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.

Analysis of Chicken IFITM3 Gene Expression and Its Effect on Avian Reovirus Replication.

Interferon-inducible transmembrane protein 3 (IFITM3) is an antiviral factor that plays an important role in the host innate immune response against viruses. Previous studies have shown that IFITM3 is upregulated in various tissues and organs after avian reovirus (ARV) infection, which suggests that IFITM3 may be involved in the antiviral response after ARV infection. In this study, the chicken IFITM3 gene was cloned and analyzed bioinformatically. Then, the role of chicken IFITM3 in ARV infection was further explored. The results showed that the molecular weight of the chicken IFITM3 protein was approximately 13 kDa. This protein was found to be localized mainly in the cytoplasm, and its protein structure contained the CD225 domain. The homology analysis and phylogenetic tree analysis showed that the IFITM3 genes of different species exhibited great variation during genetic evolution, and chicken IFITM3 shared the highest homology with that of Anas platyrhynchos and displayed relatively low homology with those of birds such as Anser cygnoides and Serinus canaria. An analysis of the distribution of chicken IFITM3 in tissues and organs revealed that the IFITM3 gene was expressed at its highest level in the intestine and in large quantities in immune organs, such as the bursa of Fabricius, thymus and spleen. Further studies showed that the overexpression of IFITM3 in chicken embryo fibroblasts (DF-1) could inhibit the replication of ARV, whereas the inhibition of IFITM3 expression in DF-1 cells promoted ARV replication. In addition, chicken IFITM3 may exert negative feedback regulatory effects on the expression of TBK1, IFN-γ and IRF1 during ARV infection, and it is speculated that IFITM3 may participate in the innate immune response after ARV infection by negatively regulating the expression of TBK1, IFN-γ and IRF1. The results of this study further enrich the understanding of the role and function of chicken IFITM3 in ARV infection and provide a theoretical basis for an in-depth understanding of the antiviral mechanism of host resistance to ARV infection.

IFI16 plays a critical role in avian reovirus induced cellular immunosuppression and suppresses virus replication.

Avian reovirus (ARV), which commonly induces viral arthritis or tenosynovitis and immunosuppression in chickens, is associated with the nonstructural protein p17 that plays a crucial role in viral replication and regulates cellular signaling pathways through its interaction with cellular proteins. In our previous study, we identified the host protein IFN-γ-inducible protein-16 (IFI16) as an interacting partner of ARV p17 through yeast two-hybrid screening. In the current study, we further confirmed the interaction between IFI16 and p17 protein using coimmunoprecipitation, glutathione S-transferase (GST)-pulldown assay, and laser confocal microscopy techniques. Additionally, we found that the amino acid of p1761-119 is responsible for mediating the interaction with the HINa and HINb domains of IFI16. Interestingly, we observed a significant increase in IFI16 expression upon ARV infection or p17 protein exposure. Moreover, the replication of ARV was found to be largely influenced by the quantity of IFI16 protein. Overexpression of IFI16 led to a significant decrease in ARV replication, while knockdown of the IFI16 expression led to the contrary result. Additionally, our findings demonstrate that IFI16 plays a crucial role in the induction of inflammatory cytokines IFN-ß and IL-1ß during ARV infection as confirmed by qRT-PCR and ELISA analyses. In conclusion, our study provides novel insights into the functional role of p17 protein and the pathogenic mechanism underlying ARV infection, particularly its association with inflammatory response. Furthermore, it offers new perspectives for identifying potential therapeutic targets against ARV infection.

Identification, pathological, and genomic characterization of novel goose reovirus associated with liver necrosis in geese, China.

Since 2021, a novel strain of goose reovirus (GRV) has emerged within the goose farming industry in Guangdong province, China. This particular viral variant is distinguished by the presence of white necrotic foci primarily localized in the liver and spleen, leading to substantial economic losses for the poultry industry. However, the etiology, prevalence and genomic characteristics of the causative agent have not been thoroughly investigated. In this study, we conducted an epidemiological inquiry employing suspected GRV samples collected from May 2021 to September 2022. The macroscopic pathological and histopathological lesions associated with GRV-infected clinical specimens were examined. Moreover, we successfully isolated the GRV strain and elucidated the complete genome sequence of the isolate GD21/88. Through phylogenetic and recombination analysis, we unveiled that the GRV strains represent a novel variant resulting from multiple reassortment events. Specifically, the µNS, λC, and σNS genes of GRV were found to have originated from chicken reovirus, while the σA gene of GRV exhibited a higher degree of similarity with a novel duck reovirus. The remaining genes of GRV were traced back to Muscovy duck reovirus. Collectively, our findings underscore the significance of GRV as a pathogenic agent impacting the goose farming industry. The insights gleaned from this study contribute to a more comprehensive understanding of the epidemiology of GRV in Southern China and shed light on the genetic reassortment events exhibited by the virus.

