQ ID NO: 2.

[0062] The group 1 avian reoviruses of the present invention can have a Cσ protein having an amino acid sequence having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 4. In some aspects, the group 1 avian reoviruses of the present invention can have a Cσ protein having the amino acid sequence of SEQ ID NO: 4. In some embodiments, the group 1 avian reoviruses as described herein have a Cσ protein having at least one substitution modification relative to SEQ ID NO: 4.

[0063] The group 1 avian reoviruses of the present invention can have a Cσ protein encoded by a nucleotide sequence having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 1. In some aspects, the group 1 avian reoviruses of the present invention can have a Cσ protein encoded by a nucleotide sequence having SEQ ID NO: 1. In some embodiments, the group 1 avian reoviruses as described herein have a Cσ protein encoded by a nucleotide sequence having at least one substitution modification relative to SEQ ID NO: 1.

[0064] The group 1 avian reoviruses of the present invention can have a Cσ protein encoded by a nucleotide sequence having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 3. In some aspects, the group 1 avian reoviruses of the present invention can have a Cσ protein encoded by a nucleotide sequence having SEQ ID NO: 3. In some embodiments, the group 1 avian reoviruses as described herein have a Cσ protein encoded by a nucleotide sequence having at least one substitution modification relative to SEQ ID NO: 3.

[0065] In some embodiments, the Group 1 avian reovirus is one of those shown in Table 21, or a progeny or derivative thereof.

[0066] In some embodiments, the group 1 avian reovirus is avian reovirus CK / 945494 / tendon / GA / 2012 (also referred to herein as "group 1 2012VA variant wild-type isolate 94594," or "94594"), or a progeny or derivative thereof.

[0067] In some embodiments, the group 1 avian reovirus is avian reovirus CK / 94826 / tendon / GA / 2012 (also referred to herein as "group 1 2012VA variant wild-type isolate 94826," or "94826"), or a progeny or derivative thereof.

[0068] The group 2 avian reoviruses of the present invention can have a Cσ protein having an amino acid sequence having at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 6. In some aspects, the group 2 avian reoviruses of the present invention can have a Cσ protein having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the group 2 avian reoviruses as described above have a Cσ protein having at least one substitution modification relative to SEQ ID NO: 6.

[0069] The group 2 avian reoviruses of the present invention can have a Cσ protein encoded by a nucleotide sequence having at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 5. In some aspects, the group 1 avian reoviruses of the present invention can have a Cσ protein encoded by a nucleotide sequence having SEQ ID NO: 5. In some embodiments, the group 1 avian reoviruses as described herein have a Cσ protein encoded by a nucleotide sequence having at least one substitution modification relative to SEQ ID NO: 5.

[0070] In some embodiments, the Group 2 avian reovirus is one of those shown in Table 21, or a progeny or derivative thereof.

[0071] In some embodiments, the group 2 avian reovirus is avian reovirus CK / 96139 / tendon / GA / 2012 (also referred to herein as "group 2 2012VA variant wild-type isolate 96139," or "96139"), or a progeny or derivative thereof.

[0072] Avian reoviruses as described herein, or cell lines infected with such viruses, may be deposited with the American Type Culture Collection. The deposit was made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0073] According to the avian reovirus of the present invention, can be prepared by conventional methods, these methods include but are not limited to any method in those methods described in the examples part included in this article.In short, the substrate that can support the replication of avian reovirus is inoculated with the avian reovirus of the present invention and bred until the virus is replicated to the infection titer or antigenic capacity of hope.Then the reovirus comprising raw material is harvested.Suitable substrate can include primary (avian) cell culture (such as, chicken embryo hepatocytes, chicken embryo fibroblasts or chicken kidney cells), mammalian cell line (such as, VERO cell line or BGM-70 cell line), or avian cell line (such as, QT-35, QM-7 or LMH).In some applications, embryonated eggs can be used.

[0074] The invention includes methods for detecting exposure to Group 1 and / or Group 2 avian reoviruses in a bird, the method comprising determining specific binding of an antiserum sample, whether or not obtained from the bird, to a virus, cell line, cell pellet, supernatant, and / or polypeptide of the invention. For example, the invention includes the use of one or more avian reoviruses of the invention in a method for detecting exposure to an avian reovirus in a bird, the method comprising determining binding of an antiserum sample obtained from the bird to an avian reovirus of the invention. Such avian reoviruses include, but are not limited to, any of those described herein. Figure 21 Any of the listed avian reovirus strains, including but not limited to avian reovirus strains 94594, 94826, or 96139. Such avian reoviruses can be inactivated, killed, and / or lyophilized. The present invention also includes diagnostic kits comprising one or more of the avian reoviruses of the present invention. Such kits can include additional components, such as, for example, a positive control virus, a negative control virus, a secondary antibody, and / or a detectable label.

[0075] The present invention includes isolated σC polypeptides having an amino acid sequence having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, and / or SEQ ID NO: 6. In some aspects, the present invention includes isolated σC polypeptides having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, and / or SEQ ID NO: 6. In some aspects, the invention includes an isolated σC polypeptide having an amino acid sequence having at least one substitution modification relative to SEQ ID NO:2, SEQ ID NO:4, and / or SEQ ID NO:6.

[0076] The present invention includes isolated σC polypeptides having an amino acid sequence encoded by a nucleotide sequence having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, and / or SEQ ID NO: 5. In some aspects, the present invention includes isolated σC polypeptides having an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, and / or SEQ ID NO: 5. In some aspects, the present invention includes an isolated σC polypeptide having an amino acid sequence encoded by a nucleotide sequence having at least one substitution modification relative to SEQ ID NO: 1, SEQ ID NO: 3, and / or SEQ ID NO: 5.

