Oncolytic avian reovirus-sensitized tumor infiltrating CD8+ T cells triggering immunogenic apoptosis in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a leading malignant disease in numerous countries, including Taiwan with limited therapeutic options. Animal viruses including oncolytic avian reovirus (ARV) have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. Here, we provide a novel insight into oncolytic ARV and UV-ARV-sensitized patient's peripheral blood mononuclear cells (P-PBMCs) and tumor infiltrating lymphocytes (TILs) killing primary GC (PGC) cells through the surface TLR3 and TRAIL/DR4/DR5 immunogenic apoptosis pathway. METHODS: We conducted a comprehensive study to reveal whether ARV- or UV-inactivated ARV (UV-ARV)-modulated P-PBMCs or TILs killing ARV- and UV-ARV-sensitized AGS cells and PGC cells derived from clinical patients and to investigate the regulation of surface TLR3 receptor and upstream signaling pathways. Apoptosis analysis by flow cytometry and Western blot, suppression of signal pathway by specific inhibitors, in situ proximity ligation assay (PLA), time-resolved flurometry and lactate dehydrogenase (LDH) cytotoxicity assays, and an in vitro co-culture model were established to study the interplay between ARV- and UV-ARV-sensitized P-PBMCs and TILs to kill PGC cells and their upstream pathways. RESULTS: Our results reveal that increased levels of DR4 and DR5 were observed in ARV and UV-ARV sensitized PGC cells through the TLR3/p38/p53 signaling pathway. Importantly, we found that the σC protein of ARV or UV-ARV interacted with surface TLR3 of CD8+ TILs, thereby triggering the TLR3/NF-κB/IFN-γ/TRAIL signaling pathway which induces immunogenic apoptosis of PGC cells. This study sheds further light on the molecular basis behind ARV oncolysis and facilitates the ARV or UV-ARV as a cancer therapeutic. CONCLUSIONS: The study provides novel insights into ARV- or UV-ARV-sensitized P-PBMCs and CD8+ TILs to kill PGC cells through the immunogenic apoptosis pathway. We conclude that P-PBMCs can easily be obtained from GC patients and provide a rich source as TILs to kill PGC cells.

tRF-His-GTG-1 enhances NETs formation and interferon-α production in lupus by extracellular vesicle.

BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a crucial role in active severe systemic lupus erythematosus (SLE). However, what triggers the imbalance in dysregulated NETs formation in SLE is elusive. Transfer RNA-derived small RNAs (tsRNAs) are novel non-coding RNAs, which participate in various cellular processes. We explore the role of tsRNAs on NETs formation in SLE. METHODS: We analyzed the levels of NETs DNA and platelet-derived extracellular vesicles (pEVs) from 50 SLE patients and 20 healthy control subjects. The effects of pEVs on NETs formation were evaluated by using immunofluorescence assay and myeloperoxidase-DNA PicoGreen assay. The regulatory mechanism of pEVs on NETs formation and inflammatory cytokines production were investigated using an in vitro cell-based assay. RESULTS: Increased circulating NETs DNA and pEVs were shown in SLE patients and were associated with disease activity (P < 0.005). We demonstrated that SLE patient-derived immune complexes (ICs) induced platelet activation, followed by pEVs release. ICs-triggered NETs formation was significantly enhanced in the presence of pEVs through Toll-like receptor (TLR) 8 activation. Increased levels of tRF-His-GTG-1 in pEVs and neutrophils of SLE patients were associated with disease activity. tRF-His-GTG-1 interacted with TLR8 to prime p47phox phosphorylation in neutrophils, resulting in reactive oxygen species production and NETs formation. Additionally, tRF-His-GTG-1 modulated NF-κB and IRF7 activation in neutrophils upon TLR8 engagement, resulting IL-1ß, IL-8, and interferon-α upregulation, respectively. CONCLUSIONS: The level of tRF-His-GTG-1 was positively correlated with NETs formation in SLE patients; tRF-His-GTG-1 inhibitor could efficiently suppress ICs-triggered NETs formation/hyperactivation, which may become a potential therapeutic target.