Genetic characterization of newly emerging avian reovirus variants in chickens with viral arthritis/tenosynovitis in Israel.

In recent years, new avian reovirus (ARV) variants caused a variety of symptoms in chickens worldwide, the most important of which was Viral arthritis/tenosynovitis which caused substantial economic losses and has become a concern to the worldwide chicken industry. In this study, we characterized emerging ARV variants in Israel and analyzed their genetic relationship with reference strains. One hundred thirty-four ARV variants were isolated from tendons and synovial fluids of commercial broiler chickens with signs of arthritis/tenosynovitis. Phylogenetic analysis of the partial segment of the sigma C (σC) gene confirmed that these field isolates from Israel could be clustered into all six known clusters. The majority of ARV isolates in Israel belonged to the genotypic cluster 5 (GC5). The strains in this study had a low sequence identity when compared to the commercial vaccine (strain S1133). The findings of this study demonstrated the genetic diversity of ARV strains in Israel from 2015 to 2022. It is reasonable to conclude from the preliminary results of this investigation that Israel has not been subject to selection pressure or the emergence of new ARV variants since the introduction of the live vaccine (ISR-7585). Due to the ongoing emergence of ARV variants, a robust epidemiological monitoring program supported by molecular biology techniques is required to track ARV strains in Israeli poultry flocks.

Research Note: Genetic characterization and pathogenicity of an epidemic variant strain of avian reovirus.

The past few years have witnessed a rapid increase in cases of viral arthritis caused by avian reovirus (ARV) in chicken farms in China, attributed to the emergence of variant strains that render traditional vaccines ineffective, leading to substantial economic losses. In this study, we successfully isolated a novel ARV strain, designated as 2023ARV-GS-SDAU-1, from chickens in a broiler flock vaccinated with an ARV vaccine in Gansu province. We performed whole-genome sequencing and assessed its pathogenicity through 2 infection routes: oral administration and intraperitoneal injection. Our analysis revealed significant variations in the σA gene, associated with the inhibition of interferon secretion, compared to known ARV strains. The highest nucleotide identity observed was below 80%. Additionally, the σC gene exhibited notable variations compared to its homologous strains within the same group. Multiple alignment of the amino acid sequences classified the 2023ARV-GS-SDAU-1 strain under genotype I. Furthermore, our pathogenicity experiments indicated that the isolated strain exhibited more severe pathogenicity when administered via intraperitoneal injection in SPF chickens. In summary, our data suggest that the 2023ARV-GS-SDAU-1 strain represents a novel variant circulating in broiler flocks in China. These findings enrich currently available genetic information on ARV strains and provide a new complete genome sequence.

Transcriptomic and Translatomic Analyses Reveal Insights into the Signaling Pathways of the Innate Immune Response in the Spleens of SPF Chickens Infected with Avian Reovirus.

Avian reovirus (ARV) infection is prevalent in farmed poultry and causes viral arthritis and severe immunosuppression. The spleen plays a very important part in protecting hosts against infectious pathogens. In this research, transcriptome and translatome sequencing technology were combined to investigate the mechanisms of transcriptional and translational regulation in the spleen after ARV infection. On a genome-wide scale, ARV infection can significantly reduce the translation efficiency (TE) of splenic genes. Differentially expressed translational efficiency genes (DTEGs) were identified, including 15 upregulated DTEGs and 396 downregulated DTEGs. These DTEGs were mainly enriched in immune regulation signaling pathways, which indicates that ARV infection reduces the innate immune response in the spleen. In addition, combined analyses revealed that the innate immune response involves the effects of transcriptional and translational regulation. Moreover, we discovered the key gene IL4I1, the most significantly upregulated gene at both the transcriptional and translational levels. Further studies in DF1 cells showed that overexpression of IL4I1 could inhibit the replication of ARV, while inhibiting the expression of endogenous IL4I1 with siRNA promoted the replication of ARV. Overexpression of IL4I1 significantly downregulated the mRNA expression of IFN-ß, LGP2, TBK1 and NF-κB; however, the expression of these genes was significantly upregulated after inhibition of IL4I1, suggesting that IL4I1 may be a negative feedback effect of innate immune signaling pathways. In addition, there may be an interaction between IL4I1 and ARV σA protein, and we speculate that the IL4I1 protein plays a regulatory role by interacting with the σA protein. This study not only provides a new perspective on the regulatory mechanisms of the innate immune response after ARV infection but also enriches the knowledge of the host defense mechanisms against ARV invasion and the outcome of ARV evasion of the host's innate immune response.

