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1.
Viruses ; 14(11)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36366482

RESUMO

Avian reoviruses (ARV) are a group of poultry pathogens that cause runting and stunting syndrome (RSS), a condition otherwise known as "frozen chicken", which are characterized by dramatically delayed growth in broilers. It has been known that p17, a nonstructural protein encoded by ARV, prohibits cellular proliferation by halting the cell cycle at the G2/M phase, the result of which is directly associated with the typical clinical sign of RSS. Nevertheless, the mechanism by which p17 modulates cell-cycle progression remains largely unknown. Here, we screened the interactome of ectopically expressed p17 through a yeast two-hybrid assay and identified Bub3, a cellular mitotic checkpoint protein, as a binding partner of p17. The infection of the Vero cells by ARV downregulated the Bub3 expression, while the knockdown of Bub3 alleviated the p17-modulated cell-cycle arrest during ARV infection. Remarkably, the suppression of Bub3 by RNAi in the Vero cells significantly reduced the viral mRNA and protein abundance, which eventually led to diminished virus replication. Altogether, our findings reveal that ARV p17 impedes host cell proliferation through a Bub3-dependent cell-cycle arrest, which eventually contributes to efficient virus replication. These results also unveil a hitherto unknown therapeutic target for RSS.


Assuntos
Orthoreovirus Aviário , Infecções por Reoviridae , Chlorocebus aethiops , Animais , Células Vero , Galinhas , Ciclo Celular , Divisão Celular
2.
Viruses ; 14(10)2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36298756

RESUMO

Caveolin-1 (Cav-1) is the basic component of caveolae, a specialized form of lipid raft that plays an essential role in endocytic viral entry. However, the evidence of direct involvement of caveolae and Cav-1 in avian reovirus (ARV) entry remains insufficient. In this study, the membrane lipid rafts were isolated as detergent-resistant microdomains (DRMs) by sucrose gradient centrifugation, and the capsid protein σB of ARV was found to associate with Cav-1 in DRMs fractions. Additionally, the interaction between ARV σB protein and Cav-1 was demonstrated by immunofluorescence co-localization and co-immunoprecipitation assays. Furthermore, we found that the internalization of ARV is sensitive to caveolae and dynamin inhibitors, while it is insensitive to clathrin inhibitors. In conclusion, these results indicate that the ARV σB protein interacts with Cav-1 during dynamin-dependent caveolae-mediated endocytosis for the entry of ARV.


Assuntos
Caveolina 1 , Orthoreovirus Aviário , Caveolina 1/metabolismo , Cavéolas/metabolismo , Detergentes , Proteínas do Capsídeo/metabolismo , Microdomínios da Membrana/metabolismo , Endocitose , Clatrina/metabolismo , Dinaminas/metabolismo , Sacarose/metabolismo , Lipídeos de Membrana/metabolismo
3.
J Virol Methods ; 310: 114613, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36087792

RESUMO

Avian reovirus (ARV) is a common pathogen in chickens and other birds causing a variety of clinical symptoms such as arthritis and tenosynovitis but also enteric and respiratory symptoms. A rapid method that detects as many ARV genotypes as possible, will contribute to the early identification and control of the virus infection that causes high economic damage to the poultry industry worldwide. In this study, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay for the detection of ARV was developed. The RT-qPCR detection threshold for ARV genomic RNA standard cases was 10 copies/µL. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assays. When the nucleic acids of different ARV genotypes and other common avian pathogens (IBDV, AIV, NDV, and IBV) were subjected to that RT-qPCR test, only ARV samples tested positive while all other pathogens tested negative. Due to the simplicity, convenience, high sensitivity, and specificity of the assay, the probe-based RT-qPCR is proposed to be used as an alternative diagnostic assay for the detection of ARVs in veterinary diagnostic laboratories.


