Infection and transmission dynamics of Turkey arthritis reovirus in different age Turkeys.

Turkey arthritis reovirus (TARV) has been established as a cause of lameness in meat type turkeys in the past decade. However, no information is available on the age susceptibility of TARV or its transmission dynamics. We conducted this study to determine the age at which turkey poults are susceptible to TARV infection and whether infected birds can horizontally transmit the virus to their non-infected pen mates (sentinels). Five groups of turkeys were orally inoculated with TARV (∼106 TCID50/ml) at 2, 7, 14, 21 and 28 days of age (DOA). Two days after each challenge, four uninfected sentinel turkeys of equal age were added to the virus-inoculated groups. At one- and two-weeks post infection, turkeys from each group, including two sentinels, were euthanized followed by necropsy. Inoculated birds in all age groups had TARV replication in the intestine and gastrocnemius tendon with no statistically significant variation at p < 0.5. Furthermore, the inoculated birds at different age groups showed consistently high gastrocnemius tendon histologic lesion scores while birds in the 28-days-old age group had numerically lower lesion scores at 14 days post inoculation (dpi). The sentinels, in turn, also showed virus replication in their intestines and tendons and histologic lesions in gastrocnemius tendons. The findings indicate that turkeys at the age of 28 days or less are susceptible to infection with TARV following oral challenge. It was also found that TARV-infected birds could transmit the infection to naïve sentinel turkeys of the same age.

Comparative pathogenesis of turkey reoviruses.

Turkey reoviruses have been implicated in multiple disease syndromes resulting in significant economic losses to the turkey industry. It has been known for decades that turkey enteric reovirus (TERV) is involved in poult enteritis complex, but turkey arthritis reovirus (TARV), the causative agent of tenosynovitis in turkeys, emerged in 2011. In 2019, we isolated reovirus from several cases of hepatitis in turkeys and tentatively named it turkey hepatitis reovirus (THRV). The comparative pathogenesis of these viruses, and correlation with their genetic make-up (if any), is not known. In this study, we inoculated nine groups of 1-week-old turkey poults with two THRV, five TARV and two TERV via oral route. A tenth group served as a negative control. A subset of birds from each group was euthanised at 3, 5, 7, 14, 21, and 28 days post-inoculation (dpi). Tissues were collected for histology and real-time RT-PCR. All nine viruses were found to be enterotropic; the virus gene copy number in the intestine reached a peak at 5 dpi followed by a sharp decline at 7 dpi. All viruses caused a significant decline in body weight gain of birds as compared to the negative control group. Both TARV and THRV strains replicated in tendons and produced histologic lesions consistent with tenosynovitis. Hepatic lesions were produced by THRV only and the virus was re-isolated from liver and spleen of inoculated birds fulfilling Koch's postulates. The results of this study should be helpful in facilitating diagnosis and designing future mitigation plans.

Efficacy and Immunogenicity of Recombinant Pichinde Virus-Vectored Turkey Arthritis Reovirus Subunit Vaccine.

We created a recombinant live pichinde virus-vectored bivalent codon optimized subunit vaccine that expresses immunogenic Sigma C and Sigma B proteins of turkey arthritis reovirus. The vaccine virus could be transmitted horizontally immunizing the non-vaccinated pen mates. The vaccine was tested for efficacy against homologous (TARV SKM121) and heterologous (TARV O'Neil) virus challenge. Immunized poults produced serum neutralizing antibodies capable of neutralizing both viruses. The vaccinated and control birds showed similar body weights indicating no adverse effect on feed efficiency. Comparison of virus gene copy numbers in intestine and histologic lesion scores in tendons of vaccinated and non-vaccinated birds showed a decrease in the replication of challenge viruses in the intestine and tendons of vaccinated birds. These results indicate the potential usefulness of this vaccine.

Development of a Recombinant Pichinde Virus-Vectored Vaccine against Turkey Arthritis Reovirus and Its Immunological Response Characterization in Vaccinated Animals.

Vaccination may be an effective way to reduce turkey arthritis reovirus (TARV)-induced lameness in turkey flocks. However, there are currently no commercial vaccines available against TARV infection. Here, we describe the use of reverse genetics technology to generate a recombinant Pichinde virus (PICV) that expresses the Sigma C and/or Sigma B proteins of TARV as antigens. Nine recombinant PICV-based TARV vaccines were developed carrying the wild-type S1 (Sigma C) and/or S3 (Sigma B) genes from three different TARV strains. In addition, three recombinant PICV-based TARV vaccines were produced carrying codon-optimized S1 and/or S3 genes of a TARV strain. The S1 and S3 genes and antigens were found to be expressed in virus-infected cells via reverse transcriptase polymerase chain reaction (RT-PCR) and the direct fluorescent antibody (DFA) technique, respectively. Turkey poults inoculated with the recombinant PICV-based TARV vaccine expressing the bivalent TARV S1 and S3 antigens developed high anti-TARV antibody titers, indicating the immunogenicity (and safety) of this vaccine. Future in vivo challenge studies using a turkey reovirus infection model will determine the optimum dose and protective efficacy of this recombinant virus-vectored candidate vaccine.

Specific-pathogen-free Turkey model for reoviral arthritis.