Whole Genomic Constellation of Avian Reovirus Strains Isolated from Broilers with Arthritis in North Carolina, USA.

Avian reovirus (ARV) is an emerging pathogen which causes significant economic challenges to the chicken and turkey industry in the USA and globally, yet the molecular characterization of most ARV strains is restricted to a single particular gene, the sigma C gene. The genome of arthrogenic reovirus field isolates (R18-37308 and R18-38167), isolated from broiler chickens in North Carolina (NC), USA in 2018, was sequenced using long-read next-generation sequencing (NGS). The isolates were genotyped based on the amino acid sequence of sigma C (σC) followed by phylogenetic and amino acid analyses of the other 11 genomically encoded proteins for whole genomic constellation and genetic variation detection. The genomic length of the NC field strains was 23,494 bp, with 10 dsRNA segments ranging from 3959 bp (L1) to 1192 bp (S4), and the 5' and 3' untranslated regions (UTRs) of all the segments were found to be conserved. R18-37308 and R18-38167 were found to belong to genotype (G) VI based on the σC analysis and showed nucleotide and amino acid sequence identity ranging from 84.91-98.47% and 83.43-98.46%, respectively, with G VI strains. Phylogenetic analyses of individual genes of the NC strains did not define a single common ancestor among the available completely sequenced ARV strains. Nevertheless, most sequences supported the Chinese strain LY383 as a probable ancestor of these isolates. Moreover, amino acid analysis revealed multiple amino acid substitution events along the entirety of the genes, some of which were unique to each strain, which suggests significant divergence owing to the accumulation of point mutations. All genes from R18-37308 and R18-38167 were found to be clustered within genotypic clusters that included only ARVs of chicken origin, which negates the possibility of genetic pooling or host variation. Collectively, this study revealed sequence divergence between the NC field strains and reference ARV strains, including the currently used vaccine strains could help updating the vaccination regime through the inclusion of these highly divergent circulating indigenous field isolates.

Avian reovirus: a furious and fast evolving pathogen.

Avian reoviruses (ARVs) have a significant economic impact on the poultry industry, affecting commercial and backyard flocks. Spread feco-orally, or vertically, many do not cause morbidity, but pathogenic strains can contribute to several diseases, including tenosynovitis/arthritis, which is clinically the most significant. The last decade has seen a surge in cases in the US, and due to ongoing evolution, seven genotypic clusters have now been identified. Control efforts include strict biosecurity and vaccination with commercial and autogenous vaccines. Research priorities include improving understanding of pathogenesis and developing new vaccines guided by ongoing molecular and serologic surveillance.

First isolation and genomic characterization of avian reovirus from black swans (Cygnus atratus) in China.

Identification and analysis of the avian reovirus from black swan. Isolation of the strain through the chorioallantoic membrane route of duck embryos, identified through transmission electron microscopy and RT-PCR based on the ARV S2 gene. The complete genome of the ARV strain was obtained using next-generation sequencing technology. The isolated strain of ARV was named CD200801 and was identified through transmission electron microscopy and RT-PCR based on the ARV S2 gene. Experimental infection with CD200801 resulted in the death of ducklings with serious spleen and liver focal necrosis. BLAST analysis of CD200801 sequences showed a 35.5 to 98.6% similarity to a novel duck reovirus that was isolated in recent years. Phylogenetic analysis revealed that CD200801 was closely related to ARV isolates YL, GX-Y7, and XT-18. We report the first avian reovirus infection in the black swan. This study provides important new insights into the evolutionary relationships among different ARV strains and highlights the need for continued surveillance and monitoring of these viruses in both domestic and wild bird flocks. These findings have significant implications for the development of effective strategies for disease prevention and control in the poultry industry.

Synergistic pathogenicity of avian orthoreovirus and Staphylococcus aureus on SPF chickens.