Assuntos
Ácidos Nucleicos , Orthoreovirus Aviário , Doenças das Aves Domésticas , Infecções por Reoviridae , Animais , Orthoreovirus Aviário/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Reprodutibilidade dos Testes , Galinhas , Doenças das Aves Domésticas/diagnóstico , Infecções por Reoviridae/diagnóstico , Infecções por Reoviridae/veterinária , Sensibilidade e Especificidade , RNA
4.
Vet Microbiol ; 273: 109545, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35998542

RESUMO

We have demonstrated previously that the σA protein of avian reovirus (ARV) functions as an activator of cellular energy, which upregulates glycolysis and the TCA cycle for virus replication. To date, there is no report with respect to σA-modulated regulation of cellular fatty acid metabolism. This study reveals that the σA protein of ARV inhibits fatty acids synthesis and enhance fatty acid oxidation by upregulating PSMB6, which suppresses Akt, sterol regulatory element-binding protein 1 (SREBP1), acetyl-coA carboxylase α (ACC1), and acetyl-coA carboxylase ß (ACC2). SREBP1 is a transcription factor involved in fatty acid and cholesterol biosynthesis. Overexpression of SREBP1 reversed σA-modulated suppression of ACC1 and ACC2. In this work, a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams, was used to study σA-modulated inhibition of fatty acids synthesis which enhances cellular ATP levels in Vero cells and human cancer cell lines (A549 and HeLa). By using Ateams, we demonstrated that σA-modulated inhibition of Akt, SREBP1, ACC1, and ACC2 leads to increased levels of ATP in mammalian and human cancer cells. Furthermore, knockdown of PSMB6 or overexpression of SREBP1 reversed σA-modulated increased levels of ATP in cells, indicating that PSMB6 and SREBP1 play important roles in ARV σA-modulated cellular fatty acid metabolism. Furthermore, we found that σA R155/273A mutant protein loses its ability to enter the nucleolus, which impairs its ability to regulate fatty acid metabolism and does not increase ATP formation, suggesting that nucleolus entry of σA is critical for regulating cellular fatty acid metabolism to generate more energy for virus replication. Collectively, this study provides novel insights into σA-modulated inhibition of fatty acid synthesis and enhancement of fatty acid oxidation to produce more energy for virus replication through the PSMB6/Akt/SREBP1/ACC pathway.


Assuntos
Orthoreovirus Aviário , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Trifosfato de Adenosina , Animais , Chlorocebus aethiops , Ácidos Graxos/metabolismo , Humanos , Mamíferos , Orthoreovirus Aviário/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Células Vero , Replicação Viral
5.
J Virol ; 96(17): e0083622, 2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-35946936

RESUMO

The mechanism by which avian reovirus (ARV)-modulated suppression of mTORC1 triggers autophagy remains largely unknown. In this work, we determined that p17 functions as a negative regulator of mTORC1. This study suggest novel mechanisms whereby p17-modulated inhibition of mTORC1 occurs via upregulation of p53, inactivation of Akt, and enhancement of binding of the endogenous mTORC1 inhibitors (PRAS40, FKBP38, and FKPP12) to mTORC1 to disrupt its assembly and accumulation on lysosomes. p17-modulated inhibition of Akt leads to activation of the downstream targets PRAS40 and TSC2, which results in mTORC1 inhibition, thereby triggering autophagy and translation shutoff, which is favorable for virus replication. p17 impairs the interaction of mTORC1 with its activator Rheb, which promotes FKBP38 interaction with mTORC1. It is worth noting that p17 activates ULK1 and Beclin1 and increases the formation of the Beclin 1/class III PI3K complex. These effects could be reversed in the presence of insulin or depletion of p53. Furthermore, we found that p17 induces autophagy in cancer cell lines by upregulating the p53/PTEN pathway, which inactivates Akt and mTORC1. This study highlights p17-modulated inhibition of Akt and mTORC1, which triggers autophagy and translation shutoff by positively modulating the tumor suppressors p53 and TSC2 and endogenous mTORC1 inhibitors. IMPORTANCE The mechanisms by which p17-modulated inhibition of mTORC1 induces autophagy and translation shutoff is elucidated. In this work, we determined that p17 serves as a negative regulator of mTORC1. This study provides several lines of conclusive evidence demonstrating that p17-modulated inhibition of mTORC1 occurs via upregulation of the p53/PTEN pathway, downregulation of the Akt/Rheb/mTORC1 pathway, enhancement of binding of the endogenous mTORC1 inhibitors to mTORC1 to disrupt its assembly, and suppression of mTORC1 accumulation on lysosomes. This work provides valuable information for better insights into p17-modulated inhibition of mTORC1, which induces autophagy and translation shutoff to benefit virus replication.


