Publications by authors named "Jiyong Zhou"

143 Publications

DeSUMOylation of Apoptosis Inhibitor 5 by VP3 Supports Virus Replication.

mBio 2021 Aug 10;12(4):e0198521. Epub 2021 Aug 10.

MOA Key Laboratory of Animal Virology, Zhejiang Universitygrid.13402.34 Center for Veterinary Sciences, Hangzhou, Zhejiang, People's Republic of China.

SUMOylation is a reversible posttranslational modification involved in the regulation of diverse biological processes. Growing evidence suggests that virus infection can interfere with the SUMOylation system. In the present study, we discovered that apoptosis inhibitor 5 (API5) is a SUMOylated protein. Amino acid substitution further identified that Lys404 of API5 was the critical residue for SUMO3 conjugation. Moreover, we found that infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. In addition, our results further revealed that viral protein VP3 inhibited the SUMOylation of API5 by targeting API5 and promoting UBC9 proteasome-dependent degradation through binding to the ubiquitin E3 ligase TRAF3. Furthermore, we revealed that wild-type but not K404R mutant API5 inhibited IBDV replication by enhancing MDA5-dependent IFN-β production. Taken together, our data demonstrate that API5 is a UBC9-dependent SUMOylated protein and deSUMOylation of API5 by viral protein VP3 aids in viral replication. Apoptosis inhibitor 5 (API5) is a nuclear protein initially identified for its antiapoptotic function. However, so far, posttranslational modification of API5 is unclear. In this study, we first identified that API5 K404 can be conjugated by SUMO3, and infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. Mechanically, viral protein VP3 directly interacts with API5 and inhibits SUMOylation of API5. Additionally, the cellular E3 ligase TNF receptor-associated factor 3 (TRAF3) is employed by VP3 to facilitate UBC9 proteasome-dependent degradation, leading to the reduction of API5 SUMOylation. Moreover, our data reveal that SUMOylation of API5 K404 promotes MDA5-dependent beta interferon (IFN-β) induction, and its deSUMOylation contributes to IBDV replication. This work highlights a critical role of conversion between SUMOylation and deSUMOylation of API5 in regulating viral replication.
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http://dx.doi.org/10.1128/mBio.01985-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406308PMC
August 2021

Interplay of the ubiquitin proteasome system and the innate immune response is essential for the replication of infectious bronchitis virus.

Arch Virol 2021 Aug 26;166(8):2173-2185. Epub 2021 May 26.

Key Laboratory of Animal Virology of Ministry of Agriculture, Department of Veterinary Medicine, College of Animal Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou, People's Republic of China.

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNβ) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKε, and IRF3, form a signaling cascade for the synthesis of IFNβ. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.
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http://dx.doi.org/10.1007/s00705-021-05073-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150628PMC
August 2021

Inhibition of Antiviral Innate Immunity by VP3 via Blocking TBK1-TRAF3 Complex Formation and IRF3 Activation.

mSystems 2021 May 11;6(3). Epub 2021 May 11.

MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, Hangzhou, People's Republic of China

The host innate immune system develops various strategies to antagonize virus infection, and the pathogen subverts or evades host innate immunity for self-replication. In the present study, we discovered that infectious bursal disease virus (IBDV) VP3 protein significantly inhibits MDA5-induced beta interferon (IFN-β) expression by blocking IRF3 activation. Binding domain mapping showed that the CC1 domain of VP3 and the residue lysine-155 of tumor necrosis factor receptor-associated factor 3 (TRAF3) are essential for the interaction. Furthermore, we found that the CC1 domain was required for VP3 to downregulate MDA5-mediated IFN-β production. A ubiquitination assay showed that lysine-155 of TRAF3 was the critical residue for K33-linked polyubiquitination, which contributes to the formation of a TRAF3-TBK1 complex. Subsequently, we revealed that VP3 blocked TRAF3-TBK1 complex formation through reducing K33-linked polyubiquitination of lysine-155 on TRAF3. Taken together, our data reveal that VP3 inhibits MDA5-dependent IRF3-mediated signaling via blocking TRAF3-TBK1 complex formation, which improves our understanding of the interplay between RNA virus infection and the innate host antiviral immune response. Type I interferon plays a critical role in the host response against virus infection, including However, many viruses have developed multiple strategies to antagonize the innate host antiviral immune response during coevolution with the host. In this study, we first identified that K33-linked polyubiquitination of lysine-155 of TRAF3 enhances the interaction with TBK1, which positively regulates the host IFN immune response. Meanwhile, we discovered that the interaction of the CC1 domain of the VP3 protein and the residue lysine-155 of TRAF3 reduced the K33-linked polyubiquitination of TRAF3 and blocked the formation of the TRAF3-TBK1 complex, which contributed to the downregulation of host IFN signaling, supporting viral replication.
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http://dx.doi.org/10.1128/mSystems.00016-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125067PMC
May 2021

Cellular hnRNPAB binding to viral nucleoprotein inhibits flu virus replication by blocking nuclear export of viral mRNA.

iScience 2021 Mar 11;24(3):102160. Epub 2021 Feb 11.

MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play critical roles in the nuclear export, splicing, and sensing of RNA. However, the role of heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB) is poorly understood. In this study, we report that hnRNPAB cooperates with nucleoprotein (NP) to restrict viral mRNA nuclear export via inhibiting viral mRNA binding to ALY and NXF1. HnRNPAB restricts mRNA transfer from ALY to NXF1, inhibiting the mRNA nuclear export. Moreover, when cells are invaded by influenza A virus, NP interacts with hnRNPAB and interrupts the ALY-UAP56 interaction, leading to repression of ALY-viral mRNA binding, and then inhibits the viral mRNA binding to NXF1, leading to nuclear stimulation of viral mRNA. Collectively, these observations provide a new role of hnRNPAB to act as an mRNA nuclear retention factor, which is also effective for viral mRNA of influenza A virus, and NP cooperates with hnRNPAB to further restrict the viral mRNA nuclear export.
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http://dx.doi.org/10.1016/j.isci.2021.102160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918295PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

The serine-48 residue of nucleolar phosphoprotein nucleophosmin-1 plays critical role in subcellular localization and interaction with porcine circovirus type 3 capsid protein.

Vet Res 2021 Jan 7;52(1). Epub 2021 Jan 7.

MOA Key Laboratory of Animal Virology, Center of Veterinary Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.

The transport of circovirus capsid protein into nucleus is essential for viral replication in infected cell. However, the role of nucleolar shuttle proteins during porcine circovirus 3 capsid protein (PCV3 Cap) import is still not understood. Here, we report a previously unidentified nucleolar localization signal (NoLS) of PCV3 Cap, which hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate nucleolar localization of PCV3 Cap. The NoLS of PCV3 Cap and serine-48 residue of N-terminal oligomerization domain of NPM1 are essential for PCV3 Cap/NPM1 interaction. In addition, charge property of serine-48 residue of NPM1 is critical for nucleolar localization and interaction with PCV3 Cap. Taken together, our findings demonstrate for the first time that NPM1 interacts with PCV3 Cap and is responsible for its nucleolar localization.
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http://dx.doi.org/10.1186/s13567-020-00876-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792357PMC
January 2021

The N-terminal residual arginine of influenza A virus NS1 protein is required for its nuclear localization and RNA binding.

Vet Microbiol 2020 Dec 14;251:108895. Epub 2020 Oct 14.

MOA Key Laboratory of Animal Virology, Center for Veterinary Sciences, Zhejiang University, Hangzhou, 310058, PR China; State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, 310003, PR China. Electronic address:

RNA binding ability and cellular distribution are important for nonstructural protein 1 (NS1) of influenza A virus to act as a viral regulatory factor to control virus life cycle. In this study, we identified that the N-terminal residues 19-21 of NS1 are a highly conserved motif depending on all the available NS1 full length sequence of H5N1 influenza A virus from NCBI database. Site-directed mutation analysis demonstrated that the R residue of NS1 is critical for its RNA binding and nuclear localization. Furthermore, the residue R of NS1 was identified to be critical for regulating M1 mRNA splicing and NS1 nuclear export. Biological analysis of the rescued viruses indicated that the RA mutation of NS1 did not interfere the replication of H5N1 virus during infection and attenuated the virulence of H5N1 virus in mice.
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http://dx.doi.org/10.1016/j.vetmic.2020.108895DOI Listing
December 2020

Influenza A Virus Induces Autophagy by Its Hemagglutinin Binding to Cell Surface Heat Shock Protein 90AA1.

Front Microbiol 2020 7;11:566348. Epub 2020 Oct 7.

MOA Key Laboratory of Animal Virology, Department of Veterinary Medicine and Center of Veterinary Medical Sciences, Zhejiang University, Hangzhou, China.

Autophagy can be utilized by the influenza A virus (IAV) to facilitate its replication. However, whether autophagy is induced at the stage of IAV entry is still unclear. Here, we report that IAV induces autophagy by hemagglutinin (HA) binding to heat shock protein 90AA1 (HSP90AA1) distributed on the cell surface. Virus overlay protein binding assay and pull-down assay indicated that IAV HA bound directly to cell surface HSP90AA1. Knockdown of HSP90AA1 weakened H1N1 infection. Incubation of IAV viral particles with recombinant HSP90AA1 or prior blockade of A549 cells with an anti-HSP90AA1 antibody could inhibit attachment of IAV. Moreover, we found that recombinant HA1 protein binding to cell surface HSP90AA1 was sufficient to induce autophagy through the AKT-MTOR pathway. Our study reveals that the HSP90AA1 on cell surface participates in IAV entry by directing binding to the HA1 subunit of IAV and subsequently induces autophagy.
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http://dx.doi.org/10.3389/fmicb.2020.566348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575715PMC
October 2020

Conformational Dynamics of Nonenveloped Circovirus Capsid to the Host Cell Receptor.

iScience 2020 Oct 11;23(10):101547. Epub 2020 Sep 11.

MOA Key Laboratory of Animal Virology, Center for Veterinary Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.