Avian arthritis is a relatively common disease in the poultry industry, the cause of which is complex. Bacterial arthritis is often caused by infection of Staphylococcus aureus (S. aureus), whereas viral arthritis is caused by avian orthoreovirus (ARV). To investigate the infection of S. aureus and ARV in cases of avian arthritis, a total of 77 samples characterized by arthritis were collected and detection. The results showed that 68.83% of the samples were positive for ARV, and 66.23% were positive for S. aureus. Among them, the ARV mono-infection rate was 22.08%, the S. aureus mono-infection rate was 19.48%, and ARV and S. aureus co-infection rate was 45.45%, indicating that ARV and S. aureus co-infection is common in arthritis cases. To further investigate the synergistic pathogenicity of ARV and S. aureus, ARV and S. aureus were used to mono-infect, co-infect, and (or) sequential infect SPF chickens and the clinical indications, pathologic changes, ARV load, S. aureus bacterial distribution, and cytokine level of the challenged chickens were evaluated. Decreased weight gain, increased mortality, and difficulties in standing were observed in all dual-infected groups and the singular-infected group. There were significantly more severe macroscopic and microscopic hock lesions, and larger amounts of a wider range of tissue distribution of ARV antigens and S. aureus bacterial distribution in the dual-infected groups compared to the single-infected and control groups. Cytokine detection showed a significant change in IFN-γ, IL-1ß, and IL-6 levels in the infected groups, especially in the ARV-S. aureus co-infection, and (or) sequential infection groups, compared with the control group. Hence, ARV and S. aureus synergistically increased mortality in infected chickens, potentiated the severity of arthritis, and increased the amount of ARV RNA in tendons.

Research Note: Genetic characterization of novel duck reoviruses from Shandong Province, China in 2022.

Since 2005, novel duck reoviruses have been outbreaks in duck breeding areas such as central China and South China. In recent years, the incidence rate of this disease is still increasing, bringing serious economic losses to waterfowl breeding industry. This study isolated 3 novel duck reoviruses (NDRV-SDLS, NDRV-SDWF, and NDRV-SDYC) from sick ducks in 3 local duck farms in Shandong Province. The study aimed to investigate the characteristics of these viruses. The virus is inoculated into duck embryo fibroblasts, where the virus replicates to produce syncytium and dies within 3 to 5 d. The viruses were also isolated from infected ducks, and RT-PCR amplified the whole genomes after passage purification in duck embryos. The resulting whole genome was analyzed for genetic evolution. The total length of the gene sequencing was 23,418 bp, divided into 10 fragments. Gene sequence comparison showed that the 3 strains had high similarity with novel duck reoviruses (NDRV) but low similarity with chicken-origin reovirus (chicken ARV) and Muscovy duck reovirus (MDRV), especially in the σC segment. Phylogenetic analysis of the 10 fragments showed that the 3 isolates constituted the same evolutionary clade as other DRV reference strains and were far related to ARV and MDRV in different evolutionary clades. The results of all 10 segments indicate that the isolates are in the evolutionary branch of NDRV, suggesting that the novel waterfowl reovirus is the dominant circulating strain in Shandong. This study complements the gene bank information of NDRV and provides references for vaccine research and disease prediction of NDRV in Shandong.

Nonenveloped Avian Reoviruses Released with Small Extracellular Vesicles Are Highly Infectious.

Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence.

Molecular characterization, complete genome sequencing, and pathogenicity of Novel Duck Reovirus from South Coastal Area in China.

Novel Duck Reovirus (NDRV) that has been found throughout the world in waterfowl, and it has been extensively described. Here, we report the complete genome sequence of a NDRV strain isolated in China called NDRV YF10. This strain was collected from 87 samples with infected ducks in South Coastal Area. The NDRV genome consists of 23,419 bp. With the assistance of computer analysis, the promoter and terminator of each gene segment and 10 viral genes segments were identified, which encode polypeptides ranging from 98 to 1,294 amino acids. All gene fragments of this virus strain were determined and compared to previously reported strains, revealing genetic variation with similarity rates ranging from 96 to 99% for each gene segment. Each gene segment formed 2 host-associated groups, the waterfowl-derived reovirus and the avian-derived reovirus, except for the S1 gene segment, which was closely related to ARV evolution and formed a host-independent subcluster. This difference may be due to Avian Reovirus (ARV) evolving in a host-dependent manner. In order to evaluate the pathogenicity of YF10, a novel isolated strain of NDRV was tested in 2 types of ducks. It was observed that the YF10 isolated strain exhibits varying degrees of virulence, highlighting the potential risk posed to different types of ducks. In conclusion, our findings emphasize the importance of epidemiology studies, molecular characterization, and prevention of NDRV in waterfowl.