Turkey arthritis reovirus (TARV) infections have been recognized since 2011 to cause disease and significant economic losses to the U.S. turkey industry. Reoviral arthritis has been reproduced in commercial-origin turkeys. However, determination of pathogenesis or vaccine efficacy in these turkeys can be complicated by enteric reovirus strains and other pathogens that ubiquitously exist at subclinical levels among commercial turkey flocks. In this study, turkeys from a specific-pathogen-free (SPF) flock were evaluated for use as a turkey reoviral arthritis model. One-day-old or 1-week-old poults were orally inoculated with TARV (O'Neil strain) and monitored for disease onset and progression. A gut isolate of turkey reovirus (MN1 strain) was also tested for comparison. Disease was observed only in TARV-infected birds. Features of reoviral arthritis in SPF turkeys included swelling of hock joints, tenosynovitis, distal tibiotarsal cartilage erosion, and gait defects (lameness). Moreover, TARV infection resulted in a significant depression of body weights during the early times post-infection. Age-dependent susceptibility to TARV infection was unclear. TARV was transmitted to all sentinel birds, which manifested high levels of tenosynovitis and tibiotarsal cartilage erosion. Simulation of stressful conditions by dexamethasone treatment did not affect the viral load or exacerbate the disease. Collectively, the clinical and pathological features of reoviral arthritis in the SPF turkey model generally resembled those induced in commercial turkeys under field and/or experimental conditions. The SPF turkey reoviral arthritis model will be instrumental in evaluation of TARV pathogenesis and reoviral vaccine efficacy.

Genomic characterization of a turkey reovirus field strain by Next-Generation Sequencing.

The genome of a turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13), isolated from a turkey flock in Pennsylvania (PA) in 2013, has been sequenced using Next-Generation Sequencing (NGS) on the Illumina MiSeq platform. The genome of the PA TARV field strain was 23,496bp in length with 10 dsRNA segments encoding 12 viral proteins. The lengths of the genomic segments ranged from 1192bp (S4) to 3959bp (L1). The 5' and 3' conserved terminal sequences of the PA TARV field strain were similar to the two Minnesota (MN) TARVs (MN9 and MN10) published recently and avian orthoreovirus (ARV) reference strains. Phylogenetic analysis of the nucleotide sequences of all 10 genome segments revealed that there was a low to significant nucleotide sequence divergence between the PA TARV field strain and reference TARV and ARV strains. Analysis of the PA TARV sequence indicates that this PA TARV field strain is a unique strain and is different from the TARV MN9 or MN10 in M2 segment genes and ARV S1133 vaccine strain.

Phylogenetic analysis, genomic diversity and classification of M class gene segments of turkey reoviruses.

From 2011 to 2014, 13 turkey arthritis reoviruses (TARVs) were isolated from cases of swollen hock joints in 2-18-week-old turkeys. In addition, two isolates from similar cases of turkey arthritis were received from another laboratory. Eight turkey enteric reoviruses (TERVs) isolated from fecal samples of turkeys were also used for comparison. The aims of this study were to characterize turkey reovirus (TRV) based on complete M class genome segments and to determine genetic diversity within TARVs in comparison to TERVs and chicken reoviruses (CRVs). Nucleotide (nt) cut off values of 84%, 83% and 85% for the M1, M2 and M3 gene segments were proposed and used for genotype classification, generating 5, 7, and 3 genotypes, respectively. Using these nt cut off values, we propose M class genotype constellations (GCs) for avian reoviruses. Of the seven GCs, GC1 and GC3 were shared between the TARVs and TERVs, indicating possible reassortment between turkey and chicken reoviruses. The TARVs and TERVs were divided into three GCs, and GC2 was unique to TARVs and TERVs. The proposed new GC approach should be useful in identifying reassortant viruses, which may ultimately be used in the design of a universal vaccine against both chicken and turkey reoviruses.

Molecular characterization of L class genome segments of a newly isolated turkey arthritis reovirus.

Seven strains of turkey arthritis reovirus (TARV) isolated from cases of turkey arthritis were characterized on the basis of their L class genome segment sequences, which were then compared with those of turkey enteric reovirus (TERV) and chicken reovirus (CRV). All three L class gene segments of TARVs and TERVs and their encoded proteins λA, λB, and λC were similar in size to those of CRV reference strain S1133. The conserved motifs such as C2H2 zinc-binding motif and conserved polymerase region were present in λA and λB, respectively. A conserved motif for ATP/GTP-binding site and an S-adenosyl-l-methionine (SAM)-binding pocket for methyltransferase were observed in λC protein of TARVs and TERVs with only one substitution as compared to that in CRV. We propose a new genotype classification system for avian reoviruses (ARVs) based on the nt identity cut-off value for each of the L class. Based on this new genotype classification, all ARVs were divided into six, seven and eight genotypes in L1, L2 and L3 genes, respectively. Interestingly TARVs and TERVs grouped with three CRVs (two arthritic strains from Taiwan and one enteritic strain from Japan) in genotype L1-I and formed a different genotypes (L2-I, L3-I) from CRVs in L2 and L3 genes. The maximum nucleotide divergence was observed in genotypes of L1 and L2 genes but less at amino acid level indicates mostly changes were synonymous type. Compared to L1 and L2 genes, the nonsynonymous changes were more in L3 gene. Point mutations and possible reassortments among TARVs, TERVs and CRVs were also observed.

Characterization of S class gene segments of a newly isolated turkey arthritis reovirus.

We report on the complete characterization of S class gene segments of 12 newly isolated turkey arthritis reoviruses (TARVs) and compare it with that of a turkey enteric reovirus (TERV). Phylogenetic analysis of S2, S3 and S4 genome segments revealed grouping of all TARVs into two lineages while, on the basis of S1 genome segment, only one lineage was found. All TARVs had 95-100% nucleotide identity based on sigma C protein sequences (S1 segment) but varied from 90-100%, 88.9-100% and 88.7-100% on the basis of S2, S3, and S4 genome segments, respectively. Point mutations as well as possible re-assortments were observed in TARVs throughout the S class indicating the need for extensive epidemiological studies on these viruses in hatcheries and commercial farms, which would be useful in determining virus variation in the field.