[0077] The present invention includes polypeptides as described herein, truncations, and fragments thereof. Truncations include, but are not limited to, amino acid sequences in which one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids are removed from the amino terminus of the amino acid sequence and / or one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids are removed from the carboxyl terminus of the amino acid sequence.

[0078] Fragments include, but are not limited to, for example, fragments having about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, and about 700 consecutive amino acid residues of a sequence described herein. Fragments also include, for example, fragments of a range of sizes having any combination of the above fragment sizes. Fragments include, but are not limited to, e.g., fragments having at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, and at least 700 contiguous amino acid residues of a sequence described herein.

[0079] The present invention includes polypeptides having amino acid sequences having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more amino acid changes from the amino acid sequences described herein, or fragments thereof. Such amino acid changes include, but are not limited to, consecutive amino acid changes.

[0080] The present invention includes the use of one or more polypeptides of the present invention in a method for detecting exposure to avian reovirus in a bird, the method comprising determining specific binding of an antiserum sample obtained from the bird to a polypeptide of the present invention. Such polypeptides include any of those described herein, including but not limited to SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, and derivatives and fragments thereof. The present invention also includes diagnostic kits comprising one or more of the polypeptides of the present invention. Such kits may include additional components such as, for example, a positive control polypeptide, a negative control polypeptide, a secondary antibody, and a detection label.

[0081] The present invention includes isolated polynucleotide sequences having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, truncations, or fragments thereof. In some aspects, the present invention includes isolated polynucleotide sequences as described herein having at least one substitution modification relative to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

[0082] The present invention includes isolated polynucleotide sequences having at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the nucleotide sequence encoding SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some aspects, the present invention includes isolated polynucleotide sequences having SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some aspects, the present invention includes isolated polynucleotide sequences as described herein that encode an amino acid sequence having at least one substitution modification relative to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[0083] The present invention includes polynucleotide sequences that hybridize under various conditions with nucleotide sequences described herein and fragments thereof. Stringent conditions include, but are not limited to, moderate stringency and high stringency. High stringency hybridization conditions can be, for example, 6X SSC, 5X Denhardt, 0.5% sodium lauryl sulfate (SDS), and 100ug / ml fragmented and denatured salmon sperm DNA at 65°C overnight hybridization, and washing at least once at room temperature for about 10 minutes in 2X SSC, 0.1% SDS, then washing at least once at 65°C for about 15 minutes, then washing at least once at room temperature for at least 3 minutes to 5 minutes in 0.2X SSC, 0.1% SDS.

[0084] The present invention includes polynucleotide sequences described herein having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, or more nucleotide substitutions. The present invention also includes polynucleotide sequences described herein in which codon usage has been adapted to optimize expression in a given host cell. For example, codon usage can be adapted to optimize expression in host cells including, but not limited to, baculovirus, yeast, E. coli, poultry, or human cells. Such adaptation can be performed by techniques known in the art.

[0085] The present invention includes primers, includes but not limited to any one in primers described herein, and can be used to generate sequence described herein or its fragmentary primer in PCR reaction.In certain embodiments, primers can include at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35 or at least 40 nucleotide residues.In certain embodiments, primers can include and be no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55 or no more than 60 nucleotide residues.This type of nucleotide residue can be a continuous sequence or its complement.Also include primer pairs, these primers are to including at least one, its complement or the primer derived from this type of sequence in the primer described herein.The present invention also includes the amplified production produced by this type of primer.

[0086] The present invention includes vectors comprising a polynucleotide sequence of the present invention. In some aspects, the vector is a vaccine vector. The present invention includes isolated polypeptides encoded by the polynucleotides of the present invention.

[0087] The present invention includes the use of one or more polynucleotide sequences of the present invention in methods for detecting an avian reovirus infectious agent in a sample. Such methods include, for example, methods comprising detecting hybridization of a polynucleotide sequence of the present invention to a sample, and methods comprising generating a polymerase chain reaction (PCR) amplification product using at least one primer derived from a polynucleotide sequence of the present invention. Such polynucleotide sequences include, for example, any of those described herein, including but not limited to SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5. The present invention also encompasses diagnostic kits comprising one or more of the polynucleotide sequences of the present invention. The present invention includes diagnostic kits comprising one or more of the viruses, cell lines, cell pellets, supernatants, polynucleotide sequences, vectors, and / or polypeptides of the present invention.

[0088] The avian reoviruses described herein can be inactivated or killed viruses. The purpose of viral inactivation is to prevent the replication of these viruses, and this can generally be achieved by chemical or physical methods. Chemical inactivation can be achieved by treating these viruses with, for example, enzymes, formaldehyde, β-propiolactone, ethyleneimine, or derivatives thereof. Physical inactivation can be achieved, for example, by subjecting these viruses to energy radiation, such as UV light.

[0089] Avian reovirus as described herein can be demonstrated to reduce virulence and serve as an attenuated virus for inoculation purposes. The attenuation of avian reovirus useful in the practice of the present invention can be obtained by methods well known in the art for this purpose. For example, the avian reovirus according to the separation of the present invention can be attenuated by passage in embryonated eggs, live animals or suitable cell lines. Such cell lines can include, for example, primary (avian) cell culture systems (such as, chicken embryo hepatocytes, chicken embryo fibroblasts or chicken kidney cells), mammalian cell lines (such as, VERO cell lines or BGM-70 cell lines), or avian cell lines (such as, QT-35, QM-7 or LMH). Such back passage viruses can, for example, be obtained by passage from about 10 to about 100 times. Such backpassage viruses can be obtained, for example, with one or more backpassages, two or more backpassages, three or more backpassages, four or more backpassages, five or more backpassages, ten or more backpassages, fifteen or more backpassages, twenty or more backpassages, twenty-five or more backpassages, fifty or more passages, seventy-five or more passages, or one hundred or more passages. Such backpassage viruses can be obtained, for example, with about one backpassage, about two backpassages, about three backpassages, about four backpassages, about five backpassages, about ten backpassages, about fifteen backpassages, about twenty backpassages, about twenty-five backpassages, about fifty passages, about seventy-five, about one hundred passages, or any range thereof.

