Analysis of Emerging Variants of Turkey Reovirus using Machine Learning.

Avian reoviruses continue to cause disease in turkeys with varied pathogenicity and tissue tropism. Turkey enteric reovirus has been identified as a causative agent of enteritis or inapparent infections in turkeys. The new emerging variants of turkey reovirus, tentatively named turkey arthritis reovirus (TARV) and turkey hepatitis reovirus (THRV), are linked to tenosynovitis/arthritis and hepatitis, respectively. Turkey arthritis and hepatitis reoviruses are causing significant economic losses to the turkey industry. These infections can lead to poor weight gain, uneven growth, poor feed conversion, increased morbidity and mortality and reduced marketability of commercial turkeys. To combat these issues, detecting and classifying the types of reoviruses in turkey populations is essential. This research aims to employ clustering methods, specifically K-means and Hierarchical clustering, to differentiate three types of turkey reoviruses and identify novel emerging variants. Additionally, it focuses on classifying variants of turkey reoviruses by leveraging various machine learning algorithms such as Support Vector Machines, Naive Bayes, Random Forest, Decision Tree, and deep learning algorithms, including convolutional neural networks (CNNs). The experiments use real turkey reovirus sequence data, allowing for robust analysis and evaluation of the proposed methods. The results indicate that machine learning methods achieve an average accuracy of 92%, F1-Macro of 93% and F1-Weighted of 92% scores in classifying reovirus types. In contrast, the CNN model demonstrates an average accuracy of 85%, F1-Macro of 71% and F1-Weighted of 84% scores in the same classification task. The superior performance of the machine learning classifiers provides valuable insights into reovirus evolution and mutation, aiding in detecting emerging variants of pathogenic TARVs and THRVs.

Reconstruction of Avian Reovirus History and Dispersal Patterns: A Phylodynamic Study.

Avian reovirus (ARV) infection can cause significant losses to the poultry industry. Disease control has traditionally been attempted mainly through vaccination. However, the increase in clinical outbreaks in the last decades demonstrated the poor effectiveness of current vaccination approaches. The present study reconstructs the evolution and molecular epidemiology of different ARV genotypes using a phylodynamic approach, benefiting from a collection of more than one thousand sigma C (σC) sequences sampled over time at a worldwide level. ARVs' origin was estimated to occur several centuries ago, largely predating the first clinical reports. The origins of all genotypes were inferred at least one century ago, and their emergence and rise reflect the intensification of the poultry industry. The introduction of vaccinations had only limited and transitory effects on viral circulation and further expansion was observed, particularly after the 1990s, likely because of the limited immunity and the suboptimal and patchy vaccination application. In parallel, strong selective pressures acted with different strengths and directionalities among genotypes, leading to the emergence of new variants. While preventing the spread of new variants with different phenotypic features would be pivotal, a phylogeographic analysis revealed an intricate network of viral migrations occurring even over long distances and reflecting well-established socio-economic relationships.

Evidence of vertical transmission of novel duck orthoreovirus in ducks.

Since 2017, duck spleen necrosis caused by new variant duck orthoreovirus (N-DRV) infection had been observed in several provinces in China. This disease retards the growth and development of ducks, thereby reducing feed return rate. N-DRV infection causes damage to duck spleen and other immune organs, leading to immunosuppression and susceptibility to other pathogens. In this study, we successfully constructed a breeding duck artificial infection model and found that N-DRV infection can cause pathologic changes, such as ovarian hemorrhage, follicle atrophy, and fallopian tube bleeding in breeding ducks, resulting in significantly reduced fertilization rate and egg hatching rate. Viral RNA was present in egg vitelline membrane, duck embryo, and duckling's spleen samples, as determined through quantitative polymerase chain reaction (qPCR). Autopsy revealed obvious pathologic changes in the spleen and other organs, although there were no obvious early clinical symptoms observed in ducklings. Sequence distance and phylogenetic analysis confirmed that N-DRV-SD19 re-isolated from the spleen samples of ducklings was consistent with the strain N-DRV-XT18 used for infecting breeding ducks. The findings in this study confirmed that N-DRV can be vertically transmitted through eggs, which provide an important reference for the disease prevention and control.