Neutrophils and platelets are key members in the immunopathogenesis of SLE. EVs play a key role in intercellular communication. Abnormal NETs formation promotes vascular complications and organ damage in SLE patients. tsRNA is a novel regulatory small non-coding RNA and participates in diverse pathological processes. Herein, we showed that SLE patient-derived ICs activates platelets directly, followed by intracellular tRF-His-GTG-1 upregulation, which is loaded into pEVs. The pEV-carried tRF-His-GTG-1 could interact with TLR8 in neutrophils, followed by activation of the downstream signaling pathway, including p47phox-NOX2-ROS, which causes NETs enhancement, while IRF7 promotes the expression of IFN-α. The tRF-His-GTG-1 inhibitor could suppress efficiently SLE ICs-induced NETs formation and pEVs primed NETs enhancement. This study offers new molecular machinery to explain the association between the platelets-derived tsRNAs, pEVs, and hyperactive NETs formation in lupus. tRF-His-GTG-1 may serve as a potential therapeutic target and help to advance our understanding of tsRNAs in SLE pathogenesis.

p17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories.

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.

Oncolytic Avian Reovirus p17-Modulated Inhibition of mTORC1 by Enhancement of Endogenous mTORC1 Inhibitors Binding to mTORC1 To Disrupt Its Assembly and Accumulation on Lysosomes.

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.

SARS-CoV-2 primed platelets-derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation.

BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive. METHODS: We explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting. RESULTS: COVID-19 patient-derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet-derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1ß/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement. CONCLUSIONS: We identified SARS-CoV-2 triggered platelets-derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17.

Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine.

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

Mechanistic insights into avian reovirus p17-modulated suppression of cell cycle CDK-cyclin complexes and enhancement of p53 and cyclin H interaction.

The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.

Avian reovirus σA-modulated suppression of lactate dehydrogenase and upregulation of glutaminolysis and the mTOC1/eIF4E/HIF-1α pathway to enhance glycolysis and the TCA cycle for virus replication.

Adenosine triphosphate (ATP) is an energy source for many types of viruses for facilitating virus replication. This is the first report to demonstrate that the structural protein σA of avian reovirus (ARV) functions as an activator of cellular energy. Three cellular factors, isocitrate dehydrogenase 3 subunit beta (IDH3B), lactate dehydrogenase A (LDHA), and vacuolar-type H+-ATPase (vATPase) co-immunoprecipitated with ARV σA and were identified by 2D-LC/MS/MS. ARV enhances glycolytic flux through upregulation of glycolytic enzymes. Increased ATP levels in both ARV-infected and σA-transfected cells were observed by a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams. Furthermore, σA upregulates IDH3B and glutamate dehydrogenase (GDH) to promote glutaminolysis, activating HIF-1α. Both HIF-1α level and viral yield in IDH3B-depleted and glutamine-deprived cells, and inhibition of glutaminolysis was significantly reduced. The σAR155/273A mutant loses its ability to enter the nucleolus, impairing its ability to regulate glycolysis. In addition, we have identified the conserved untranslated regions (UTR) of the 5'- and 3'-termini of the ARV genome segments that are required for viral protein synthesis in an ATP-dependent manner. Deletion of either the 5'- or 3'-UTR impaired viral protein synthesis. Knockdown of σA reduced the ATP level and significantly decreased virus yield, suggesting that σA enhances ATP formation to promote virus replication. Collectively, this study provides novel insights into σA-modulated suppression of LDHA and activation of IDH3B and GDH to activate the mTORC1/eIF4E/HIF-1α pathways to upregulate glycolysis and the TCA cycle for virus replication.

Bovine ephemeral fever virus triggers autophagy enhancing virus replication via upregulation of the Src/JNK/AP1 and PI3K/Akt/NF-κB pathways and suppression of the PI3K/Akt/mTOR pathway.