[0090] For example, the present invention includes avian reoviruses that are attenuated by passage in primary chicken embryo fibroblasts followed by plaque purification. This process can be repeated, for example, from about 10 to about 100 times to attenuate the virus. Pathogenicity testing can be performed at different time points, for example, at passage 25, at passage 50, at passage 75, at passage 100, and at additional time points.

[0091] The present invention includes methods for generating an immune response against Group 1 and / or Group 2 avian reoviruses in poultry, the methods comprising administering an isolated virus, cell line, cell pellet, supernatant, polynucleotide sequence, vector, and / or polypeptide of the present invention. In some aspects, the immunity comprises humoral immunity and / or cellular immunity. In some aspects, the immunity comprises mucosal immunity.

[0092] The present invention includes methods for preventing avian reovirus infection in poultry comprising administering a composition comprising the isolated virus, cell line, cell pellet, supernatant, polynucleotide sequence, vector, and / or polypeptide of the present invention.

[0093] In some aspects of the method of the present invention, administration comprises injection, spraying, oral administration, or respiratory administration. In some aspects of the method of the present invention, administration induces mucosal immunity. In some aspects of the method of the present invention, administration comprises in ovo administration. In some aspects, in ovo administration comprises administration at about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or any range thereof.

[0094] The present invention includes immunogenic compositions and vaccines comprising one or more of the isolated viruses, polynucleotide sequences, vectors, and / or polypeptides described herein. In some embodiments, the virus is live. In some embodiments, the virus is attenuated or inactivated. In some embodiments, the virus is inactivated or killed. In some embodiments, the virus, composition, or vaccine is lyophilized. In some embodiments, the virus, composition, or vaccine is frozen.

[0095] Such compositions and vaccines can be administered as active ingredients to immunize birds to elicit an immune response to group 1 and / or group 2 avian reoviruses and / or to induce immunity against such avian reoviruses. Immunity can include inducing a higher level of protection in a bird population after vaccination compared to an unvaccinated population. The immune response may or may not confer protective immunity. The immune response can, for example, include one or more of a cell-mediated immune response and / or a humoral immune response, wherein the cell-mediated immune response comprises the production of lymphocytes in response to antigen exposure, and the humoral immune response comprises the production of plasma lymphocytes (B cells) in response to continuous antigen exposure, followed by antibody production. The immunization can result in alleviating, suppressing or preventing one or more of the symptoms of viral arthritis (VA) / tenosynovitis associated with avian reoviruses. Such symptoms may include one or more of body weight suppression, decreased egg production, mortality, gross lesions (including but not limited to tendon swelling, tenosynovitis, tendon rupture, and pericardial effusion), and histological changes (including but not limited to lymphocytic tenosynovitis, lymphocytic epicarditis, and lymphocytic myocarditis).

[0096] The immunogenic 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, based on, for example, mineral oils (such as Bayol F TM or Marcol 52 TM , complete Freund's adjuvant, incomplete Freund's adjuvant), or vegetable oil (such as vitamin E acetate) in water-in-oil or water-in-oil emulsions, and saponins.

[0097] The immunogenic compositions or vaccines of the present invention may include one or more suitable pharmaceutically acceptable carriers or diluents. The immunogenic compositions or vaccines of the present invention may also include one or more stabilizers. Any suitable stabilizer may be used, including carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, or glucose; proteins such as albumin or casein; and buffers such as alkali metal phosphates. Stabilizers are particularly advantageous when preparing dry vaccine products by lyophilization.

[0098] The immunogenic compositions or vaccines of the present invention may further include one or more immunogens derived from other pathogens that infect 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, chicken parvovirus, and avian nephritis virus (including but not limited to ANV-1 and ANV-2).

[0099] The immunogenic compositions or vaccines of the present invention can be administered by any suitable known method for inoculating poultry, including nasal, ocular, by injection, in drinking water, in feed, by exposure, in ovo, maternal, by respiratory inhalation, and the like. The immunogenic compositions or vaccines can be administered by a number of administration techniques, such as by placing the vaccine in drinking water or by a spray environment. When administered by injection, the immunogenic compositions or vaccines can be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.

[0100] In some embodiments, live vaccines can be administered at a dose of not less than 10 2 Titering units (wherein titering units are defined in the Code of Federal Regulations, Section 113.332, Title 9) are administered at doses, and the inactivated vaccine may comprise 10 4 -10 10 TCID 50 The antigen equivalent of TCID is the abbreviation of tissue culture infectious dose.

[0101] The compositions and vaccines of the present invention can be substantially pure. As used herein, "substantially pure" means that the material is essentially free of any macromolecules or other biological entities similar to those normally found in nature.

[0102] The compositions and vaccines of the present invention can be given to birds in any of many avian species, including but not limited to poultry, birds of the order Galliformes and exotic bird species, which are susceptible to avian reovirus. Birds of the order Galliformes include but are not limited to chickens, turkeys, grouse, quail and pheasants. As used herein, poultry includes domesticated birds that are raised for the purpose of collecting their eggs or killed for the purpose of their meat and / or feathers. These are most typically the Galloanserae superorder (poultry), especially the Galliformes (including, for example, chickens, quail, turkeys and grouse) and the Anatidae family (in the order Anseriformes), commonly referred to as members of "waterfowl" (including, for example, ducks, geese and swans). Poultry can also include other birds that are killed for their meat, such as pigeons or doves or birds considered to be prey, such as pheasants. Chickens include but are not limited to hens, roosters, broilers, roasters, breeders, the offspring of breeders, and layer hens. As used herein, the term "susceptible" refers to the likelihood or reality of an adverse response to a control microorganism, such as, for example, reduced viability or failure to thrive when compared to non-susceptible individuals or populations, and / or one or more pathological conditions indicative of avian viral infection.