Genotypic characterization and molecular evolution of avian reovirus in poultry flocks from Brazil.

Avian reovirus (ARV) is one of the main causes of infectious arthritis/tenosynovitis and malabsorption syndrome (MAS) in poultry. ARVs have been disseminated in Brazilian poultry flocks in the last years. This study aimed to genotype ARVs and to evaluate the molecular evolution of the more frequent ARV lineages detected in Brazilian poultry-producing farms. A total of 100 poultry flocks with clinical signs of tenosynovitis/MAS, from all Brazilian poultry-producing regions were positive for ARV by PCR. Seventeen bird tissues were submitted to cell culture and ARV RNA detection/genotyping by two PCRs. The phylogenetic classification was based on σC gene alignment using a dataset with other Brazilian and worldwide ARVs sequences. ARVs were specifically detected by both PCRs from the 17 cell cultures, and σC gene partial fragments were sequenced. All these sequences were aligned with a total of 451 ARV σC gene data available in GenBank. Phylogenetic analysis demonstrated five well-defined clusters that were classified into lineages I, II, III, IV, and V. Three lineages could be further divided into sub-lineages: I (I vaccine, Ia, Ib), II (IIa, IIb, IIc) and IV (IVa and IVb). Brazilian ARVs were from four lineages/sub-lineages: Ib (48.2%), IIb (22.2%), III (3.7%) and V (25.9%). The Bayesian analysis demonstrated that the most frequent sub-lineage Ib emerged in the world around 1968 and it was introduced into Brazil in 2010, with increasing spread soon after. In conclusion, four different ARV lineages are circulating in Brazilian poultry flocks, all associated with clinical diseases. RESEARCH HIGHLIGHTS One-hundred ARV-positive flocks were detected in all main poultry-producing regions from Brazil. A large dataset of 468 S1 sequences was constructed and divided ARVs into five lineages. Four lineages/sub-lineages (Ib, IIb, III and V) were detected in commercial poultry flocks from Brazil. Brazilian lineages shared a low identity with the commercial vaccine lineage (I vaccine). Sub-lineage Ib emerged around 1968 and was introduced into Brazil in 2010.

The genomic constellation of a novel duck reovirus strain associated with hemorrhagic necrotizing hepatitis and splenitis in Muscovy ducklings in Fujian, China.

The complete sequence of a reovirus, strain NP03 associated with necrotic focus formation in the liver and spleen of Muscovy ducklings in Fujian Province, China in 2009, was determined and compared with sequences of other waterfowl and chicken-origin avian reoviruses (ARVs). Sequencing of the complete genomes of strain NP03 showed that they consisted of 23,418 bp and were divided into 10 segments, ranging from 1191 bp (S4) to 3959 bp (L1) in length, and all segments contained conserved sequences in the 5' non-coding region (GCUUUU) and 3' non-coding region (UCAUC). Pairwise sequence comparisons demonstrated that NP03 strain showed the highest similarity with novel waterfowl origin reoviruses (WRVs). The genome analysis revealed that the S1 segment of novel WRV is a tricistronic gene, encoding the overlapping open reading frames (ORFs) for p10, p18, and σC, similar to the ARV S1 gene, but distinct from classical WRV S4 genome segment, which contained two overlapping ORFs encoding p10 and σC. Phylogenetic analyses of the nucleotide sequences of all 10 segments revealed that NP03 strain was clustered together with other novel WRVs and were distinct from classical WRVs and chicken-origin ARVs. The analyses also showed possible intra-segmental reassortment events in the segments encoding λA, λB, µB, µNS, σA, and σNS between novel and classical WRVs. Potential recombination events detection in segment L1 suggests that NP03 strain may be recombinants of novel WRVs. Based on our genetic analyses, multiple reassortment events, intra-segmental recombination, and accumulation of point mutations have possibly contributed to the emergence of this novel genotype of WRV, identified in China.