Autophagy plays an important role in cellular response to pathogens. However, the impact of the autophagy machinery on bovine ephemeral fever virus (BEFV) infection is not yet determined. A recent study in our laboratory demonstrated that BEFV triggers simultaneously the PI3K/Akt/NF-κB and Src/JNK-AP1 pathways in the stage of virus binding to enhance virus entry. In this work, we report that BEFV induces autophagy via upregulation of the PI3K/Akt/NF-κB and Src/JNK/AP1 pathways in the early to middle stages of infection and suppresses the PI3K/Akt/mTOR pathway at the late stage of infection. To activate NF-κB, BEFV promotes degradation of IκBα and activates Akt to stimulate NF-κB translocation into the nucleus. Immunoprecipitation assays revealed that BEFV disrupts Beclin 1 and Bcl-2 interaction by JNK-mediated Bcl-2 phosphorylation, thereby activating autophagy. Overexpression of Bcl-2 reversed the BEFV-induced increase in the LC3 II levels. Suppression of autophagy either by knockdown of autophagy-related genes with shRNAs or treatment with a pharmacological inhibitor 3-MA reduced BEFV replication, suggesting that BEFV-induced autophagy benefits virus replication. Our results revealed that the BEFV M protein is one of the viral proteins involved in inducing autophagy via suppression of the PI3K/Akt/mTORC1 pathway. Furthermore, degradation of p62 was observed by immunoblotting, suggesting that BEFV infection triggers a complete autophagic response. Disruption of autophagosome-lysosome fusion by depleting LAMP2 resulted in reduction of virus yield, suggesting that formation of autolysosome benefits virus production.

Muscovy duck reovirus σNS protein triggers autophagy enhancing virus replication.

BACKGROUND: Muscovy duck reovirus (MDRV) causes high morbidity and mortality in Muscovy ducklings at 10 days old and can persist in an infected flock until the ducklings of 6 weeks old. It shares common physicochemical properties with avian reovirus (ARV) and differs in coding assignment and pathogenicity. The ARV p17 protein has been shown to trigger autophagy via activation multiple signaling pathways, which benefits virus replication. Since MDRV lacks the p17 protein, whether and how MDRV induces autophagy remains unknown. The aim of this study was to explore whether MDRV induces autophagy and which viral proteins are involved in MDRV-induced autophagy. METHODS: The autophagosome-like structures in MDRV-infected cells was observed under transmission electron microscopy. MDRV-induced autophagy was examined by analyzing the LC3-II level and phosphorylated form of mammalian target of rapamycin (mTOR) by Western blot assays. The effects of 3-methyladenine, rapamycin, chloroquine on viral yields were measured with quantitative(q) real-time reverse transcription (RT)-polymerase chain reaction (PCR) and 50% tissue culture infective dose (TCID50) assays, respectively. Additionally, to determine which viral protein is responsible for MDRV-induced autophagy, both p10.8- and σNS-encoding genes of MDRV were cloned into the pCI-neo-flag vector and transfected into DF-1 cells for detection of LC3-II. RESULTS: The typical double-membrane vesicles containing cytoplasmic inclusions were visible in MDRV-infected immortalized chicken embryo fibroblast (DF-1) cells under transmission electron microscopy. Both primary Muscovy duck embryo fibroblasts (MDEF) and DF-1 cells infected with MDRV exhibited a significant increased levels of LC3-II accompanied with downregulation of phosphorylated form of mTOR, further confirming that MDRV is capable of inducing autophagy. Autophagy could be suppressed by 3-methylademine and induced by rapamycin and chloroquine. Furthermore, we found that σNS induces an increased levels of LC3-II, suggesting that the MDRV σNS protein is one of viral proteins involved in induction of autophagy. Both qRT-PCR and TCID50 assays showed that virus yield was increased in rapamycin treated DF-1 cells following MDRV infection. Conversely, when infected cells were pretreated with chloroquine, virus yield was decreased. CONCLUSIONS: The MDRV σNS nonstructural protein is responsible for MDRV-induced autophagy and benefits virus replication.