[0103] The vaccines of the present invention can be administered to poultry before or after hatching. Poultry can receive the vaccine at a variety of ages. For example, broiler chickens can be vaccinated in ovo at one day old, or at 2-3 weeks old. Egg-laying or breeding livestock can be vaccinated, for example, at about 6 to 12 weeks old and boosted at about 16 to 20 weeks old. Such egg-laying or breeding livestock can be vaccinated at about 6, at about 7, at about 8, at about 9, at about 10, at about 11, or at about 12 weeks old. Such egg-laying or breeding livestock can be boosted at about 16, at about 17, at about 18, at about 19, or at about 20 weeks old. The offspring of such egg-laying or breeding livestock can exhibit an antibody titer to the polypeptide as described herein, which will prevent or alleviate the symptoms of avian reovirus infection in the offspring. In ovo vaccination can occur, for example, at about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or any range therein.

[0104] Chickens can be vaccinated at any suitable age and generally about one to three days before the first vaccination. The chickens can be vaccinated only once. Alternatively, if a two-dose vaccine is used, the first dose is given when the chickens are, for example, three days to one week old and the second dose is subsequently given after another 1-10 weeks.

[0105] Multiple doses of the composition may be administered throughout the life of the chicken.Because maternal immunity is the primary source of protection provided to broiler offspring, breeder chickens are typically vaccinated, although broiler chickens can also be vaccinated if desired.

[0106] The present invention also includes antisera and antibodies that bind to group 1 avian reovirus and / or group 2 avian reovirus. In some aspects, the antisera or antibodies are not directed against currently commercially available vaccine strains of avian reovirus, such as, for example, avian reovirus strains S1133, 1733, 2408, and / or 2177. Antibodies useful in the practice of the present invention include monoclonal antibodies, polyclonal antibodies, single-chain antibodies, humanized antibodies, chimeric antibodies, or fragments thereof, and also include bispecific antibodies, synthetic antibodies, antibody fragments (e.g., Fab, FV, F(ab)2, or scFv fragments), single-chain antibodies, or chemically modified derivatives of any of these. Such antibodies can be used in diagnostic methods and diagnostic kits. In some aspects, the anti-avian reovirus antibodies can be attached to a solid substrate.

[0107] The present invention provides methods for detecting and / or measuring the amount of group 1 or group 2 avian reovirus in a sample obtained from a bird. In some aspects, the group 1 avian reovirus comprises a σC protein having an amino acid sequence as described herein, including but not limited to a σC protein having SEQ ID NO: 2 or SEQ ID NO 4, or a fragment thereof, or a derivative thereof. In some aspects, the group 2 avian reovirus comprises a σC protein having an amino acid sequence as described herein, including but not limited to a σC protein having SEQ ID NO: 6, or a fragment thereof, or a derivative thereof. Such methods can include contacting the sample with an antibody that selectively binds to an avian reovirus as described herein and / or a σC protein as described herein, and measuring the amount of antibody bound to the virus or protein in the sample. The sample can be any biological material, such as tissue, bone, blood, urine, or feces. The methods of this aspect of the invention are useful, for example, for determining whether poultry are infected with an avian reovirus of the present invention. In order to prevent the reovirus from spreading to other animals, the infected animal can be killed.

[0108] As used herein, "isolated" refers to material that has been removed from its original environment (eg, the natural environment if it is naturally occurring) and thus altered "by the hand of man" from its natural state.

[0109] The term "and / or" means one or all of the listed elements or any combination of two or more of the listed elements.

[0110] The words "preferred" and "preferably" refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present invention.

[0111] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0112] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0113] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0114] For any method disclosed herein that includes separate steps, the steps can be performed in any practicable order. Also, any combination of two or more steps can be performed simultaneously, if appropriate.

[0115] Unless otherwise indicated, all numbers expressing components, 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". Therefore, unless otherwise indicated to the contrary, the numerical parameters set forth in this specification and claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by the present invention. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the present claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0116] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, all numerical values ​​inherently contain the necessary range resulting from the standard deviation found in their respective testing measurements.

[0117] All headings are intended for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless so indicated.

[0118] Throughout this application, in several places, guidance is provided through a series of examples, which can be used in various combinations. In each case, the series serves only as a representative group and should not be construed as an exclusive series. It should be understood that the specific examples, materials, amounts, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0119] The present invention is illustrated by the following examples. It should be understood that the specific examples, materials, amounts, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0120] Examples

[0121] Example 1

[0122] Isolation and characterization of reovirus variants

[0123] This example describes the isolation of reovirus variants from the tendons of numerous clinical cases of tenosynovitis and / or lameness in broiler chickens ranging in age from 2.5 to 8 weeks old submitted to the Poultry Diagnostic and Research Center. These avian reoviruses were characterized by sequencing the σC protein and serological assays.

[0124] Materials and methods

[0125] Virus Isolation. Wild-type reovirus isolates Ck / 94594 Tendon / GA / 2012, Ck / 94826 Tendon / GA / 2012, and Ck / 96139 Tendon / GA / 2012 were isolated from tendons of commercial broiler chickens showing clinical symptoms of viral arthritis and tenosynovitis at the Poultry Diagnostic and Research Center at the University of Georgia. Tendons and synovial aspirates from clinically infected chickens were homogenized in virus transport medium with antibiotics. The homogenized tissue was filtered through a 0.45 μm syringe filter. The filtrate was incubated with reovirus S1133 antiserum (Charles River, SPAFAS, Wilmington, MA) at 37°C for 1 hour. Subsequently, 0.2 ml of the homogenate was cultured in primary chicken embryo hepatocytes for a total of four generations. With the development of 70%-80% cytopathic effect, when syncytia formation was observed, the cell culture was frozen and stored at -80° C. Before subsequent cell subculture, the cell culture was frozen and thawed three times.