Molecular characterization of avian reovirus causing tenosynovitis outbreaks in broiler flocks, Iran.

Avian reoviruses (ARVs) cause arthritis, tenosynovitis, retarded growth, and malabsorption syndrome. After a long time of effective prevention and low rates of viral arthritis/ tenosynovitis in Iran, outbreaks of tenosynovitis in broiler flocks have increased in recent years. Lameness, splay legs, high rate of cull birds, poor performance, uneven birds at harvest, and condemnation at processing cause huge economic losses. In this study, ARVs from the tendons of birds from 23 broiler flocks with marked tenosynovitis were characterized, and their genetic relationship was examined. Analysis of the amino acid sequence of Sigma C protein revealed that all ARVs detected in affected broiler flocks shared genetic homogeneity and this suggests that a single genotype is involved in recent outbreaks. This genotype, so-called "Ardehal strain", is grouped in cluster I with vaccine strains. The amino acid sequence similarity between Ardehal and vaccine strains, including S1133, 1733, and 2408 was less than 80%. As the outbreaks have occurred in progenies of vaccinated flocks, it is proposed here that the difference between vaccine and field strains might contribute to the failure of currently available vaccines to induce protective immunity against Ardehal strain and this led to widespread viral tenosynovitis in Iran.

Molecular characterization of two novel reoviruses isolated from Muscovy ducklings in Guangdong, China.

BACKGROUND: Novel Muscovy duck reovirus (N-MDRV), emerged in southeast China in 2002, which can infect a wide range of waterfowl and induces clinical signs and cytopathic effects that are distinct from those of classical MDRV, and continues to cause high morbidity and 5-50% mortality in ducklings. The present study aimed to investigate the characteristics of two novel reoviruses isolated from Muscovy ducklings in Guangdong, China. RESULTS: Two novel MDRV strains, designated as MDRV-SH12 and MDRV-DH13, were isolated from two diseased Muscovy ducklings in Guangdong province, China in June 2012 and September 2013, respectively. Sequencing of the complete genomes of these two viruses showed that they consisted of 23,418 bp and were divided into 10 segments, ranging from 1191 bp (S4) to 3959 bp (L1) in length, and all segments contained conserved sequences in the 5' non-coding region (GCUUUU) and 3' non-coding region (UCAUC). Pairwise sequence comparisons demonstrated that MDRV-SH12 and MDRV-DH13 showed the highest similarity with novel MDRVs. Phylogenetic analyses of the nucleotide sequences of all 10 segments revealed that MDRV-SH12 and MDRV-DH13 were clustered together with other novel waterfowl-origin reoviruses and were distinct from classical waterfowl-origin and chicken-origin reoviruses. The analyses also showed possible genetic re-assortment events in segment M2 between waterfowl-origin and chicken-origin reoviruses and the segments encoding λA, µA, µNS, σA, and σNS between classical and novel waterfowl-origin reoviruses. Potential recombination events detection in segment S2 suggests that MDRV-SH12 and MDRV-DH13 may be recombinants of classical and novel WRVs. CONCLUSIONS: The results presented in this study, the full genomic data for two novel MDRV strains, will improve our understanding of the evolutionary relationships among the waterfowl-origin reoviruses circulating in China, and may aid in the development of more effective vaccines against various waterfowl-origin reoviruses.

Pathogenicity and genomic characterization of a novel avian orthoreovius variant isolated from a vaccinated broiler flock in China.

Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5 dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China. RESEARCH HIGHLIGHTS A variant avian orthoreovirus was isolated from a vaccinated broiler flock in North China. The ARV field strain was distinct from previous China-origin ARV isolates and vaccine strains. The current commercial ARV vaccine could not provide effective protection of broilers against the field isolate infection. These findings indicated that variant ARV field strains might become frequent in broiler flocks in China and effective measures should be conducted to prevent and control the disease.