Cell entry of bovine ephemeral fever virus requires activation of Src-JNK-AP1 and PI3K-Akt-NF-κB pathways as well as Cox-2-mediated PGE2 /EP receptor signalling to enhance clathrin-mediated virus endocytosis.

Although we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin-mediated and dynamin 2-dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src-JNK-AP1 and PI3K-Akt-NF-κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src-JNK signalling to enhance its entry. Activation of these pathways by ultraviolet-inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV-induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3'-dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox-2-catalysed prostaglandin E2 (PGE2) synthesis and induces expressions of G-protein-coupled E-prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src-JNK-AP1 and PI3K-Akt-NF-κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV-induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox-2-mediated PGE2/EP receptor signalling pathways, further enhancing Src-JNK-AP1 in a cAMP-dependent manner and PI3K-Akt-NF-κB in a cAMP-independent manner. Accordingly, BEFV stimulates PGE2/EP receptor signalling amplifying Src-JNK-AP1 and PI3K-Akt-NF-κB pathways in an autocrine or paracrine fashion to enhance virus entry.

Avian reovirus S1133-induced apoptosis is associated with Bip/GRP79-mediated Bim translocation to the endoplasmic reticulum.

In this study the mechanism of avian reovirus (ARV) S1133-induced pathogenesis was investigated, with a focus on the contribution of ER stress to apoptosis. Our results showed that upregulation of the ER stress response protein, as well as caspase-3 activation, occurred in ARV S1133-infected cultured cells and in SPF White Leghorn chicks organs. Upon infection, Bim was translocated specifically to the ER, but not mitochondria, in the middle to late infectious stages. In addition, ARV S1133 induced JNK phosphorylation and promoted JNK-Bim complex formation, which correlated with the Bim translocation and apoptosis induction that was observed at the same time point. Knockdown of BiP/GRP78 by siRNA and inhibition of BiP/GRP78 using EGCG both abolished the formation of the JNK-Bim complex, caspase-3 activation, and subsequent apoptosis induction by ARV S1133 efficiently. These results suggest that BiP/GRP78 played critical roles and works upstream of JNK-Bim in response to the ARV S1133-mediated apoptosis process.

Recurrent positive selection and heterogeneous codon usage bias events leading to coexistence of divergent pigeon circoviruses.

The capsid genes from 14 pigeon circovirus (PiCV) sequences, collected from Taiwan between 2009 and 2010, were sequenced and compared with 14 PiCV capsid gene sequences from GenBank. Based on pairwise comparison, PiCV strains from Taiwan shared 73.9-100% nucleotide identity and 72-100% amino acid identity with those of the 14 reported PiCV sequences. Phylogenetic analyses revealed that Taiwanese PiCV isolates can be grouped into two clades: clade 1 comprising isolates from Belgium, Australia, USA, Italy and China, and clade 2 showing close relation to isolates from Germany and France. Recurrent positive selection was detected in clade 1 PiCV lineages, which may contribute to the diversification of predominant PiCV sequences in Taiwan. Further observations suggest that synonymous codon usage variations between PiCV clade 1 and clade 2 may reflect the adaptive divergence on translation efficiency of capsid genes in infectious hosts. Variation in selective pressures acting on the evolutionary divergence and codon usage bias of both clades explains the regional coexistence of virus sequences congeners prevented from competitive exclusion within an island such as Taiwan. Our genotyping results also provide insight into the aetiological agents of PiCV outbreak in Taiwan and we present a comparative analysis of the central coding region of PiCV genome. From the sequence comparison results of 28 PiCVs which differs in regard to the geographical origin and columbid species, we identified conserved regions within the capsid gene that are likely to be suitable for primer selection and vaccine development.

Characterization of emerging Newcastle disease virus isolates in China.