Assuntos
Lisossomos , Alvo Mecanístico do Complexo 1 de Rapamicina , Orthoreovirus Aviário , Proteínas Adaptadoras de Transdução de Sinal , Autofagia , Linhagem Celular Tumoral , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Orthoreovirus Aviário/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a Tacrolimo , Proteína 2 do Complexo Esclerose Tuberosa , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
Transbound Emerg Dis ; 69(5): e3386-e3392, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35810357

RESUMO

A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production. Histopathological lesions were seen in the oviduct and in the lungs of birds sent for diagnostic investigation. The fusogenic agent was characterized as an orthoreovirus by viral metagenomics. The assembled viral genome was composed of 10 genomic segments and was 23,433 nucleotides (nt) in length. The study strain, designated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%-72.33%) as well as with representative strains of neoavian orthoreoviruses (nt, 40.07%-68.23%). On the contrary, the study strain shared 86.48%-95.01% pairwise nt sequence identities with recent German and Chinese reovirus isolates, D2533/6 and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl pathogens on a monophyletic branch, indicating a common evolutionary origin of Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the past few years. The finding that a candidate new orthoreovirus species, tentatively called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in moribund ducks seems an alarming sign that needs to be better evaluated by extending laboratory diagnosis of viral pathogens in countries where the waterfowl industry is important.


Assuntos
Orthoreovirus Aviário , Orthoreovirus , Infecções por Reoviridae , Animais , Aves , Patos , Genoma Viral , Nucleotídeos , Orthoreovirus/genética , Orthoreovirus Aviário/genética , Filogenia , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/veterinária , Análise de Sequência de DNA/veterinária
7.
J Virol ; 96(14): e0075922, 2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35867570

RESUMO

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. In this study, we show that ARV infection markedly increased gga-miR-30c-5p expression in DF-1 cells and that transfection of cells with gga-miR-30c-5p inhibited ARV replication while knockdown of endogenous gga-miR-30c-5p enhanced viral growth in cells. Importantly, we identified the autophagy related 5 (ATG5), an important proautophagic protein, as a bona fide target of gga-miR-30c-5p. Transfection of DF-1 cells with gga-miR-30c-5p markedly reduced ATG5 expression accompanied with reduced conversion of ARV-induced-microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, an indicator of autophagy in host cell, while knockdown of endogenous gga-miR-30c-5p enhanced ATG5 expression as well as ARV-induced conversion of LC3-II, facilitating viral growth in cells. Furthermore, knockdown of ATG5 by RNA interference (RNAi) or treatment of cells with autophagy inhibitors (3-MA and wortmannin) markedly reduced ARV-induced LC3-II and syncytium formation, suppressing viral growth in cells, while overexpression of ATG5 increased ARV-induced LC3-II and syncytium formation, promoting viral growth in cells. Thus, gga-miR-30c-5p suppressed viral replication by inhibition of ARV-induced autophagy via targeting ATG5. These findings unraveled the mechanism of how host cells combat against ARV infection by self-encoded small RNA and furthered our understanding of the role of microRNAs in host response to pathogenic infection. IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, and retarded growth, leading to considerable economic losses to the poultry industry across the globe. Elucidation of the pathogenesis of ARV infection is crucial to guiding the development of novel vaccines or drugs for the effective control of these diseases. Here, we investigated the role of miRNAs in host response to ARV infection. We found that infection of host cells by ARV remarkably upregulated gga-miR-30c-5p expression. Importantly, gga-miR-30c-5p suppressed ARV replication by inhibition of ARV-induced autophagy via targeting autophagy related 5 (ATG5) accompanied by suppression of virus-induced syncytium formation, thus serving as an important antivirus factor in host response against ARV infection. These findings will further our understanding of how host cells combat against ARV infection by self-encoded small RNAs and may be used as a potential target for intervening ARV infection.