Circovirus, comprising one capsid protein, is the smallest nonenveloped virus and induces lymphopenia. Circovirus can be used to explore the cell adhesion mechanism of nonenveloped viruses. We developed a single-molecule fluorescence resonance energy transfer (smFRET) assay to directly visualize the capsid's conformational feature. The capsid underwent reversible dynamic transformation between three conformations. The cell surface receptor heparan sulfate (HS) altered the dynamic equilibrium of the capsid to the high-FRET state, revealing the HS-binding region. Neutralizing antibodies restricted capsid transition to a low-FRET state, masking the HS-binding domain. The lack of positively charged amino acids in the HS-binding site reduced cell surface affinity and attenuated virus infectivity via conformational changes. These intrinsic characteristics of the capsid suggested that conformational dynamics is critical for the structural changes occurring upon cell surface receptor binding, supporting a dynamics-based mechanism of receptor binding.
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http://dx.doi.org/10.1016/j.isci.2020.101547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519355PMC
October 2020

Nucleolar protein NPM1 is essential for circovirus replication by binding to viral capsid.

Virulence 2020 12;11(1):1379-1393

MOA Key Laboratory of Animal Virology, Center of Veterinary Sciences, Zhejiang University , Hangzhou, Zhejiang, PR China.

Entry of circovirus into the host cell nucleus is essential for viral replication during the early stage of infection. However, the mechanisms by which nucleolar shuttle proteins are used during viral replication is still not well understood. Here, we report a previously unidentified nucleolar localization signal in circovirus capsid protein (Cap), and that circovirus hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate its replication. Colocalization analysis showed that NPM1 translocates from the nucleolus to the nucleoplasm and cytoplasm during viral infection. Coimmunoprecipitation and glutathione -transferase pull-down assays showed that Cap interacts directly with NPM1. Binding domain mapping showed that the arginine-rich N-terminal motif MTYPYHPSHLG of Cap, and residue serine-48 of the N-terminal oligomerization domain of NPM1, are essential for the interaction. Virus rescue experiments showed that all arginine to alanine substitution in the N-terminal arginine-rich motif of Cap resulted in diminished viral replication. Knockdown of and substitution of serine-48 in NPM1 to glutamic acid also decreased viral replication. In addition, binding assays showed that the arginine-rich motif of Cap is a nucleolar localization signal. Taken together, our findings demonstrate that circovirus protein Cap is a nucleolus-located, and regulates viral replication by directly binding to NPM1.
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http://dx.doi.org/10.1080/21505594.2020.1832366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575006PMC
December 2020

Iron status is linked to disease severity after avian influenza virus H7N9 infection.

Asia Pac J Clin Nutr 2020 ;29(3):593-602

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, PR China. Email:

Background And Objectives: The high mortality rate of H7N9 strain of avian influenza virus (AIV) infected patients has been a major clinical concern. Iron overload increases the susceptibility of host for several kinds of microbial infection. However, the study on patients' iron and ferritin status associated with clinical outcome of AIVH7N9 virus infection is poorly understood, and in order to explain the linkage we carried out this study.

Methods And Study Design: We retrospectively collected serum from 46 patients infected with H7N9 virus from the hospital in Hangzhou city, Zhejiang province of China in 2013. We measured the level of serum iron and ferritin by Enzyme-Linked Immunosorbent Assay (ELISA). The correlation analysis of iron and ferritin with disease severity was done by SPSS 16.0 and MedCalc Software.

Results: After H7N9 infection, there is a reduction in iron level and an increase in ferritin, hepcidin and C-reactive protein (CRP) level in patient's serum compared to those of the control (p<0.001), and there's little correlation between procalcitonin (PCT) level and H7N9 infection. At week 1 and week 2 post-infection, serum iron level is much lower and ferritin level is much higher in the patients who died later than those in the patients who survived. The sensitivity, specificity, and Area Under the Curve (AUC) of the assay was calculated with MedCalc software and they were 85.5%, 65.9% and 0.803 for iron and 84.9%, 80.7% and 0.900 for ferritin, 95.2%, 51.1% and 0.684 for PCT and 100%, 94.6% and 0.988 for CRP, respectively.

Conclusions: Our study found that low serum iron and high serum ferritin levels are correlated with the disease severity of H7N9-infected patients and can predict fatal outcomes.
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http://dx.doi.org/10.6133/apjcn.202009_29(3).0019DOI Listing
July 2021

Cytoplasmic Cargo Receptor p62 Inhibits Avibirnavirus Replication by Mediating Autophagic Degradation of Viral Protein VP2.

J Virol 2020 11 23;94(24). Epub 2020 Nov 23.