Partial Molecular Characterization and Pathogenicity Study of an Avian Reovirus Causing Tenosynovitis in Commercial Broilers.

This study describes the molecular characterization of avian reoviruses (ARVs) isolated during an outbreak in commercial chickens between 2015 and 2016. In addition, a pathogenicity study of a selected ARV strain isolated from a field case of viral tenosynovitis in commercial broiler chickens was performed. On the basis of phylogenetic analysis of a 1088-bp fragment of the ARV S1 gene, the investigated sequences were differentiated into five distinct genotypic clusters (GCs), namely GC1, GC2, GC3, GC4, and GC6. Specific-pathogen-free (SPF) and commercial broiler chickens were challenged with the GC1 genetic type MK247011, at 14 days of age via the interdigital toe web. No significant effects in body weight gain and feed conversion were detected in both chicken types. The Δ interdigital web thickness was most severe at 4 days postchallenge (DPC) in both the SPF and broiler subgroups. The inflammation in SPF birds was slightly more severe compared with broilers. Neither mortality nor clinical signs occurred in the infected groups for the duration of the experiment, despite the presence of significant microscopic lesions in challenged birds. Microscopic changes of tenosynovitis became evident at 3 DPC, with the highest incidence and severity detected at 14 and 21 DPC, respectively. Seroconversion against ARV occurred 3 wk postchallenge, and the microscopic lesions detected in tendon and heart sections were highly compatible with those described in the field. Increased severity of tenosynovitis and epicarditis lesions were noted in the ARV-challenged groups compared with the control groups. Although SPF and broiler chickens showed comparable responses to the challenge with an ARV genetic variant, detected lesions were subclinical, denoting the limitations of our challenge approach. The age selected in this experiment possibly influenced the course of the infection. Data from this study highlight the genotypic diversity of isolates in California, and the outcome of the pathogenicity study can be used as a basis to improve protocols for pathogenicity studies to characterize ARV variants causing clinical disease in the field.

Caracterización molecular parcial y estudio de patogenicidad de un reovirus aviar que causa tenosinovitis en pollos de engorde comerciales. Este estudio describe la caracterización molecular de reovirus aviares (ARV) aislados durante un brote en pollos comerciales entre los años 2015 y 2016. Además, se realizó un estudio de patogenicidad de una cepa de reovirus seleccionada que fue aislada de un caso de campo de tenosinovitis viral en pollos de engorde comerciales. Con base en el análisis filogenético de un fragmento de 1088 pb del gene S1 de reovirus, las secuencias investigadas se diferenciaron en cinco grupos genotípicos distintos (GCs), denominados, GC1, GC2, GC3, GC4 y GC6. Aves libres de patógenos específicos (SPF) y pollos de engorde comerciales se desafiaron con el tipo genético GC1 MK247011 a los 14 días de edad a través de la membrana interdigital. No se detectaron efectos significativos en el aumento de peso corporal ni en la conversión de alimento en ambos tipos de aves. El grosor de la banda interdigital diferencial fue más severa a los cuatro días posteriores al desafío en las aves libres de patógenos específicos y en los pollos de engorde. La inflamación en las aves libres de patógenos específicos fue ligeramente más severa en comparación con los pollos de engorde. No se presentó mortalidad ni signos clínicos en los grupos infectados durante la duración del experimento, a pesar de la presencia de lesiones microscópicas significativas en las aves desafiadas. Los cambios microscópicos de la tenosinovitis se hicieron evidentes a los tres días postinoculación, con la mayor incidencia y severidad detectadas a los 14 y 21días postinoculación, respectivamente. La seroconversión para reovirus ocurrió tres semanas después del desafío, y las lesiones microscópicas detectadas en secciones de tendón y corazón fueron altamente compatibles con las descritas en el campo. El aumento en la severidad de las lesiones de tenosinovitis y epicarditis se observó en los grupos expuestos a reovirus aviar en comparación con los grupos de control. Aunque las aves libres de patógenos específicos y los pollos de engorde mostraron respuestas comparables ante el desafío con una variante genética de reovirus, las lesiones detectadas fueron subclínicas, lo que denota las limitaciones de nuestro enfoque de desafío. La edad seleccionada en este experimento posiblemente influyó en el curso de la infección. Los datos de este estudio resaltan la diversidad genotípica de los aislamientos en California y el resultado del estudio de patogenicidad se puede usar como base para mejorar los protocolos de los estudios de patogenicidad para caracterizar las variantes de reovirus que causan enfermedades clínicas en el campo.