Assuntos
Artrite Infecciosa , MicroRNAs , Orthoreovirus Aviário , Animais , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Galinhas/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Orthoreovirus Aviário/fisiologia , Replicação Viral/fisiologia
8.
Pol J Vet Sci ; 25(1): 109-118, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35575862

RESUMO

A novel avian orthoreovirus (N-ARV) variant characterized with obvious arthritis and synovial inflammation, was isolated from Shandong, China in May 2016. It caused chicken poor growth and enormous economic losses to the poultry industry of China. However, there are few effective methods for detecting the antibody levels of N-ARV. In this study, a viral structural protein σC was expressed using the prokaryotic expression vector pET32a (+). The target protein was obtained by inducing for 6 hours at an IPTG concentration of 0.6mM. The optimal dilution of the coating antigen and serum antibody were determined to be 1000 fold and 10 fold respectively. A specificity test showed that there was no positive reactivity between N-ARV and other pathogens, and when the positive serum was diluted 100 times detection results were still checkable. The repeatability of this method was determined by the inter assay and intra assay tests with variability ranging from 4.85% to 7.93%. In conclusion, this indirect enzyme linked immunosorbent assay (ELISA) will be useful for large-scale serological surveys and monitoring antibody levels in N-ARV infection.


Assuntos
Orthoreovirus Aviário , Orthoreovirus , Doenças das Aves Domésticas , Infecções por Reoviridae , Animais , Anticorpos Antivirais , Galinhas , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças das Aves Domésticas/diagnóstico , Infecções por Reoviridae/diagnóstico , Infecções por Reoviridae/veterinária , Sensibilidade e Especificidade , Proteínas Virais
9.
Viruses ; 14(5)2022 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-35632635

RESUMO

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. However, the precise molecular mechanism remains unclear. Here, we report the host cellular proteins that interact with ARV p17 by yeast two-hybrid screening. In this study, the p17 gene was cloned into pGBKT7 to obtain the bait plasmid pGBKT7-p17. After several rounds of screening of a chicken cDNA library, 43 positive clones were identified as possible host factors that interacted with p17. A BLAST search of the sequences was performed on the NCBI website, which ultimately revealed 19 interacting proteins. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genome analyses indicated that the acquired proteins were involved in multicellular organismal processes, metabolic processes, and biological regulation. When the subcellular localization of the host protein and ARV p17 protein was investigated, we observed colocalization of p17-GFP with IGF2BP1-RED and PQBP1-RED in the transfected cells but not with FGF1-RED. The direct interaction of ARV p17 protein with IGF2BP1 and PQBP1 was confirmed by coimmunoprecipitation and GST pulldown assays. We used RT-qPCR to assess the expression variation during ARV infection. The results showed that IGF2BP1, PAPSS2, RPL5, NEDD4L, PRPS2 and IFI16 were significantly upregulated, whereas the expression of FGF1, CDH2 and PQBP1 was markedly decreased in DF-1 cells infected with ARV. Finally, we demonstrated that IGF2BP1 had a positive effect on ARV replication, while PQBP1 had the opposite effect. Our findings provide valuable information for better insights into ARV's pathogenesis and the role of the p17 protein in this process.