BACKGROUND: Newcastle disease (ND) is a devastating worldwide disease of poultry characterized by increased respiration, circulatory disturbances, hemorrhagic enteritis, and nervous signs. Sequence analysis shows several amino acid residue substitutions at neutralizing epitopes on the F and HN proteins of recent Shaanxi strains. Both Cross protection and cross serum neutralization tests revealed that the traditional vaccine strains were unable to provide full protection for the flocks. METHODS: To better understand the epidemiology of Newcastle disease outbreak, a portion of the F gene and the full-length HN gene were amplified from Shaanxi isolates by reverse transcription-polymerase chain reaction (RT-PCR) and then conducted sequence and phylogenetic analyzes. In pathogenicity analysis, both high intra-cerebral pathogenicity index (ICPI) and mean death time (MDT) tests of chicken embryo were carried out. Furthermore, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with a LaSota vaccine strain were challenged by the recent Shaanxi strain was also performed. RESULTS: Nine Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken flocks in China were genotypically and pathotypically characterized. Amino acid sequence analysis revealed that all the recent Shaanxi-isolated NDVs have (112)R-R-Q-K-R-F(117) for the C-terminus of the F2 protein and exhibit high ICPI and MDT of chicken embryos, suggesting that they were all classified as velogenic type of NDVs. Phylogenetic analysis of these isolates showed that they belong to subgenotype VIId that have been implicated in the recent outbreaks in northwestern China. The percentage of amino acid sequence identity of F protein between recent Shaanxi stains and five vaccine strains was in the range of 81.9 %-88.1 %, while the percentage of amino acid sequence identity of HN protein between recent Shaanxi strains and vaccine strains was in the range of 87.4 %-91.2 %. Furthermore, a number of amino acid residue substitutions at neutralizing epitopes on the F and HN proteins of these isolates were observed, which may lead to the change of antibody recognition and neutralization capacity. A cross-protection experiment indicated that specific-pathogen-free chickens vaccinated with a LaSota vaccine strain was not capable of providing full protection for the flocks that were challenged by the recent Shaanxi strain. CONCLUSIONS: Taken together, our findings reveal that recent Shannxi NDVstrains exhibit antigenic variations that could be responsible for recent outbreaks of NDVs in northwestern China.

The distribution of sialic acid receptors of avian influenza virus in the reproductive tract of laying hens.

The susceptibility of the host to influenza virus is determined by the distribution of the sialic acid (SA) receptors on host cell membrane. Avian influenza virus (AIV) preferentially binds to SA α-2,3-galactose (SA α2,3-gal) linked receptors, while human strains bind to sialic acid α2,6-galactose (SA α2,6-gal) linked receptors. Here, we describe the SA patterns and distributions in the reproductive tract of hens by employing two specific lectins, Maackia amurensis agglutinin (MAA) for SA α2,3-gal and sambucus nigra agglutinin (SNA) for SA α 2,6-gal receptors. Our results revealed that both SA α2,3-gal and SA α2,6-gal receptors exist in the reproductive tract of hens, including magnum, isthmus, uterus and vagina except for infundibulum. The distribution of SAα-2,3-gal receptor was more abundantly in the columnar epithelium cells of magnum, isthmus and uterus. Only minimal positive results for SA α-2,6-gal receptors were detected in the columnar epithelium cells of magnum, isthmus, uterus and vagina. Furthermore, AIV in tissues of the reproductive tract tissues of laying hens were detected by SYBR green-based reverse transcription and polymerase chain reaction (RT-PCR). Results showed that both viral loads and pathological changes in different parts of the reproductive tract were positively correlated with the expression of both receptors. Our results revealed that the reproductive tract of hens may provide an environment for the replication of both avian and human influenza viruses.

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.

Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity.

Nucleocytoplasmic shuttling of BEFV M protein-modulated by lamin A/C and chromosome maintenance region 1 through a transcription-, carrier- and energy-dependent pathway.

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.