Complete genome sequence of a novel avian orthoreovirus isolated from gosling, China.

Avian orthoreovirus (ARV) has been considered as a significant pathogen causing great infectious diseases to the avian, like broiler and waterfowl. The genome of this novel ARV(Reo/SDPY/Goose) was completely sequenced by next-generation sequencing. The complete genome was found to be 23517 bp in length with 10 segments. Although the Reo/SDPY/Goose was isolated from the gosling, it shares great similarity, no matter which segment within the genome, with those published as avian-origin reovirus. Genomic analysis revealed that this virus was distinct from published ARV strains and met criteria to become a novel ARV strain.

Genomic sequence and phylogenetic analyses of two novel orthoreovirus strains isolated from Pekin ducks in 2014 in Germany.

Complete genomic sequences of two orthoreovirus strains, D2533/4/1-10 and D2533/6/1-10, isolated from Pekin ducklings in Germany have been determined. Pairwise sequence comparisons and phylogenetic analyses indicated that strain D2533/4/1-10 might have acquired its genomic segments from three different origins, from classical and novel waterfowl reoviruses, and a yet unknown orthoreovirus strain. D2533/6/1-10 proved to be only distantly related to previously described orthoreoviruses. Reassortment, host species transmission events, and successful adaptation of novel variants may signify a challenge for animal health and maintenance of economic production.

Molecular characterization of emerging avian reovirus variants isolated from viral arthritis cases in Western Canada 2012-2017 based on partial sigma (σ)C gene.

Viral Arthritis (VA), a disease caused by Avian Reovirus (ARV), has emerged as a significant cause of economic losses in broiler chicken flocks in Western Canada. These outbreaks were characterized by 4-13% morbidity, followed by a spike in mortality/culling that in extreme cases required total flock depopulation. From 2012-2017, 38 ARV isolates were recovered. Molecular characterization of a partial segment of the sigma (σ)C gene shows all six previously known ARV clusters in Western Canadian broiler chickens. The most numerous clusters were Cluster#4 and Cluster #5 while the most variable clusters were Cluster#1 (76.7-100% identity), Cluster#2 (66-99.3%), and Cluster#4 (62-100%). This variation suggests that an autogenous vaccine may not protect against a same-cluster challenge virus. This is the first publication showing the wide genetic diversity of ARV Cluster#4, the circulation of all six worldwide reported ARV clusters in Canada, and important differences in ARV Cluster classification among researchers.

Isolation and genomic characterization of a novel avian orthoreovirus strain in Korea, 2014.

In this study, we isolated a novel avian reovirus (ARV) strain, K738/14, from a broiler chicken with viral arthritis in South Korea. Genome sequence comparisons showed relatively low nucleotide identity with previously identified ARV strains. Phylogenetic analyses suggested multiple reassortment events between reovirus strain S1133 and reoviruses of Hungarian, Chinese, and US origin had occurred. In addition, recombination analyses showed evidence of intra-segmental recombination in the M2 and S2 genes. Based on our genetic analyses, multiple reassortment events, intra-segmental recombination, and accumulation of point mutations have possibly contributed to the emergence of this novel genotype of ARV, identified in Korea.

A double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus.