Assuntos
Orthoreovirus Aviário , Animais , Galinhas , Fator 1 de Crescimento de Fibroblastos/metabolismo , Imunoprecipitação , Orthoreovirus Aviário/genética
10.
J Virol ; 96(6): e0007422, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35107368

RESUMO

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and µNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.


Assuntos
Proteínas de Ciclo Celular , Chaperoninas , Proteínas de Choque Térmico HSP90 , Orthoreovirus Aviário , Proteínas Virais , Replicação Viral , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Genoma Viral , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Orthoreovirus Aviário/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genética
11.
Methods Mol Biol ; 2465: 27-40, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35118614

RESUMO

Particulate material is more efficient in eliciting immune responses. Here we describe the production of micro- and nanospheres formed by protein muNS-Mi from avian reoviruses, loaded with foreign epitopes for their use as vaccines.


Assuntos
Orthoreovirus Aviário , Vacinas , Adjuvantes Imunológicos , Epitopos/metabolismo , Orthoreovirus Aviário/metabolismo , Vacinas/metabolismo , Vacinas de Subunidades , Proteínas não Estruturais Virais/metabolismo
12.
J Virol Methods ; 303: 114479, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35114290

RESUMO

The novel duck reovirus (NDRV) disease first appeared in China in 2011. Infected ducks may be of various ages and breeds. Sigma B protein, which is the key component of NDRV's outer capsid, may trigger group-specific neutralizing antibodies linked to NDRV infection, pathogenicity, and immune defense. The sigma B protein gene of fourteen NDRV field strains was amplified by RT-PCR and cloned into the pMD-18 T vector for sequencing to examine the genetic variation in sigma B proteins of NDRVs in southeastern China between 2011 and 2020. The sigma B protein gene of the fourteen NDRV southeastern strains included in this analysis had 96.3 %-99.8 % nucleotide, and 96.2 %-99.7 % deduced amino acid sequence homology. Phylogenetic analysis revealed that the fourteen southeastern strains belonged to a well-supported lineage that included NDRV and Muscovy duck reovirus (MDRV) strains. However, Avian reovirus (ARV) formed a distinct genetic lineage in the gene tree. The sigma B protein gene sequences of NDRV strains found in southeastern China are substantially conserved, according to these findings. There is no significant geographical difference between NDRV southeastern strains and DRV strains in other regions of China. Our findings will add to the molecular epidemiological picture of NDRV strain spread in southeastern China between 2011 and 2020, laying the groundwork for potential in-depth research on vaccine collection and comprehensive prevention.


Assuntos
Orthoreovirus Aviário , Doenças das Aves Domésticas , Infecções por Reoviridae , Animais , China/epidemiologia , Variação Genética , Orthoreovirus Aviário/genética , Filogenia , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/veterinária
13.
J Vet Med Sci ; 84(2): 238-243, 2022 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-34980758

RESUMO

Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of 104 chicken fecal samples were collected from 7 farms of breeder chickens (layers and broilers) in Japan from 2019 to 2021, and yielded 26 FAdV plus 14 ARV isolates. By sequencing, FAdV isolates were classified as FAdV-1, 5 and 8b. ARV isolates were classified as genotype II, IV and V. These results suggest that FAdVs and ARVs are resident in the breeder chicken farms in Japan.


Assuntos
Infecções por Adenoviridae , Aviadenovirus , Orthoreovirus Aviário , Doenças das Aves Domésticas , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/veterinária , Animais , Aviadenovirus/genética , Galinhas , Japão/epidemiologia , Orthoreovirus Aviário/genética , Filogenia
14.
Vet Microbiol ; 264: 109294, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34847454