[0126] RNA extraction. Total viral RNA was extracted from primary chicken embryo liver cells using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's recommendations. Briefly, 48 hours after infection, the cell culture medium was removed and the monolayers were overlaid with 300 μl of RLT buffer containing 0.143 M β-mercaptoethanol and scraped out using a cell scraper as recommended by the manufacturer.

[0127] RT-PCR. The SUPERSRIPT sequence was constructed by reverse transcription and PCR using the previously published primers P1 (AGTATTTGTGAGTACG ATTG (SEQ ID NO: 7)) and P4 (GGCGCCACACCTTAGGT (SEQ ID NO: 8)). TM III RNase H - Reverse Transcriptase and Taq DNA polymerase (Invitrogen, Carlsbad, CA) was used to generate cDNA corresponding to the S1 gene (Kant et al., 2003, Vet Res; 34: 203-212). The 1.1 kb amplification product was separated on a 1.0% agarose gel, stained with ethidium bromide, and visualized with a UV transilluminator. The fragment was excised and purified using a QIAEX II gel extraction kit (Qiagen Inc., Valencia, CA), eluted in diethylpyrocarbonate (DEPC)-treated water, and stored at -80°C.

[0128] The nucleotide sequence of amplified product and S1 clone is determined. Gel-purified PCR product is directly used to carry out sequencing using double-stranded DNA sequencing with fluorescently labeled dideoxynucleotides and Taq polymerase and is operated on an ABI 9700 automatic sequencer (Applied Biosystems Inc., Foster City, California). PCR primers P1 and P4 are used to carry out sequencing, and conservative internal S1 gene primers are also used as needed to complete sequencing. Primer sequences for sequencing can be obtained after request. In addition, gel-purified product is cloned into plasmid pCR2TOPO (Invitrogen) and transformed into Escherichia coli according to the manufacturer's advice. Several clones from every kind of isolate are identified by PCR using M13 forward and reverse primers, and these isolates comprise a plasmid with a 1.1kb insert. These clones are amplified and plasmid microprep kit (Qiagen, Valencia, California) is used to purify the plasmid according to the manufacturer's method. Sequencing was performed using M13 universal forward and reverse primers, and internal S1 primers were used as needed to complete the sequencing. At least three clones or PCR products from the three amplifications containing the S1 gene were sequenced in both directions and used to obtain the consensus region. The nucleotide and amino acid sequences for isolates 94594, 94826, and 96139 were obtained in Figure 1-6 to be identified in.

[0129] Sequence Analysis: Nucleotide and predicted amino acid sequence analysis and multiple alignment of the S1 gene and σC protein were performed using CLUSTAL W (Lasergene, v.5.0, DNASTAR, Madison, Wisconsin). The previously published sequence of the S1 gene of avian reovirus was obtained from GenBank.

[0130] Aligned sequences were compared and a phylogenetic tree was generated using Parsimony v 4.10b software (PAUP) using neighbor-joining clustering and 1000 bootstrap replicates (confidence levels are listed in parentheses) with phylogenetic analysis in a heuristic search.

[0131] GenBank Accession Nos. Sequences obtained from chicken isolates 94594, 94826, and 96139 have been submitted to GenBank and assigned the following accession numbers: KJ803966, KJ803967, and KJ803990, respectively.

[0132] Results and discussion

[0133] To date, two primary variant genotypes of wild-type reovirus isolates have been reported in clinical cases of VA / tenosynovitis and have been identified due to their association with current reovirus vaccine strains (S1133, 1733, 2408, 2177) and other viruses in the database ( Figure 7 ) lack similarity and are characterized as variant genotypes. Most wild-type isolates belong to cluster 1 (identified as cluster 1 2012VA variants). When compared with the public domain and PDRC database ( Figure 8 ), the σC amino acid sequences of reoviruses belonging to this genotype have >99% similarity to each other and <50% similarity to the vaccine strain. The reoviruses in cluster 2 (identified as cluster 2 2012VA variant) have >99% similarity to each other and <50% similarity to cluster 1 2012VA variant ( Figure 8 ) < 50% similarity. The σC similarity between cluster 2 2012VA variant and vaccine strain is 80% ( Figure 8 Although the groups share high sequence similarity, it is unclear whether this 80% similarity translates into a degree of cross-protection with current vaccine strains. Serological evaluation of wild-type isolates from these two groups was performed to provide the necessary information on the serological relatedness of the viruses.

[0134] Representative viruses from cluster 1 and cluster 2 variants were evaluated in a one-way cross-neutralization assay using antisera against S1133 and 2408, and hyperimmune sera were prepared against cluster 1 2012VA variant. VN titers between the wild-type isolate and the serum panel ranged from 2 to 16, providing evidence that the wild-type isolate is not serologically related to S1133, 2408, or each other.

[0135] Example 2

[0136] Study on the pathogenicity and progeny protection of group 1 variant reovirus

[0137] This example determines the pathogenicity of reovirus variant serogroup 1 2012VA isolates 94826 and 94594 in chickens.

[0138] Experiment #1

[0139] This experiment was designed to determine whether Company A's vaccination program provided adequate protection against the variant serogroup 1 2012VA isolate in progeny challenged with the wild-type reovirus isolate 94826.

[0140] Chickens. 80 one-day-old commercial broiler chickens from vaccinated breeder stock from Company A were used.

[0141] Table 1. Treatment groups

[0142] Number of chicks Group ID Group Processing 20 chicks 1 Phlebotomy for reovirus ELISA (IDEXX) 20 chicks 2 Inoculation with S1133 20 chicks 3 Inoculation with isolate 94826 20 chicks 4 Inoculate with sterile PBS

[0143] The wild-type isolate 94826 was propagated and titered in primary chicken embryo liver cells and used to inoculate day-old chicks at 10 3 The S1133 stock, also titrated in primary chicken embryo hepatocytes, will be used to inoculate day-old chicks at a challenge dose of 10 TCID50 / bird. 3 Negative controls will be mock challenged via the footpad with sterile PBS.