MOA Key Laboratory of Animal Virology, Center for Veterinary Sciences, Zhejiang University, Hangzhou, China

Selective autophagy regulates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and aggregated proteins. Furthermore, autophagy is capable of degrading avibirnavirus, but the mechanism responsible for this process is unclear. Here, we show that autophagy cargo receptor p62 regulates the degradation of the avibirnavirus capsid protein VP2. Binding of p62 to VP2 enhances autophagic induction and promotes autophagic degradation of viral protein VP2. Further study showed that the interaction of p62 with viral protein VP2 is dependent on ubiquitination at the K411 site of VP2 and the ubiquitin-associated domain of p62. Mutation analysis showed that the K411R mutation of viral protein VP2 prohibits its p62-mediated degradation. Consistent with this finding, p62 lacking the ubiquitin-associated domain or the LC3-interacting region no longer promoted the degradation of VP2. Virus production revealed that the knockout of p62 but not the overexpression of p62 promotes the replication of avibirnavirus. Collectively, our findings suggest that p62 mediates selective autophagic degradation of avibirnavirus protein VP2 in a ubiquitin-dependent manner and is an inhibitor of avibirnavirus replication. Avibirnavirus causes severe immunosuppression and mortality in young chickens. VP2, the capsid protein of avibirnavirus, is responsible for virus assembly, maturation, and replication. Previous study showed that avibirnavirus particles could be engulfed into the autophagosome and degradation of virus particles took apart. Selective autophagy is a highly specific and regulated degradation pathway for the clearance of damaged or unwanted cytosolic components and superfluous organelles as well as invading microbes. However, whether and how selective autophagy removes avibirnavirus capsids is largely unknown. Here, we have shown that selective autophagy specifically clears ubiquitinated avibirnavirus protein VP2 by p62 recognition and that p62 is an inhibitor of avibirnavirus replication, highlighting the role of p62 as a potential drug target for mediating the removal of ubiquitinated virus components from cells.
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http://dx.doi.org/10.1128/JVI.01255-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925189PMC
November 2020

Rabies virus phosphoprotein P5 binding to BECN1 regulates self-replication by BECN1-mediated autophagy signaling pathway.

Cell Commun Signal 2020 09 18;18(1):153. Epub 2020 Sep 18.

MOA Key Laboratory of Animal Virology, Center for Veterinary Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, PR China.

Background: Rabies virus (RABV) is reported to encode five phosphoproteins (P), which are involved in viral genomic replication, axonal transport, oxidative stress, interferon antagonism, and autophagy induction. However, the functions of the different P proteins are poorly understood.

Methods: Immunofluorescence staining and western blot were performed to detect the autophagy activity, the form of ring-like structure, and the colocalization of BECN1 and P. Co-immunoprecipitation was performed to detect the interaction between P and BECN1. QRT-PCR and TCID assay were performed to detect the replication level of RABV. Small interfering RNA was used to detect the autophagy signaling pathway.

Results: We found that P5 attaches to N-terminal residues 1-139 of BECN1 (beclin1) on the BECN1 ring-like structure through amino acid residues 173-222 of P5. Subsequently, we found that P5-induced autophagosomes did not fuse with lysosomes. Becn1 silencing did not recover P5 overexpression-induced promotion of RABV replication. Mechanistically, RABV protein PΔN82 (P5) induced incomplete autophagy via the BECN1-mediated signaling pathway.

Conclusions: Our data indicate that P5 binding to the BECN1 ring benefits RABV replication by inducing BECN1 signaling pathway-dependent incomplete autophagy, which provides a potential target for antiviral drugs against RABV. Video abstract.
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http://dx.doi.org/10.1186/s12964-020-00644-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499888PMC
September 2020

PDPK1 regulates autophagosome biogenesis by binding to PIK3C3.

Autophagy 2020 Sep 10:1-18. Epub 2020 Sep 10.

MOA Key Laboratory of Animal Virology, Center for Veterinary Sciences, Zhejiang University, Hangzhou, China.

PDPK1 (3-phosphoinositide dependent protein kinase 1) is a phosphorylation-regulated kinase that plays a central role in activating multiple signaling pathways and cellular processes. Here, this study shows that PDPK1 turns on macroautophagy/autophagy as a SUMOylation-regulated kinase. data demonstrate that the SUMO modification of PDPK1 is a physiological feature in the brain and that it can be induced by viral infections. The SUMOylated PDPK1 regulates its own phosphorylation and subsequent activation of the AKT1 (AKT serine/threonine kinase 1)-MTOR (mechanistic target of rapamycin kinase) pathway. However, SUMOylation of PDPK1 is inhibited by binding to PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3). The nonSUMOylated PDPK1 then tethers LC3 to the endoplasmic reticulum to initiate autophagy, and it acts as a key component in forming the autophagic vacuole. Collectively, this study reveals the intricate molecular regulation of PDPK1 by post-translational modification in controlling autophagosome biogenesis, and it highlights the role of PDPK1 as a sensor of cellular stress and regulator of autophagosome biogenesis. AKT1: AKT serine/threonine kinase 1; ATG14: autophagy related 14; Co-IP: co-immunoprecipitation; ER: endoplasmic reticulum; hpi: hours post-infection; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; pAb: polyclonal antibody; PDPK1: 3-phosphoinositide dependent protein kinase 1; PI3K: phosphoinositide 3-kinase; PIK3C3: phosphatidylinositol 3-kinase catalytic, subunit type 3; RPS6KB1: ribosomal protein S6 kinase B1; SGK: serum/glucocorticoid regulated kinase; SQSTM1: sequestosome 1; SUMO: small ubiquitin like modifier; UBE2I/UBC9: ubiquitin conjugating enzyme E2 I; UVRAG: UV radiation resistance associated.
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http://dx.doi.org/10.1080/15548627.2020.1817279DOI Listing
September 2020

Construction of an infectious bronchitis virus vaccine strain carrying chimeric S1 gene of a virulent isolate and its pathogenicity analysis.

