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.

Diagnostic investigation of avian reovirus field variants circulating in broiler chickens in Pennsylvania of United States between 2017 and 2022.

Avian reovirus (ARV) has been continuously affecting the poultry industry in Pennsylvania (PA) in recent years. This report provides our diagnostic investigation on monitoring ARV field variants from broiler chickens in Pennsylvania. Genomic characterization findings of 72 ARV field isolates obtained from broiler cases during the last 6 years indicated that six distinct cluster variant strains (genotype I-VI), which were genetically diverse and distant from the vaccine and vaccine-related field strains, continuously circulated in PA poultry. Most of the variants clustered within genotype V (24/72, 33.3%), followed by genotype II (16/72, 22.2%), genotype IV (13/72, 18.1%), genotype III (13/72, 18.1%), genotype VI (05/72, 6.94%), and genotype I (1/72, 1.38%). The amino acid identity between 72 field variants and the vaccine strains (1133, 1733, 2408, 2177) varied from 45.3% to 99.7%, while the difference in amino acid counts ranged from 1-164. Among the field variants, the amino acid identity and count difference ranged from 43.3% to 100% and 0 to 170, respectively. Variants within genotype V had maximum amino acid identity (94.7-100%), whereas none of the variants within genotypes II and VI were alike. These findings indicate the continuing occurrence of multiple ARV genotypes in the environment.

Emerging new avian reovirus variants from cases of enteric disorders and arthritis/tenosynovitis in Brazilian poultry flocks.

1. The objective of this study was to characterise circulating Brazilian avian reovirus (ARV) strains by genetic analysis of the σC protein encoded by segment 1 of the viral genome and compare these with those of viral strains used for immunising commercial poultry.2. The analysis detected the presence of ARV genomes by quantitative reverse transcriptase PCR (RT-qPCR) in the enteric samples and the joint tissues (JT) of birds with signs of viral arthritis/tenosynovitis. Nucleotide sequencing used 16 strains (three commercial vaccines, 10 from enteric tissues and three from JT). The results indicated high variability in the amino acid sequences of 13 wild strains, showing between 40% and 75% similarity compared with the vaccine strains (S1133 and 2177).3. The sequences were grouped into three well-defined clusters in a phylogenetic tree, two of these clusters together with previous Brazilian σC ARV sequences, and one cluster (VII) that was novel for Brazilian strains. Antigenic analysis showed that there were amino acids within putative epitopes located on the surface of the receptor-binding region of the σC protein with a high degree of variability.4. The study confirmed the presence of ARV genetic variants circulating in commercial birds in Brazil, and according to the antigenic prediction, the possibility of antigenic variants appears to be high.

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.

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.

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.

Stability of local secondary structure determines selectivity of viral RNA chaperones.

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

Characterization of a P18 protein in the S1 segment of the novel duck reovirus genome.

Novel duck reovirus (NDRV), the prototype strain of avian orthoreoviruses, continues to circulate among ducks. Analysis of its genome suggested that a putative second open reading frame in the S1 segment encodes a 162-amino acid nonstructural protein with size of 18 kDa, provisionally designated P18. This protein is different from the 17 kDa nonstructural protein encoded in the same open reading frame in other avian orthoreoviruses, which is designated P17 and consists of 146 amino acids. There is no corresponding protein in Muscovy duck reovirus. Antibodies raised to the purified recombinant protein reacted with viral P18 both in vitro and in vivo. In cells, P18 was located predominantly in the nucleus at 6-12 h post-infection, with negligible levels in the cytoplasm. However, the protein accumulated both in the nucleus and cytoplasm at 24 to 36 h post-infection. Immunohistochemistry indicated that P18 strongly accumulates in spleen tissues of infected ducklings. Collectively, the data provide the direct experimental evidence that P18 is expressed by novel duck reovirus both in vivo and in vitro. Keywords: duck reovirus; expression; characterization; novel P18 protein.

First report of seroprevalence and genetic characterization of avian orthoreovirus in Egypt.

Recently, the Egyptian broiler industry has experienced an increased incidence of avian reovirus (ARV) infections. However, to date, no studies have been carried out to investigate the epidemiologic status of ARV infections as well as the genetic characteristics of the currently circulating ARV strains. The present study estimates the seroprevalence of ARV infections in Alexandria, El-Behera, Giza, Kafr El-Sheikh, and Gharbia governorates, Egypt, during the period 2017-2018. A total of 150 serum samples from 15 unvaccinated broiler flocks with suspicious ARV infection were screened using a commercial enzyme-linked immunosorbent assay kit. All the tested flocks were found to be positive for ARV-specific antibodies, and the overall seropositivity rate was 80.6%. Meanwhile, 5 (33.3%) flocks were confirmed for the presence of ARV through a reverse transcription-polymerase chain reaction (RT-PCR) assay based on the σA-encoding gene. Phylogenetic analysis based on the nucleotide sequences of the σA-encoding gene revealed that the obtained ARV isolate, designated EGY1, was grouped in the S1113-like cluster of ARV and displayed 100% and 98.7% nucleotide identity with the Chinese MSO1 isolate and the S1133 vaccine strain, respectively. In addition, amino acid alignments with the S1133 vaccine strain revealed that the σA protein of the EGY1 isolate carried the substitutions G81S and A118V. In conclusion, the present study provides the evidence for a ubiquitous distribution of ARV infection in Egypt as well as represents a starting point for genetic characterization of the currently circulating ARV strains.