We applied a probe-based real-time loop-mediated isothermal amplification (Cy5-RTqLAMP) technique targeting the avian reovirus (ARV) S3 gene to develop a rapid, sensitive, and specific method for virus detection and quantification. This test specifically detected the presence of ARV, but not other viruses or bacteria present in clinical or artificially spiked samples, including Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, Marek's disease virus, Escherichia coli, and Salmonella spp. This test can detect ARV in less than one hour with an analytical sensitivity of 10 viral gene copies and 1 fg of total cDNA. The Cy5-RTqLAMP does not yield false positive results and is 100 times more sensitive than conventional PCR. This test was shown to be able to detect the presence of ARV in clinical samples. A similar strategy may be used for detection of other important human and animal viral pathogens.

Detection and molecular characterization of enteric viruses in enteritis-affected commercial broiler chickens in India.

A study was conducted to detect and characterize the enteric viruses (chicken astrovirus, avian nephritis virus and avian orthoreovirus) present in flocks of commercial broiler chickens suffering from enteritis in Haryana, India. The intestinal contents were collected from 65 enteritis-affected flocks (cases) and tested by reverse transcription PCR (RT-PCR). Of these 65 cases, 35 (53.80%) were positive for a single virus and 26 (40.00%) for two viruses. The remaining four samples were negative for all three viruses tested. Of the 65 cases, 57 were positive for chicken astrovirus (CAstV) while 30 cases had avian nephritis virus (ANV). None of the cases were positive for orthoreovirus. Comparison of 12 CAstVs of this study with previously published CAstV sequences revealed nucleotide identities ranging from 73.20 to 98.00%. The nucleotide identities ranged between 83.10-95.50% when nine ANVs of this study were compared with previously reported ANV sequences. The amino acid sequences of CAstVs in comparison to previously published sequences revealed certain unique changes. Phylogeny based on polymerase gene revealed that CAstVs and ANVs of this study were under the same monophyletic clade. In conclusion, a large number of broiler chicken flocks experiencing enteritis were positive for CAstV and ANV by RT-PCR. The presence of more than one enteric virus in enteritis-affected flocks and changes at the genetic level in these viruses may affect the severity of disease.

Lineage diversification, homo- and heterologous reassortment and recombination shape the evolution of chicken orthoreoviruses.

The near complete genome sequences of ten field avian orthoreovirus (ARV) strains collected from young chicken between 2002 and 2011 in Hungary have been determined in order to explore the genetic diversity and evolutionary mechanisms affecting ARVs in this region. Sequence analyses and phylogenetic calculations revealed similar geographic distribution and distinct evolution in case of eight studied strains that were closely related to the recently described Hungarian strain T1781. The remaining two strains showed the highest similarity with the US origin AVS-B. The topology of the phylogenetic trees of certain segments was affected by several potential homologous reassortment events between strains of Hungarian, Chinese and US origin. Analyzing the µB gene a possible heterologous reassortment event was identified in three Hungarian strains. Recombination events were detected in as much as a dozen cases implying that beside point mutations and reassorment this mechanism also plays an important role in the diversification of ARVs. All these mechanisms in concert may explain the reduced effectiveness of immunization using commercial vaccine strains.

A duck reovirus variant with a unique deletion in the sigma C gene exhibiting high pathogenicity in Pekin ducklings.

A novel strain of duck reovirus (DRV) associated with a high mortality in Pekin ducklings in China, 2013, was isolated and characterized. This strain (designated as HN5d) grew well in Vero cells and produced marked cytopathic effects. HN5d contains 10 dsRNA genome segments, a typical feature of avian orthoreovirus. Following cloning, sequencing, and sequence analysis of the genome segments, a unique deletion of 18 amino acids was found in the sigma C protein of HN5d when compared with that of the recent Chinese waterfowl reoviruses (e.g., DRV 091). Phylogenetic analysis of cDNA amplicons of segments encoding for the outer capsid proteins revealed that HN5d is a novel genotype 2 waterfowl reovirus isolate. Inoculation of Pekin ducklings with HN5d resulted in splenic necrosis, a typical feature of "Duck spleen necrosis disease" (DSND) discovered in China in 2006. Unlike the typical DSND, HN5d produced severer hemorrhagic and/or necrotic lesions in livers of experimentally infected ducklings. 20-30% of death was observed during the first 7 day in the experimentally exposed birds. These findings suggest that HN5d is a novel duck reovirus isolate with severer pathogenicity in Pekin ducklings.