Appl Microbiol Biotechnol 2020 Oct 19;104(19):8427-8437. Epub 2020 Aug 19.

Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, People's Republic of China.

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5' end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3' end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity. KEY POINTS: • BAC vector was used to construct IBV full-length cDNA by homologous recombination. • Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued. • Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.
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http://dx.doi.org/10.1007/s00253-020-10834-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434845PMC
October 2020

Heat Shock Protein Member 8 Is an Attachment Factor for Infectious Bronchitis Virus.

Front Microbiol 2020 10;11:1630. Epub 2020 Jul 10.

Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, China.

Although infectious bronchitis virus (IBV) is the first coronavirus identified, little is known about which membrane protein of host cells could interact with IBV spike protein and facilitate the infection by the virus. In this study, by using a monoclonal antibody to the S1 protein of IBV M41 strain, we found that heat shock protein member 8 (HSPA8) could interact with spike protein of IBV. HSPA8 was found to be present on the cell membrane and chicken tissues, with highest expression level in the kidney. Results of co-IP and GST-pull-down assays indicated that the receptor binding domain (RBD) of IBV M41 could interact with HSPA8. The results of binding blocking assay and infection inhibition assay showed that recombinant protein HSPA8 and antibody to HSPA8 could inhibit IBV M41 infection of chicken embryonic kidney (CEK) cells. Further, we found that HSPA8 interacted with the N-terminal 19-272 amino acids of S1 of IBV Beaudette, H120 and QX strains and HSPA8 from human and pig also interacted with IBV M41-RBD. Finally the results of binding blocking assay and infection inhibition assay showed that recombinant HSPA8 protein and antibody to HSPA8 could inhibit IBV Beaudette strain infection of Vero cells that were treated with heparanase to remove heparan sulfate from the cell surface. Taken together, our results indicate that HSPA8 is a novel host factor involved in IBV infection.
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http://dx.doi.org/10.3389/fmicb.2020.01630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381282PMC
July 2020

Molecular characterization of an emerging reassortant mammalian orthoreovirus in China.

Arch Virol 2020 Oct 5;165(10):2367-2372. Epub 2020 Aug 5.

Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 427 Maduan Street, Nangang District, Harbin, 150001, China.

Mammalian orthoreoviruses (MRVs) infect almost all mammals, and there are some reports on MRVs in China. In this study, a novel strain was identified, which was designated as HLJYC2017. The results of genetic analysis showed that MRV HLJYC2017 is a reassortant strain. According to biological information analysis, different serotypes of MRV contain specific amino acid insertions and deletions in the σ1 protein. Neutralizing antibody epitope analysis revealed partial cross-protection among MRV1, MRV2, and MRV3 isolates from China. L3 gene recombination in MRV was identified for the first time in this study. The results of this study provide valuable information on MRV reassortment and evolution.
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http://dx.doi.org/10.1007/s00705-020-04712-5DOI Listing
October 2020

HFE inhibits type I IFNs signaling by targeting the SQSTM1-mediated MAVS autophagic degradation.

Autophagy 2021 08 18;17(8):1962-1977. Epub 2020 Aug 18.

MOA Key Laboratory of Animal Virology, Center for Veterinary Sciences, Zhejiang University, Hangzhou, PR China.

Iron metabolism is involved in numerous physiological processes such as erythropoiesis, oxidative metabolism. However, the physiological functions of the iron metabolism-related gene in immune response during viral infection remain poorly understood. Here, we identified 5 iron metabolism-associated genes specifically affected during RNA virus infection by a high-throughput assay and further found that HFE was a key negative regulator of RIG-I-like receptors (RLR)-mediated type I interferons (IFNs) signaling. RNA virus infection inhibited the binding of HFE to MAVS (mitochondrial antiviral signaling protein) and blocked MAVS degradation via selective autophagy. HFE mediated MAVS autophagic degradation by binding to SQSTM1/p62. Depletion of abrogated the autophagic degradation of MAVS, leading to the stronger antiviral immune response. These findings established a novel regulatory role of selective autophagy in innate antiviral immune response by the iron metabolism-related gene . These data further provided insights into the crosstalk among iron metabolism, autophagy, and innate immune response. ATG: autophagy-related; BAL: bronchoalveolar lavage fluid; BMDMs: bone marrow-derived macrophages; CGAS: cyclic GMP-AMP synthase; CQ: chloroquine; Dpi: days post-infection; ELISA: enzyme-linked immunosorbent assay; GFP: green fluorescent protein; HAMP: hepcidin antimicrobial peptide; Hpi: hours post-infection; HJV: hemojuvelin BMP co-receptor; IFNs: interferons; IL6: interleukin 6; IRF3: interferon regulatory factor 3; ISRE: interferon-stimulated response element; Lipo: clodronate liposomes; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MEFs: mouse embryonic fibroblasts; SLC40A1/FPN1: solute carrier family 40 (iron-regulated transporter), member 1; flatiron; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1/STING: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TFRC/TfR1: transferrin receptor; TNF/TNFα: tumor necrosis factor; WT: wild type.
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http://dx.doi.org/10.1080/15548627.2020.1804683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386699PMC
August 2021

Snapshot of the evolution and mutation patterns of SARS-CoV-2.

bioRxiv 2020 Jul 5. Epub 2020 Jul 5.