RESUMO

Avian reovirus (ARV) is an important pathogen causing multiple types of clinical diseases in chickens, including viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. Here, we show that infection of DF-1 cells (a chicken fibroblast cell line) with ARV markedly altered the expressions of 583 chicken miRNAs(gga-miR), and that transfection of DF-1 cells with gga-miR-29a-3p, an upregulated miRNA in ARV-infected cells, significantly suppressed ARV-induced apoptosis via directly targeting Caspase-3, retarding ARV growth in cells. In contrast, knockdown of endogenous gga-miR-29a-3p in DF-1 cells by specific miRNA inhibitor enhanced ARV-induced apoptosis and increased the content and activity of caspase-3, facilitating viral growth in cells. Consistently, inhibition of Caspase-3 activity by inhibitors decreased viral titers in cell cultures. Thus, gga-miR-29a-3p plays an important antiviral role in host response to ARV infection by suppression of apoptosis via targeting Caspase-3. This information will further our understandings of how host cells combat against ARV infection by self-encoded small RNA and increase our knowledge of the role of microRNAs in host response to pathogenic infection.


Assuntos
Apoptose , Caspase 3 , MicroRNAs , Orthoreovirus Aviário , Replicação Viral , Animais , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Galinhas , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Orthoreovirus Aviário/fisiologia , Replicação Viral/genética
15.
Vet Microbiol ; 264: 109277, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34826648

RESUMO

Avian reoviruses (ARVs) are important pathogens that cause considerable economic losses in poultry farming. To date, host factors that control stabilization of ARV proteins remain largely unknown. In this work we determined that the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC) is essential for avian reovirus (ARV) replication by stabilizing outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV. TriC serves as a chaperone of viral proteins and prevent their degradation via the ubiquitin-proteasome pathway. Furthermore, reciprocal co-immunoprecipitation assays confirmed the association of viral proteins (σA, σC, and σNS) with TRiC. Immunofluorescence staining indicated that the TRiC chaperonins (CCT2 and CCT5) are colocalized with viral proteins σC, σA, and σNS of ARV. In this study, inhibition of TRiC chaperonins (CCT2 and CCT5) by the inhibitor HSF1A or shRNAs significantly reduced expression levels of the σC, σA, and σNS proteins of ARV as well as virus yield, suggesting that the TRiC complex functions in stabilization of viral proteins and virus replication. This study provides novel insights into TRiC chaperonin governing virus replication via stabilization of outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV.


Assuntos
Chaperonina com TCP-1 , Orthoreovirus Aviário , Proteínas Virais , Replicação Viral , Animais , Proteínas do Capsídeo/metabolismo , Chaperonina com TCP-1/metabolismo , Orthoreovirus Aviário/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral/genética
16.
J Virol Methods ; 299: 114332, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34655690

RESUMO

Reovirus fusion-associated small transmembrane (FAST) proteins induce syncytium formation. Recently, several studies have shown that the use of recombinant vectors engineered to express fusion proteins is becoming attractive for the development of enhanced oncolytic viruses. In this study, we investigated the cytotoxic effect of four different FAST proteins (p10 FAST of Avian reovirus [ARV], p10 FAST of Pulau virus [PuV], p13 FAST of Broome virus [BroV], and p14 FAST of reptilian reovirus [RRV]). Plasmids encoding FASTs were transfected into Vero cells. All FAST proteins induced syncytium formation at varying intensities. To achieve high levels of FAST expression, four different FAST genes were inserted into the murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vector. Two days after transfection in 293 T cells, only the MoMLV-10A1-p10(PuV) RCR vector showed syncytia formation. Based on these results, p10(Puv) was selected from the four FASTs. Next, we investigated the cytotoxicity of p10(PuV) on HeLa cervical carcinoma cells, HT1080 human fibrosarcoma cells, and U87 human glioma cells. Although three human cancer cell lines induced syncytium formation, U87 cells were highly susceptible to syncytia formation by transfection with p10(PuV). In addition, the viral supernatants from MoMLV-10A-p10(PuV) RCR vector-transfected 293 T cells also induced syncytium formation in HT1080, TE671, and U87 cells. This RCR vector encoding p10(PuV) is a promising candidate for cancer gene therapy.