[0144] Reovirus challenge. Sixty commercial broiler chickens were received on the day of hatching and divided into 3 groups of 20 birds each and placed in Horsfall Bauer separation units. Chicks from Group 1 were bled for Reovirus ELISA (IDEXX) and terminated. Chicks from Groups 2-4 were inoculated via the footpad with 1) sterile PBS, 2) S1133, or 3) 94826. This is detailed in Table 1. The birds were observed daily and hock swelling in each treatment group was measured with a digital caliper and recorded every 2 days. On day 14, all birds were euthanized, weighed, and necropsy performed. Birds were inspected for any macroscopic lesions and for hock swelling, hemorrhage, and / or rupture. Protective parameters included serology on the day of hatching, clinical signs, mortality, body weight, macroscopic lesions, and assessment / measurement of hock swelling throughout the study.

[0145] Experiment #2

[0146] This study aimed to determine whether Company B's vaccination program provided adequate protection against viral arthritis in offspring challenged with a wild-type isolate of reovirus from Company B's farm, and whether the use of a commercial reovirus vaccine at day old age provided any protection.

[0147] Chickens. One hundred one-day-old commercial broiler chickens from Company B vaccinated breeding stock were used.

[0148] Table 2. Treatment groups

[0149] Number of chicks Group ID Group Processing 20 chicks 1 Phlebotomy for reovirus ELISA (IDEXX) 20 chicks 2 Inoculate with sterile PBS 20 chicks 3 Full dose of VA ChickVac on day 1, then challenge w / 94594 20 chicks 4 VA ChickVac 1 / 2 dose on day 1, then challenge w / 94594 20 chicks 5 Challenge with Sanderson wild-type isolate 94594

[0150] Virus. Wild-type reovirus isolate 94594 was propagated and titered in primary chicken embryo hepatocytes prior to the study. VA ChickVac (Zoetis) was used for vaccination on the day of incubation, as recommended by the manufacturer. Negative controls were mock challenged with sterile PBS.

[0151] Reovirus challenge. One hundred commercial chicks were received on the day of hatching and divided into 5 equal groups of 20 chicks each. Group 1 was bled for Reovirus ELISA (IDEXX). The remaining 4 groups of chicks were placed in Horsfall Bauer separation units under positive pressure of forced air. On the day of hatching, Group 3 was vaccinated with a full dose of VA ChickVac by subcutaneous administration while Group 4 was vaccinated with a half dose of VA ChickVac by subcutaneous administration. This is detailed in Table 2. On day 10, footpad measurements were obtained for all chicks. On day 10, Group 2 was inoculated with 0.1 ml sterile phosphate buffered saline through the footpad while chicks in Groups 3-5 were vaccinated with wild type isolate 94594 through the footpad at 10 3 TCID50 / bird challenge. Starting at 10 days of age, chicks were monitored daily and hock measurements were obtained every two days. The study was terminated at 22 days of age. Parameters used for protection were serology on the day of hatch, clinical signs, mortality, body weight, and macroscopic lesions.

[0152] Results and discussion

[0153] Pathogenicity and progeny protection studies in commercial broiler chickens were performed using representative strains from the Cluster 1 2012VA variant genotype. In the first study, day-of-hatch chicks were challenged via the footpad with either the S1133 or Cluster 1 2012VA variant isolates and one group was placed for negative control. In addition, a separate group of twenty day-of-hatch chicks were bled for reovirus ELISA. All chicks were placed in separate cages on fresh wood shavings and given free access to feed and water. Footpad measurements were taken every 2 days and the birds were monitored daily for clinical signs until the experiment was terminated at 14 days ( Figure 9 The group's reovirus ELISA geometric mean titer (GMT) was 1,212, a value considered low for chicks on the day of hatch. Clinical tenosynovitis and significant suppression of weight loss were observed in both the S1133 and variant reovirus challenge groups ( Figure 10 and 12 While S1133 chicks were more severely affected by lameness than those challenged with the variant, there was a higher incidence of swelling around the digital flexor tendons in the variant-challenged group ( Figure 11 Other clinical signs were observed in the challenge group, including pericardial effusion and tarsal joint inflammation ( Figure 12 ). Reovirus titers were low on the day of incubation, and therefore maternal antibodies from Company A's vaccination program did not provide adequate protection against challenge with group 1 variants. In addition, this wild-type isolate reproduced the clinical disease observed in the wild-type strain and was confirmed to be the causative agent of disease.

[0154] In the second study, commercial broiler chicks were challenged with a group 1 variant via the footpad at 12 days of age and the study was terminated at 23 days of age. The Reovirus ELISA GMT obtained from sera collected on the day of hatch was 4,900. In this study, the mean body weight in the challenged group was only slightly lower ( Figure 13 However, the challenge group showed clinical signs and macroscopic lesions characteristic of viral arthritis / tenosynovitis ( Figure 14 As in the first study, swelling around the digital flexor tendons along with pericardial effusion was observed in the provoked birds ( Figure 12 and Figure 13 ). It was concluded from this study that chicks with higher levels of maternal reovirus antibodies were not protected against challenge with group 1 variants.

[0155] In addition, the use of a commercial reovirus vaccine administered subcutaneously at full and half doses on the day of hatch in commercial broiler chickens, followed by a challenge with a group 1 variant at 12 days of age, was evaluated. Using the same parameters as in the previous study, we observed clinical signs and macroscopic lesions characteristic of VA / tenosynovitis in all challenged groups. Interestingly, at the neglected dose, the incidence of pericardial effusion and swollen hocks was higher in the vaccinated and challenged groups compared to the challenged-only group ( Figure 14 ).