The COVID-19 pandemic is the most important public health threat in recent history. Here we study how its causal agent, SARS-CoV-2, has diversified genetically since its first emergence in December 2019. We have created a pipeline combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in a data set consisting of 4,894 SARS-CoV-2 complete genome sequences. Although the phylogenetic diversity of SARS-CoV-2 is low, the whole genome phylogenetic tree can be divided into five clusters/clades based on the tree topology and clustering of specific mutations, but its branches exhibit low genetic distance and bootstrap support values. We also identified 11 residues that are high-frequency substitutions, with four of them currently showing some signal for potential positive selection. These fast-evolving sites are in the non-structural proteins nsp2, nsp5 (3CL-protease), nsp6, nsp12 (polymerase) and nsp13 (helicase), in accessory proteins (ORF3a, ORF8) and in the structural proteins N and S. Temporal and spatial analysis of these potentially adaptive mutations revealed that the incidence of some of these sites was declining after having reached an (often local) peak, whereas the frequency of other sites is continually increasing and now exhibit a worldwide distribution. Structural analysis revealed that the mutations are located on the surface of the proteins that modulate biochemical properties. We speculate that this improves binding to cellular proteins and hence represents fine-tuning of adaptation to human cells. Our study has implications for the design of biochemical and clinical experiments to assess whether important properties of SARS-CoV-2 have changed during the epidemic.
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http://dx.doi.org/10.1101/2020.07.04.187435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337380PMC
July 2020

Differential CircRNA Expression Profiles in PK-15 Cells Infected with Pseudorabies Virus Type II.

Virol Sin 2021 Feb 2;36(1):75-84. Epub 2020 Jul 2.

MOA Key Laboratory of Animal Virology, Center of Veterinary Sciences, Zhejiang University, Hangzhou, 310058, China.

Circular RNAs (circRNAs) belong to a class of non-coding RNAs with diverse biological functions. However, little is known about their roles in case of pseudorabies virus (PrV) infection. Here, we analyzed the expression profile of host circRNAs from a virulent PrV type II strain DX (PrV-DX) infected and an attenuated gE/TK deficient (gETKPrV) strain of PrV infected PK-15 cells. CircRNAs were identified by find_circ and analyzed with DESeq 2. Compared with the mock cells, 449 differentially expressed (DE) circRNAs (233 down-regulated and 216 up-regulated) from PrV-DX infected and 578 DE circRNAs (331 down-regulated and 247 up-regulated) from gETK PrV infected PK-15 cells were identified. In addition, 459 DE circRNAs (164 down-regulated and 295 up-regulated) between the PrV-DX and gETKPrV infected cells were identified. The expression patterns of 13 circRNAs were validated by reverse transcription quantitative real-time PCR (RT-qPCR) and results were similar as of RNA-seq. The putative target miRNA binding sites of DE circRNAs were predicted by using miRanda and psRobot. The circRNA-miRNA-mRNA network was constructed and certain miRNAs that have possible roles in antiviral immune response, such as miR-210 and miR-340, were predicted. GO and KEGG pathway analysis demonstrated that DE circRNAs were enriched in the processes such as cellular metabolism, protein binding, RNA degradation and regulation of actin cytoskeleton. Collectively, these findings might provide the useful information for a better understanding of mechanisms underlying the interaction between PrV-II and host cells.
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http://dx.doi.org/10.1007/s12250-020-00255-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973350PMC
February 2021

NAP1L4 inhibits porcine circovirus type 2 replication via IFN-β signaling pathway.

Vet Microbiol 2020 Jul 17;246:108692. Epub 2020 May 17.

Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. Electronic address:

Porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to interact with nucleosome assembly protein 1-like 4 (NAP1L4). The biological function of Cap-NAP1L4 interaction is unknown. Here, we demonstrated that PCV2 Cap could directly interact with NAP1L4, which the amino acid residues 124-279 of NAP1L4 were responsible for the interaction. Furthermore, over-expression of NAP1L4 down-regulated the expression of PCV2 Cap and Rep. DNA copies and virus titers were also decreased in NAP1L4 over-expressed PK15 cells. While, knockout of NAP1L4 through CRISPR/Cas9 technology in PK15 cells could up-regulate the mRNA and protein levels of PCV2 Cap and Rep. PCV2 genomic DNA copies and virus titers were also increased in NAP1L4-knockdown/-knockout PK15 cells compared with wild type PK15 cells. In addition, NAP1L4 depletion was demonstrated to facilitate cytosolic carboxypeptidase-like protein 5 (CCP5) and cytosolic carboxypeptidase 6 (CCP6) expression, which could activate cGAS to promote IFN-β production. Indeed, knockout of NAP1L4 was also confirmed to increase IFN-β expression. And IFN-β stimulation could promote PCV2 replication in PK15 cells. Taken together, our findings suggest that NAP1L4 interacts with PCV2 Cap and inhibits PCV2 replication through regulating IFN-β production. Our study provides theoretical basis for further study of PCV2.
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http://dx.doi.org/10.1016/j.vetmic.2020.108692DOI Listing
July 2020

Protein Interactions Network of Porcine Circovirus Type 2 Capsid With Host Proteins.