Assuntos
Neoplasias , Orthoreovirus Aviário , Animais , Chlorocebus aethiops , Genes Neoplásicos , Vetores Genéticos/genética , Células HEK293 , Humanos , Camundongos , Células Vero
17.
Vet Microbiol ; 261: 109214, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34461358

RESUMO

Novel duck reovirus (NDRV) causes high morbidity in ducklings, and recovered ducklings are often remarkably stunted in growth. In this study, four NDRV strains were isolated from the NDRV outbreaks that occurred in different regions of Shandong province, China. The biological characteristics and pathogenicity of the four NDRV strains were elucidated, and the N20 was identified as a naturally attenuated strain. Three-day-old ducklings were immunized with live N20 strain (100 ELD50/duck), and challenged with 104.52 ELD50 of virulent N19 strain at 7 days post immunization. The vaccinated ducklings showed no evidence of clinical signs, gross and histopathological lesions, or loss of body weight, and 100 % protection against the virulent NDRV N19 infection. The NDRV-specific antibodies were generated in the immunized ducklings and could neutralize different NDRV strains. These results indicated that the N20 strain was a promising live attenuated vaccine candidate against highly pathogenic NDRV infection.


Assuntos
Orthoreovirus Aviário/imunologia , Doenças das Aves Domésticas/prevenção & controle , Infecções por Reoviridae/veterinária , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/sangue , China , Patos , Orthoreovirus Aviário/genética , Orthoreovirus Aviário/isolamento & purificação , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/prevenção & controle , Vacinas Atenuadas/imunologia
18.
Avian Dis ; 65(3): 346-350, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34427406

RESUMO

Sigma C protein-coding sequences have been used to phylogenetically classify avian reovirus (ARV) strains. However, the relationship between serotype and phylogenetic cluster classification of the five prototype serotype strains of ARV in Japan has not been established. Thus, we used sigma C protein-coding sequences to characterize avian reoviruses (ARVs) isolated from chickens with tendonitis in Japan together with the five prototype serotype strains of ARV in Japan. Phylogenetic analysis of ARVs based on the sigma C protein-coding sequences revealed that the five prototype serotype strains of ARV were each classified into different, independent clusters. Two field isolates (JP/Tottori/2016 and JP/Nagasaki/2017) that were isolated from chickens with arthritis/tenosynovitis were classified into different clusters. JP/Tottori/2016 was classified into cluster VI with the CS-108 strain, and JP/Nagasaki/2017 was classified into cluster I with strain TS-142. Serologically, JP/Tottori/2016 was well-neutralized by antisera against the CS-108 strain, whereas JP/Nagasaki/2017 cross-reacted with antisera against both the CS-108 and TS-142 strains. Embryo lethality test revealed that the two field isolates induced 80% and 67% embryo mortality, respectively, whereas the five prototype strains induced 0%-33% embryo mortality. Our findings will contribute to understanding the characteristics of ARV strains in Japan.


Nota de investigación­Análisis genético de reovirus aviares aislados de pollos en Japón. Se han utilizado secuencias de proteína Sigma C para clasificar filogenéticamente cepas de reovirus aviar (ARV). Sin embargo, no se ha establecido la relación entre el serotipo y la clasificación de grupos filogenéticos de las cinco cepas prototipo de serotipo de reovirus aviares en Japón. Por lo tanto, se utilizó la secuenciación de la proteína sigma C para caracterizar los reovirus aviares (ARV) aislados de pollos con tendinitis en Japón junto con las cinco cepas prototipos de serotipos de reovirus aviares en Japón. El análisis filogenético de los reovirus extranjeros basado en el gene sigma C reveló que las cinco cepas prototipo de serotipo de reoviruses se clasificaron cada una en grupos diferentes e independientes. Dos aislamientos de campo (JP/Tottori/2016 y JP/Nagasaki/2017) que se aislaron de pollos con artritis/tenosinovitis se clasificaron en diferentes grupos. El aislamiento JP/Tottori/2016 se clasificó en el grupo VI con la cepa CS-108, y el aislamiento JP/Nagasaki/2017 se clasificó en el grupo I con la cepa TS-142. Serológicamente, el aislamiento JP/Tottori/2016 fue completamente neutralizado por antisueros contra la cepa CS-108, mientras que el virus JP/Nagasaki/2017 reaccionó de forma cruzada con antisueros contra las cepas CS-108 y TS-142. Las pruebas de patogenicidad de embriones revelaron que los dos aislados de campo indujeron 80% y 67% de mortalidad embrionaria, respectivamente, mientras que las cinco cepas prototipo indujeron 0% -33% de mortalidad embrionaria. Estos hallazgos contribuirán a comprender las características de las cepas de reovirus aviares en Japón.