First Seroprevalence Survey of Avian Reovirus in Broiler Breeders Chicken Flocks in Morocco.

Avian reovirus (ARV) is a prevalent infectious agent that has the potential to cause respiratory and gastrointestinal illnesses in poultry, leading to substantial financial losses in the poultry sector. Until now, there have been no investigations conducted to examine the epidemiological status of ARV infections in Morocco. The aim of this study was to investigate the seroprevalence of ARV infections with respect to area, types of chickens (broiler breeder, and broiler), vaccination status, and age of chickens. A total of 826 serum samples were collected from 36 broiler and broiler breeder flocks, with 14 of them being unvaccinated, fromsix different regions of Morocco, namely Casablanca-Settat, Rabat-Salé-Kénitra, Tanger-Tétouan-Al Hoceïma, Oriental, Marrakech-Safi, and Fez-Meknès between 2021 and 2022.These serum samples were screened using a commercial indirect ELISA ARV antibody test kit (IDEXX REO). The study found that all tested flocks were positive for ARV-specific antibodies, indicating that the virus was present in these flocks. Out of the 826 serum samples tested, 782 were positive for ARV-specific antibodies. The overall prevalence of ARV infections in breeder and broiler flocks was calculated to be 94.6% ± 0.78. To summarize, the current study provides evidence of the widespread distribution of ARV infections in Morocco, suggesting that the poultry industry in the country is highly infected with ARV.

PQBP1 regulates the cellular inflammation induced by avian reovirus and interacts with the viral p17 protein.

Avian reovirus (ARV) can commonly infect a flock and cause immunosuppressive diseases in poultry. The nonstructural protein p17 is involved in viral replication, and significant progress has been made in showing its ability to regulate cellular signaling pathways. In our previous study, to further investigate the effect of ARV p17 protein on viral replication, the host protein polyglu-tamine binding protein 1 (PQBP1) was identified to interact with p17 by a yeast two-hybrid system. In the current study, the interaction between PQBP1 and p17 protein was further confirmed by laser confocal microscopy and coimmunoprecipitation assays. In addition, the N-terminal WWD of PQBP1 was found to mediate the process of binding to the p17 protein. Interestingly, we found that ARV infection significantly inhibited PQBP1 expression. While the quantity of ARV replication was largely influenced by PQBP1, PQBP1 overexpression decreased ARV replication. In contrast, upon PQBP1 knockdown, the quantity of ARV was notably increased. ARV infection and p17 protein expression were both proven to induce PQBP1 to mediate cellular inflammation. In the current study, we revealed through qRT‒PCR, ELISA and Western blotting methods that PQBP1 plays a positive role in ARV-induced inflammation. Furthermore, the mechanism of this process was shown to involve the NFκB-dependent transcription of inflammatory genes. In addition, PQBP1 was shown to regulate the phosphorylation of p65 protein. In conclusion, this research provides clues to elucidating the function of the p17 protein and the pathogenic mechanism of ARV, especially the cause of the inflammatory response. It also provides new ideas for the study of therapeutic targets of ARV.

Cell Entry of Avian Reovirus Modulated by Cell-Surface Annexin A2 and Adhesion G Protein-Coupled Receptor Latrophilin-2 Triggers Src and p38 MAPK Signaling Enhancing Caveolin-1- and Dynamin 2-Dependent Endocytosis.