Isolation and molecular characterization of newly emerging avian reovirus variants and novel strains in Pennsylvania, USA, 2011-2014.

Avian reovirus (ARV) infections of broiler and turkey flocks have caused significant clinical disease and economic losses in Pennsylvania (PA) since 2011. Most of the ARV-infected birds suffered from severe arthritis, tenosynovitis, pericarditis and depressed growth or runting-stunting syndrome (RSS). A high morbidity (up to 20% to 40%) was observed in ARV-affected flocks, and the flock mortality was occasionally as high as 10%. ARV infections in turkeys were diagnosed for the first time in PA in 2011. From 2011 to 2014, a total of 301 ARV isolations were made from affected PA poultry. The molecular characterization of the Sigma C gene of 114 field isolates, representing most ARV outbreaks, revealed that only 21.93% of the 114 sequenced ARV isolates were in the same genotyping cluster (cluster 1) as the ARV vaccine strains (S1133, 1733, and 2048), whereas 78.07% of the sequenced isolates were in genotyping clusters 2, 3, 4, 5, and 6 (which were distinct from the vaccine strains) and represented newly emerging ARV variants. In particular, genotyping cluster 6 was a new ARV genotype that was identified for the first time in 10 novel PA ARV variants of field isolates.

Genotyping and Classification of Tunisian Strains of Avian Reovirus using RT-PCR and RFLP Analysis.

Since 1998, avian reovirus (ARV) infection has been detected in broiler and breeding chicken flocks in Tunisia. The genotype of avian reoviruses was established using simple and rapid approaches. Reverse transcription PCR (RT-PCR) on both sigma C (σC) and sigma B (σB)-encoding genes followed by restriction fragment length polymorphism (RFLP) analyses were used to better characterize Tunisian isolated strains. The RT-PCR amplified fragments of 738 and 540 bp for σC- and σB-encoding genes, respectively, of 15 ARV Tunisian strains. DNA fragments amplified from S 1133 vaccine and isolated strains were digested with different restrictions enzymes. RFLP on the σC gene indicated that the field isolates and the S 1133 vaccine strain have identical profiles when separately digested with TaqI, PstI, DdeI, and HincII. Considering the σB gene, RFLP profiles were identical with RsaI, BclI, DpnII, and NciI restriction enzymes for all the strains. However, using MseI and AciI enzymes, it was shown that all tested isolates could be clearly distinguished from the vaccine strain. ARV strains could be classified in groups with strong relatedness. Strain-typing based on cleavage site results are in agreement with ARV clustering based on nucleotide sequences of both the σC and σB genes. RT-PCR-RFLP provides a simple and a rapid approach for genotyping ARV isolates, especially when a large number of isolates are being studied. Additionally, this approach may also determine whether a new variant strain has been introduced into a flock or if a given virus strain is being spread from one flock to another.

Efficacy of Five Commonly Used Disinfectants Against Turkey Arthritis Reovirus.

Since late 2009, an unusual problem of reovirus-related lameness has been seen in market-age tom turkeys in the upper Midwest area of the United States. In this study, we determined the efficacy of five commonly used disinfectants (Virocid, Keno X5, Synergize, One Stroke, and Tek Trol) against turkey arthritis reoviruses (TARVs). For comparison, turkey enteric reovirus (TERV) and chicken arthritis reovirus (CARV) were also included. At their recommended concentrations, all five disinfectants were found to be effective virucidals, inactivating 99.99% of all viruses within 10 min. However, oxidizing agents and quaternary ammonium compounds + aldehyde types of disinfectants were more effective, killing the viruses in a shorter time (2-5 min) than the other types of disinfectants. These results indicate that these disinfectants can be an effective tool in the control of these viruses.