Front Microbiol 2020 3;11:1129. Epub 2020 Jun 3.

MOA Key Laboratory of Animal Virology, Department of Veterinary Medicine and Center of Veterinary Medical Sciences, Zhejiang University, Hangzhou, China.

Virus-host interaction is a tug of war between pathogenesis and immunity, followed by either activating the host immune defense system to eliminate virus or manipulating host immune control mechanisms to survive and facilitate virus propagation. Comprehensive knowledge of interactions between host and viral proteins might provide hints for developing novel antiviral strategies. To gain a more detailed knowledge of the interactions with porcine circovirus type 2 capsid protein, we employed a coimmunoprecipitation combined with liquid chromatography mass spectrometry (LC-MS) approach and 222 putative PCV2 Cap-interacting host proteins were identified in the infected porcine kidney (PK-15) cells. Further, a protein-protein interactions (PPIs) network was plotted, and the PCV2 Cap-interacting host proteins were potentially involved in protein binding, DNA transcription, metabolism and innate immune response based on the gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes database enrichment. Verification assay demonstrated that eight cellular proteins, namely heterogeneous nuclear ribonucleoprotein C, nucleophosmin-1, DEAD-box RNA helicase 21, importin β3, eukaryotic translation initiation factor 4A2, snail family transcriptional repressor 2, MX dynamin like GTPase 2, and intermediate chain 1 interacted with PCV2 Cap. Thus, this work effectively provides useful protein-related information to facilitate further investigation of the underlying mechanism of PCV2 infection and pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2020.01129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283462PMC
June 2020

ATM-mediated DNA double-strand break response facilitated oncolytic Newcastle disease virus replication and promoted syncytium formation in tumor cells.

PLoS Pathog 2020 06 1;16(6):e1008514. Epub 2020 Jun 1.

Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute. Chinese Academy of Agricultural Science, Shanghai, P.R. China.

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.
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http://dx.doi.org/10.1371/journal.ppat.1008514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263568PMC
June 2020

Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses.

J Virol 2020 07 30;94(16). Epub 2020 Jul 30.

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an , as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the -adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses. Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.
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http://dx.doi.org/10.1128/JVI.00447-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394890PMC
July 2020

COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives.

Trends Mol Med 2020 05 21;26(5):483-495. Epub 2020 Mar 21.

MOE Joint International Research Laboratory of Animal Health and Food Safety, Jiangsu Engineering Laboratory of Animal Immunology, Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. Electronic address:

The recent outbreak of COVID-19 in Wuhan turned into a public health emergency of international concern. With no antiviral drugs nor vaccines, and the presence of carriers without obvious symptoms, traditional public health intervention measures are significantly less effective. Here, we report the epidemiological and virological characteristics of the COVID-19 outbreak. Originated in bats, 2019-nCoV/ severe acute respiratory syndrome coronavirus (SARS-CoV)-2 likely experienced adaptive evolution in intermediate hosts before transfer to humans at a concentrated source of transmission. Similarities of receptor sequence binding to 2019-nCoV between humans and animals suggest a low species barrier for transmission of the virus to farm animals. We propose, based on the One Health model, that veterinarians and animal specialists should be involved in a cross-disciplinary collaboration in the fight against this epidemic.
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http://dx.doi.org/10.1016/j.molmed.2020.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118693PMC
May 2020

Preparation of Monoclonal Antibody Against EMA-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China.

J Parasitol 2020 04;106(2):283-290

Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, 310058, P. R. China.

The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area.
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http://dx.doi.org/10.1645/19-98DOI Listing
April 2020

Identification of functional lncRNAs in pseudorabies virus type II infected cells.

Vet Microbiol 2020 Mar 23;242:108564. Epub 2019 Dec 23.

MOA Key Laboratory of Animal Virology, Department of Veterinary Sciences, Zhejiang University, Hangzhou 310058, China; Institute of Animal Preventive Sciences, Department of Veterinary Sciences, Zhejiang University, Hangzhou 310058, China; Collaborative Innovation Center and State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou 310058, China. Electronic address:

Long noncoding RNAs (lncRNAs) play important roles in the antiviral responses. However, little is known about the identification and functions of swine lncRNAs in response to pseudorabies virus type II (PRV-II). Here, we detected the expression profiles of host lncRNAs from a wild-type (PRV-II DX) and gE/TK deficient (gE-TK-PRV) PRV-II infected cells. RNA-seq identified 664 differentially expressed (DE) lncRNAs from PRV-DX infected cells, 654 DE lncRNAs from gE-TK-PRV infected cells and 276 DE lncRNAs between PRV-DX and gE-TK-PRV infected cells. The potential functions of the significant differentially expressed (SDE) lncRNAs were involved in interleukin secretion, axon extension and metabolic process based on the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, the expression patterns of sixteen SDE lncRNAs determined by RT-qPCR exhibited high correlation (r > 0.95) with those by RNA-seq results. Western blotting assay displayed the lncA02830 did not code for protein, and the silencing of lncA02830 could significantly up-regulate the transcription levels of IRF3, IFNβ as well as MX1 and inhibit the replication of PRV-II. Taken together, these data highlighted the potentials of lncRNA as targets for antiviral therapy and provided some novel knowledge of the mechanisms underlying the host interaction with PRV-II.
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http://dx.doi.org/10.1016/j.vetmic.2019.108564DOI Listing
March 2020

Genotyping (PCV-3) Nowadays: Does It Make Sense?