[0156] In summary, variant reoviruses have been isolated from clinical cases of viral arthritis / tenosynovitis and are genetically and antigenically distinct from current reovirus vaccine strains. Based on genetic and serological characterization, two distinct groups have been identified as group 1 and group 2 2012 variant VA reoviruses. In vivo studies have shown that currently commercially available vaccines do not provide protection against challenge with the group 1 2012 variant in chicks with maternal reovirus antibodies. In vivo studies using representative group 2 isolates are given in Example 3 below. Group 1 and group 2 variant reoviruses have been isolated or detected from clinical cases of tenosynovitis in numerous states within the United States and Canada, with isolates belonging to group 1 outnumbering those from group 2. The origin of these viruses is unknown.

[0157] Example 3

[0158] Study on the pathogenicity of group 2 variant reovirus

[0159] The procedure described in more detail in Example 2 was used to characterize the pathogenicity of group 2 variant reovirus in commercial broiler chickens. A representative strain from the group 2 2012VA variant genotype (Ck / 96139 tendon / GA / 2012) was used. The treatment groups are detailed in Table 3.

[0160] Table 3. Treatment groups

[0161]

[0162] The chicks were treated with 0.1 ml sterile phosphate buffered saline or wild-type isolate 996139 at 10 3 TCID50 / bird inoculated. Parameters used for protection were serology on the day of hatching, clinical signs, mortality, body weight, and macroscopic lesions. Footpad swelling and tendon swelling were assessed and measured throughout the study.

[0163] Results and discussion

[0164] Reovirus ELISA (IDEXX) serology on the day of incubation is shown in Figure 17 Specifically, the mean was 511, the GMT was 385, the SD was 343, and the %CV was 66.8%.

[0165] like Figure 18 As shown, Cluster 2 challenge reduced body weight. Specifically, an 8% decrease in body weight was observed in the Cluster 2 challenged birds.

[0166] Figure 19 Footpad swelling measurements as a percentage of body weight are shown on day 14, with Figure 20 Tendon swelling measurements as a percentage of body weight are shown for birds challenged with Cohort 2.

[0167] Necropsy and histological findings for birds challenged with cluster 2 were as follows: 2 / 10 birds had ruptured tendons; 5 / 10 birds had lymphocytic tenosynovitis (tendon sheath); 2 / 10 birds had pericardial effusion; and 2 / 10 birds had lymphocytic epicarditis and myocarditis. No significant lesions were observed in the liver or duodenum.

[0168] Thus, cluster 2 challenge resulted in mild weight loss. Pericardial effusion, swollen tendons, and several tendon ruptures were observed in cluster 2 challenged birds. Furthermore, tissue damage consistent with reovirus was observed in the heart and tendons.

[0169] In summary, both group 1 and group 2 variants were isolated from clinical cases of viral arthritis in commercial broiler chickens. Both group 1 and group 2 variants reproduced tenosynovitis, including grossly observed pericardial effusion and tendon swelling, and histological changes consistent with reovirus were observed in the heart and tendons. Group 1 variants are more prevalent. Currently commercial vaccines do not appear to provide adequate protection for broiler chickens. Therefore, autologous vaccination appears to be the only current option for control.

[0170] Example 4

[0171] Plaque assay for avian reovirus

[0172] Seed chicken embryo liver cells in 35 mm plates:

[0173] - One day before the assay, chicken embryonic hepatocytes (CELiC) were added to 35 culture dishes (2 ml / dish).

[0174] - Place these 35 mm plates in an incubator at 37°C (overnight, at least more than 12 hours).

[0175] Plaque assay procedure:

[0176] - Remove the culture medium (M199 / F10 + 10% FBS) from these confluent CELiCs.

[0177] - Add 200 μl of the virus (10 2 TCID 50 ) to the plate.

[0178] - Thirty minutes later, add 2 ml of M199 / F10 + 2% CS with diluted virus to the plate and vortex the plate gently.

[0179] - Incubate the plate at 37°C for 3-4 hours.

[0180] - During that time prepare 2 ml per plate: 1 ml (2x concentration of M199 / F10 + 5% CS) + and 1 ml of 3% SeaPlaque agarose in cell culture gradient H2O.

[0181] - After 3-4 hours, remove the plate from the incubator and remove the supernatant (1,200 μl).

[0182] - Now layer 2 ml / well of the prepared and incubate at 37°C for 2-3 days.

[0183] Stain after 2-3 days (usually try staining after 2 days of culture; if it's longer than 3 days, the cells will start to die and the plaques may not be good):

[0184] - Prepare 10% filtered neutral red diluted in cell culture gradient water.

[0185] -Add 1 ml / plate.

[0186] - Incubate in the incubator for at least 2 hours (2-4 hours).

[0187] -After a few hours, plaques will clearly appear.

[0188] Result: Hepatocytes will stain red. Plaques will remain unstained.

[0189] Example 5

[0190] Cluster I and cluster 2σC protein sequences

[0191] Following the procedures described in more detail in Example 1, nucleotide sequences encoding the σC protein and encoded σC amino acid sequences of 122 group 1 and group 2 variant avian reoviruses were tested. Figure 21 Shown are the GenBank accession number, sequence ID, isolate design, tissue type from which the virus was isolated, date of collection, state and country of isolation, and source of isolation for the 122 isolates. Chicken embryo liver cells served as the laboratory host for all 122 isolates.