The specifics of cell receptor-modulated avian reovirus (ARV) entry remain unknown. By using a viral overlay protein-binding assay (VOPBA) and an in-gel digestion coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we determined that cell-surface annexin A2 (AnxA2) and adhesion G protein-coupled receptor Latrophilin-2 (ADGRL2) modulate ARV entry. Direct interaction between the ARV σC protein and AnxA2 and ADGRL2 in Vero and DF-1 cells was demonstrated in situ by proximity ligation assays. By using short hairpin RNAs (shRNAs) to silence the endogenous AnxA2 and ADGRL2 genes, ARV entry could be efficiently blocked. A significant decrease in virus yields and the intracellular specific signal for σC protein was observed in Vero cells preincubated with the specific AnxA2 and ADGRL2 monoclonal antibodies, indicating that AnxA2 and ADGRL2 are involved in modulating ARV entry. Furthermore, we found that cells pretreated with the AnxA2/S100A10 heterotetramer (A2t) inhibitor A2ti-1 suppressed ARV-mediated activation of Src and p38 mitogen-activated protein kinase (MAPK), demonstrating that Src and p38 MAPK serve as downstream molecules of cell-surface AnxA2 signaling. Our results reveal that suppression of cell-surface AnxA2 with the A2ti-1 inhibitor increased Csk-Cbp interaction, suggesting that ARV entry suppresses Cbp-mediated relocation of Csk to the membrane, thereby activating Src. Furthermore, reciprocal coimmunoprecipitation assays revealed that σC can interact with signaling molecules, lipid raft, and vimentin. The current study provides novel insights into cell-surface AnxA2- and ADGRL2-modulated cell entry of ARV which triggers Src and p38 MAPK signaling to enhance caveolin-1-, dynamin 2-, and lipid raft-dependent endocytosis. IMPORTANCE By analyzing results from VOPBA and LC-MS/MS, we have determined that cell-surface AnxA2 and ADGRL2 modulate ARV entry. After ARV binding to receptors, Src and p38 MAPK signaling were triggered and, in turn, increased the phosphorylation of caveolin-1 (Tyr14) and upregulated dynamin 2 expression to facilitate caveolin-1-mediated and dynamin 2-dependent endocytosis. In this work, we demonstrated that ARV triggers Src activation by impeding Cbp-mediated relocation of Csk to the membrane in the early stages of the life cycle. This work provides better insight into cell-surface AnxA2 and ADGRL2, which upregulate Src and p38MAPK signaling pathways to enhance ARV entry and productive infection.

Oncolytic Avian Reovirus σA-Modulated Upregulation of the HIF-1α/C-myc/glut1 Pathway to Produce More Energy in Different Cancer Cell Lines Benefiting Virus Replication.

Our previous reports proved that the structural protein σA of avian reovirus (ARV) is an energy activator which can regulate cellular metabolism that is essential for virus replication. This study has further demonstrated that the ARV protein σA is able to upregulate the HIF-1α/myc/glut1 pathway in three cancer cell lines (A549, B16-F10, and HeLa) to alter the metabolic pathway of host cells. Quantitative real-time RT-PCR and Western blotting results have revealed that σA protein could enhance both mRNA and the protein levels of HIF-1α, c-myc, and glut1 in these cancer cell lines. In this work, ATeam immunofluorescence staining was used to reveal that knockdown of HIF-1α, c-myc, and glut1 by shRNAs decreased cellular ATP levels. Our data reveal that the ARV σA protein can downregulate lactate fermentation and upregulate glutaminolysis. The σA protein upregulates glutaminase, which converts glutamate into the TCA cycle intermediate α-ketoglutarate, activating the TCA cycle. In the lactate fermentation pathway, ARV σA protein suppresses lactate dehydrogenase A (LDHA), implying the Warburg effect does not occur in these cancer cell lines. This study provides a novel finding revealing that ARV σA protein upregulates glycolysis and glutaminolysis to produce energy using the HIF-1α/c-myc/glut1 pathway to benefit virus replication in these cancer cell lines.

Recombinant characteristics, pathogenicity, and transmissibility of a variant goose orthoreovirus derived from inter-lineage recombination.

Since March 2021, an infectious characterized by white necrotic foci throughout the goose body has appeared in the major goose-producing regions in China. This disease has caused economic hardship for goose farms in many regions of China with approximately 50 % mortality. A novel goose-origin orthoreovirus was isolated from the spleen of diseased geese and designated as N-GRV/HN/Goose/2021/China (N-GRV-HN21) strain. Next-generation sequencing and phylogenetic analysis revealed that the isolate was a reassortant virus containing viral gene segments from three ARV serotypes that infect duck, muscovy duck, and goose. Geese infection test showed that both N-GRV-HN21-infected and contacted geese displayed whole-body white necrotic foci. N-GRV RNA was detected in different organs of both infected and contacted geese, indicating that the N-GRV isolate is pathogenic and transmissible in geese. Seroconversion was also observed in experimentally infected and contacted geese. A prevalence study of 323 goose serum samples collected from different goose breeding areas showed that 86 % of the geese were positive for N-GRV. In conclusion, all results warrant the necessity to monitor orthoreovirus epidemiology and reassortment as the orthoreovirus could be an important pathogen for the waterfowl industry and a novel orthoreovirus might emerge to threaten animal and public health.