Assuntos
Orthoreovirus Aviário , Doenças das Aves Domésticas , Infecções por Reoviridae , Animais , Galinhas , Japão/epidemiologia , Orthoreovirus Aviário/genética , Filogenia , Doenças das Aves Domésticas/epidemiologia , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/veterinária
19.
PLoS One ; 16(8): e0256137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34411166

RESUMO

Inclusion body hepatitis (IBH) is, in some cases, a fatal disease affecting fowl by adenovirus strains which are subdivided into 5 species (A-E). In the current study, we investigated sequences from the Loop L1 region of the hexon gene of sequences of adenovirus field stains 1/A and 11/D isolated from a poultry flock co-infected with IBH and avian reoviruses ARVs. In early 2021, an epidemiologic survey highlighted the coinfection adenoviruses with other viruses (orthoreovirus infection) as being particularly deleterious within the poultry industry. Here, we investigated the Loop L1 HVR1-4 region of the hexon gene with relative synonymous codon usage (RSCU) designation and RSCU inclusive of all the mutations. These are the first results that have been presented on fowl adenovirus species A and D with simultaneous reovirus infection in 38-days old broiler chickens in Poland.


Assuntos
Orthoreovirus Aviário/isolamento & purificação , Infecções por Reoviridae/virologia , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/genética , Galinhas/genética , Uso do Códon/genética , Coinfecção , Orthoreovirus Aviário/genética , Orthoreovirus Aviário/patogenicidade , Filogenia , Polônia , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/veterinária , Sorogrupo
20.
Vet Microbiol ; 260: 109094, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34271302

RESUMO

In mid-2020, using next-generation sequencing (NGS) technology, we identified a recombinant cluster 2 avian orthoreovirus (ARV) variant named PHC-2020-0545, isolated from tendons of 33-day-old broilers with leg swelling in China. Complete genomic sequencing and analyses demonstrated that the isolate was genetically significantly distinct from known ARV strains in M1 and M3 genes and its σC coding gene had an extremely high variability, compared with the identified ARV strains grouped into other genotyping cluster. Further analysis showed that many base substitutions were silent and non-silent substitutions are most likely to occur in the first positions of codons. Multiple segmental recombination, intra-segmental recombination and accumulation of point mutations might contribute to the emergence of this isolate. The PHC-2020-0545 strain had a strong replication ability in 1-day-old broilers, and mainly affected the movement, digestion and metabolism of broilers. In addition, the infection route of the isolate is related to its pathogenicity to broilers. Therefore, combined with its unique genetic characteristics and potential origin, we determined that the PHC-2020-0545 field strain is a novel recombinant ARV strain, which has certain reference value for the preparation and evaluation of new vaccines.


Assuntos
Galinhas/virologia , Genoma Viral/genética , Orthoreovirus Aviário/genética , Doenças das Aves Domésticas/virologia , Recombinação Genética , Infecções por Reoviridae/veterinária , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , China , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Masculino , Mutação , Orthoreovirus Aviário/patogenicidade , Filogenia , Infecções por Reoviridae/virologia , Alinhamento de Sequência/veterinária
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