Viruses 2020 02 28;12(3). Epub 2020 Feb 28.

Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, Spain.

The discovery of a globally distributed porcine circovirus (; PCV-3) has led to intense research activity and the production of a large amount of molecular data. Different research groups have proposed several, not always concordant, genotypes for this virus. While such categories could aid an easier interpretation of PCV-3 molecular epidemiology, any classification, to be useful in practical settings, must be univocal and of help in the understanding of underlying biological features and epidemiology. Based on these premises, the possibility of defining PCV-3 genotypes was evaluated on the broadest available dataset of PCV-3 complete genome ( = 357) and open reading frame 2 (ORF2, = 653) sequences. Genetic distance and phylogenetic clustering were selected as the main objective criteria. Additional factors, including the number of within-cluster sequences, host and geographic clustering, concordance between different genomic regions, and analysis method were also taken in account to generate a classification that could be effectively applied in research and diagnostic settings. A maximum within-genotype genetic distance of 3% at the complete genome and 6% at the ORF2 levels, bootstrap support higher than 90%, and concordance between analysis methods allowed us to clearly define two clades which could be potentially defined as genotypes. Further subdivision was not suggested due to the absence of a meaningful association between PCV-3 and its biological/epidemiological features. Nevertheless, since one of the clades included two strains only, thus far we formally propose the definition of only one PCV-3 genotype (PCV-3a). The established criteria will allow us to automatically recognize other genotypes when more strain sequences are characterized.
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http://dx.doi.org/10.3390/v12030265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150946PMC
February 2020

Comprehensive Genomic Characterization Analysis of lncRNAs in Cells With Porcine Delta Coronavirus Infection.

Front Microbiol 2019 28;10:3036. Epub 2020 Jan 28.

MOE International Joint Collaborative Research Laboratory for Animal Health and Food Safety, Institute of Immunology, Nanjing Agricultural University, Nanjing, China.

Porcine delta coronavirus (PDCoV) is a novel emerging enterocytetropic virus causing diarrhea, vomiting, dehydration, and mortality in suckling piglets. Long non-coding RNAs (lncRNAs) are known to be important regulators during virus infection. Here, we describe a comprehensive transcriptome profile of lncRNA in PDCoV-infected swine testicular (ST) cells. In total, 1,308 annotated and 1,190 novel lncRNA candidate sequences were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these lncRNAs might be involved in numerous biological processes. Clustering analysis of differentially expressed lncRNAs showed that 454 annotated and 376 novel lncRNAs were regulated after PDCoV infection. Furthermore, we constructed a lncRNA-protein-coding gene co-expression interaction network. The KEGG analysis of the co-expressed genes showed that these differentially expressed lncRNAs were enriched in pathways related to metabolism and TNF signaling. Our study provided comprehensive information about lncRNAs that would be a useful resource for studying the pathogenesis of and designing antiviral therapy for PDCoV infection.
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http://dx.doi.org/10.3389/fmicb.2019.03036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999024PMC
January 2020

Antiviral Effect of Epigallocatechin Gallate via Impairing Porcine Circovirus Type 2 Attachment to Host Cell Receptor.

Viruses 2020 02 4;12(2). Epub 2020 Feb 4.

Institute of Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.

The green tea catechin epigallocatechin gallate (EGCG) exhibits antiviral activity against various viruses. Whether EGCG also inhibits the infectivity of circovirus remains unclear. In this study, we demonstrated the antiviral effect of EGCG on porcine circovirus type 2 (PCV2). EGCG targets PCV2 virions directly and blocks the attachment of virions to host cells. The microscale thermophoresis assay showed EGCG could interact with PCV2 capsid protein in vitro with considerable affinity (Kd = 98.03 ± 4.76 μM), thereby interfering with the binding of the capsid to the cell surface receptor heparan sulfate. The molecular docking analysis of capsid-EGCG interaction identified the key amino acids which formed the binding pocket accommodating EGCG. Amino acids ARG51, ASP70, ARG73 and ASP78 of capsid were found to be critical for maintaining the binding, and the arginine residues were also essential for the electrostatic interaction with heparan sulfate. The rescued mutant viruses also confirm the importance of the key amino acids of the capsid to the antiviral effect of EGCG. Our findings suggest that catechins could act as anti-infective agents against circovirus invasion, as well as provide the basic information for the development and synthesis of structure-based anti-circovirus drugs.
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http://dx.doi.org/10.3390/v12020176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077276PMC
February 2020
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