[0192] Example 5

[0193] Attenuation of group 1 and group 2 variants of avian reovirus

[0194] Listed in Figure 21 Group 1 and group 2 avian reovirus isolates, including but not limited to isolates 94826 and 94594 (both group 1 isolates) and isolate 96139 (group 2 isolate), will be attenuated by passage in eggs or in cell lines such as, for example, chicken embryo liver cells (CELiC). Cloned, plaque-purified virus preparations will be prepared after each passage and used for the next round of attenuation. The isolates will be tested for pathogenicity and vaccine effectiveness after approximately every 25 passages, for example, after about 25, about 50, about 75, about 100, and about 125 passages. The amino acid sequence of the Cσ protein can be obtained for use in attenuated strains.

[0195] The complete disclosures of all patents, patent applications, and publications and electronically available materials cited herein (including, for example, nucleotide sequences submitted to, for example, GenBank and RefSeq, and amino acid sequences submitted to, for example, SwissProt databases, PIR, PRF, PDB, and translations of annotated coding regions from GenBank and RefSeq) are incorporated by reference. In the event of any inconsistency between the disclosure of this application and one or more disclosures of any document incorporated herein by reference, the disclosure of this application will control. The above detailed description and examples are given only for the sake of clarity of understanding. No unnecessary limitations should be construed therefrom. The present invention is not limited to the precise details shown and described, as variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0196] Free Sequence List

[0197] SEQ ID NO: 1

[0198] S1 nucleotide sequence encoding σC protein (base pairs 1-931) from cluster 1 2012VA variant wild-type isolate 94594

[0199] SEQ ID NO:2

[0200] S1 amino acid sequence of σC (amino acids 1-310) from cluster 1 2012VA variant wild-type isolate 94594

[0201] SEQ ID NO:3

[0202] S1 nucleotide sequence encoding σC protein (base pairs 1-931) from cluster 1 2012VA variant wild-type isolate 94826

[0203] SEQ ID NO:4

[0204] S1 amino acid sequence of σC (amino acids 1-310) from cluster 1 2012VA variant wild-type isolate 94826

[0205] SEQ ID NO:5

[0206] Nucleotide sequence of S1 encoding σC protein (base pairs 1-931) from cluster 2 2012VA variant wild-type isolate 96139

[0207] SEQ ID NO:6

[0208] S1 amino acid sequence of the σC protein (amino acids 1-310) from cluster 2 2012VA variant wild-type isolate 96139

[0209] SEQ ID NO:7P1PCR primer

[0210] SEQ ID NO:8P4 PCR primer

Claims

1. An isolated avian reovirus, wherein the avian reovirus comprises: a σC protein consisting of the amino acid sequence of SEQ ID NO:4; or a σC protein consisting of the amino acid sequence of SEQ ID NO:

6.

2. The avian reovirus according to claim 1, wherein the avian reovirus is attenuated by at least 25 passages in eggs or a host cell line.

3. The avian reovirus according to claim 1, wherein the avian reovirus is attenuated, inactivated, or killed.

4. A composition comprising the avian reovirus according to any one of claims 1 to 3.

5. A vaccine comprising the avian reovirus according to any one of claims 1 to 3.

6. An immunological composition for raising antibodies in poultry, the immunological composition comprising the avian reovirus according to any one of claims 1 to 3.

7. The composition, vaccine, or immunological composition according to any one of claims 4 to 6, further comprising an adjuvant.

8. The composition, vaccine, or immunological composition according to any one of claims 4 to 6, further comprising antigenic determinants from one or more additional pathogens infecting poultry.

9. The composition, vaccine, or immunological composition according to claim 7, further comprising antigenic determinants from one or more additional pathogens infecting poultry.

10. A diagnostic kit comprising the virus according to any one of claims 1 to 3.

11. Use of the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine, or immunological composition according to any one of claims 4 to 9 in the preparation of a product for generating anti-reovirus antibodies in poultry, comprising administering to the poultry the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine, or immunological composition according to any one of claims 4 to 9.

12. Use of the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine, or immunological composition according to any one of claims 4 to 9 in the preparation of a product for protecting birds of the Galliformes against lesions or diseases induced by avian reovirus, comprising administering to the bird the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine, or immunological composition according to any one of claims 4 to 9.

13. Use of the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine, or immunological composition according to any one of claims 4 to 9 in the preparation of a product for reducing the susceptibility of birds of the Galliformes to lesions or diseases induced by avian reovirus, comprising administering to the bird the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine, or immunological composition according to any one of claims 4 to 9.

14. Use of the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine or immunological composition according to any one of claims 4 to 9 in the preparation of a product for reducing viral arthritis and / or tenosynovitis induced by avian reovirus in birds of the Galliformes order, comprising administering to the bird the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine or immunological composition according to any one of claims 4 to 9.

15. Use of the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine or immunological composition according to any one of claims 4 to 9 in the preparation of a product for preventing viral arthritis and / or tenosynovitis induced by avian reovirus in birds of the Galliformes order, comprising administering to the bird the isolated avian reovirus according to any one of claims 1 to 3 or the composition, vaccine or immunological composition according to any one of claims 4 to 9.

16. Use according to any one of claims 11 to 15, wherein the poultry or the bird is a chicken or a turkey.

17. Use according to any one of claims 11 to 15, wherein the administration comprises administration before or after hatching.

18. Use according to any one of claims 11 to 15, wherein the administration comprises in ovo administration.

19. Use according to claim 18, wherein the in ovo administration comprises administration at 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or any range thereof.

20. Use according to any one of claims 11 to 15, wherein the administration comprises administration to the breeder hen.

21. Use of the avian reovirus according to any one of claims 1 to 3 in the preparation of a product for detecting avian reovirus exposure in a bird, comprising determining the specific binding of an antiserum sample obtained from the bird to the avian reovirus according to any one of claims 1 to 3.

22. An isolated σC protein from the avian reovirus according to claim 1.

23. Use of the isolated σC protein according to claim 22 in the preparation of a product for detecting avian reovirus exposure in a bird, comprising determining the specific binding of an antiserum sample obtained from the bird to the isolated σC protein according to claim 22.

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