The E3 Ubiquitin Ligase Siah-1 Suppresses Avian Reovirus Infection by Targeting p10 for Degradation.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. The ARV p10 protein, a viroporin responsible for the induction of cell syncytium formation and apoptosis, is rapidly degraded in host cells. Our previous report demonstrated that cellular lysosome-associated membrane protein 1 (LAMP-1) interacted with p10 and was involved in its degradation. However, the molecular mechanism underlying LAMP-1-mediated p10 degradation remains elusive. We report here that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) is critical for p10 ubiquitylation. Our data show that Siah-1 ubiquitylated p10 and targeted it for proteasome degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 promotes the proteasomal degradation of p10 via interacting with both p10 and the E3 ligase Siah-1. These data establish a novel host defense mechanism where LAMP-1 serves as a scaffold for both Siah-1 and p10 that allows the E3 ligase targeting p10 for ubiquitylation and degradation to suppress ARV infection.IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. The ARV p10 protein is a virulence factor responsible for the induction of cell syncytium formation and apoptosis and is rapidly degraded in host cells. We previously found that cellular lysosome-associated membrane protein 1 (LAMP-1) interacts with p10 and is involved in its degradation. Here we report that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) ubiquitylated p10 and targeted it for proteasomal degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 serves as an adaptor to allow Siah-1 to target p10 for degradation, thereby suppressing ARV growth in host cells.

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.

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.

Construction and characterization of an infectious molecular clone of novel duck reovirus.

Novel duck reovirus (NDRV), the prototype strain of the species Avian orthoreovirus (ARV), is currently an infectious agent for ducks. Studies on NDRV replication and pathogenesis have been hampered by the lack of an available reverse-genetics system. In this study, a plasmid-based reverse-genetics system that is free of helper viruses has been developed. In this system, 10 full-length gene segments of wild-type NDRV TH11 strain are transfected into BSR-T7/5 cells that express bacteriophage T7 RNA polymerase. Production of infectious virus was shown by the inoculation of cell lysate derived from transfected cells into 10-day-old duck embryos. The in vivo growth kinetics and infectivity of the recombinant strains were identical to those of the wild-type strain. These viruses grew well and were genetically stable both in vitro and in vivo. Altogether, these results show the successful production of an infectious clone for NDRV. The infectious clone reported will be further used to elucidate the mechanisms of host tropism, viral replication and pathogenesis, as well as immunological changes induced by NDRV.

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.

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.

Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA-RNA interactions between genomic precursors during segment assortment and packaging.

A critical role of LAMP-1 in avian reovirus P10 degradation associated with inhibition of apoptosis and virus release.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. The ARV p10 protein, a viroporin responsible for the induction of cell syncytium formation and apoptosis, is rapidly degraded in host cells. However, the mechanism of p10 degradation and its relevance are still unclear. We report here the identification of cellular lysosome-associated membrane protein 1 (LAMP-1) as an interaction partner of p10 by yeast two-hybrid screening, immunoprecipitation and confocal microscopy assays. We found that rapid degradation of p10 was associated with ubiquitination. Importantly, ARV p10 degradation in host cells could be completely abolished by knockdown of LAMP-1 by siRNA, indicating that LAMP-1 is required for ARV p10 degradation in host cells. In contrast, overexpression of LAMP-1 facilitated p10 degradation. Furthermore, knockdown of LAMP-1 allowed p10 accumulation, enhancing p10-induced apoptosis and viral release. Thus, LAMP-1 plays a critical role in ARV p10 degradation associated with inhibition of apoptosis and viral release.

Avian reovirus σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent Akt signalling pathway.

The present study was conducted to identify avian reovirus (ARV) proteins that can activate the phosphatidylinositol 3-kinase (PI3K)-dependent Akt pathway. Based on ARV protein amino acid sequence analysis, σA, σNS, µA, µB and µNS were identified as putative proteins capable of mediating PI3K/Akt pathway activation. The recombinant plasmids σA-pcAGEN, σNS-pcAGEN, µA-pcAGEN, µB-pcAGEN and µNS-pcAGEN were constructed and used to transfect Vero cells, and the expression levels of the corresponding genes were quantified by immunofluorescence and Western blot analysis. Phosphorylated Akt (P-Akt) levels in the transfected cells were measured by flow cytometry and Western blot analysis. The results showed that the σA, σNS, µA, µB and µNS genes were expressed in Vero cells. σA-expressing and σNS-expressing cells had higher P-Akt levels than negative control cells, pcAGEN-expressing cells and cells designed to express other proteins (i.e., µA, µB and µNS). Pre-treatment with the PI3K inhibitor LY294002 inhibited Akt phosphorylation in σA- and σNS-expressing cells. These results indicate that the σA and σNS proteins can activate the PI3K/Akt pathway.

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.