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H7N9 virus infection triggers lethal cytokine storm by activating gasdermin E-mediated pyroptosis of lung alveolar epithelial cells

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National Science Review 2022年第1期9卷 47-55页

作者： Xiaopeng Wan Jiqing Li Yupeng Wang Xiaofei Yu Xijun He Jianzhong Shi Guohua Deng Xianying Zeng Guobin Tian Yanbing Li Yongping Jiang Yuntao Guan Chengjun Li Feng Shao Hualan Chen State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences National institute of Biological Sciences

The H7 N9 influenza virus emerged in China in 2013,causing more than 1560 human infections,39% of which were fatal.A ’cytokine storm’ in the lungs of H7 N9 patients has been linked to a poor prognosis and death;however,the underlying mechanism that triggers the cytokine storm is unknown.Here,we found that efficient replication of the H7 N9 virus in mouse lungs activates gasdermin E(GSDME)-mediated pyroptosis in alveolar epithelial cells,and that the released cytosolic contents then trigger a cytokine storm.Knockout of Gsdme switched the manner of death of A549 and human primary alveolar epithelial cells from pyroptosis to apoptosis upon H7 N9 virus infection,and Gsdme knockout mice survived H7 N9 virus lethal infection.Our findings reveal that GSDME activation is a key and unique mechanism for the pulmonary cytokine storm and lethal outcome of H7 N9 virus infection and thus opens a new door for the development of antivirals against the H7 N9 virus.

关键词： H7N9 virus cytokine storm gasdermin E pyroptosis

Prevalence and molecular epidemiology of equine piroplasmosis in China:a neglected tick-borne disease

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Science China(Life Sciences) 2022年第9期65卷 445-447页

作者： Kewei Chen Zhe Hu Jingkun Li Jingfei Wang Diqiu Liu Ting Qi Wei Guo Cheng Du Xiaojun Wang State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China Department of Biostatistics School of Public HealthHarbin Medical UniversityHarbin 150081China

Dear Editor,Equine piroplasmosis(EP)is an infectious,tick-borne dis-ease caused by the hemoprotozoan parasites Theileria equi(T.equi)and Babesia caballi(B.caballi).EP affects all wild and domestic equids,and clinical presentation is related to intravascular hemolysis and associated systemic illness(Scoles and Ueti,2015).This disease has considerable ve-terinary and economic impact on the equine industry worldwide,and is listed as a reportable disease by the World Organization for Animal Health(OIE).Importantly,a ne-gative EP test is mandatory for animals participating in equestrian sports and the horse trade.It is estimated that 90%of the global horse population resides in EP-endemic areas.Many risk factors are associated with the infection rate,in-cluding animal species,age,gender,breed,activities,and environmental factors(Onyiche et al.,2019).

关键词： horse epidemiology listed

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M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes

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Science China(Life Sciences) 2024年第3期67卷 579-595页

作者： Yuzhen Hu Li Jiang Guangwen Wang Yangming Song Zhibo Shan Xuyuan Wang Guohua Deng Jianzhong Shi Guobin Tian Xianying Zeng Liling Liu Hualan Chen Chengjun Li State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China

Influenza A virus(IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor(M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR,the nuclear accumulation of viral nucleoprotein(NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking,or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin(HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes,thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR–HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

关键词： influenza A virus M6PR HA membrane fusion late endosome

Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

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Journal of Integrative Agriculture 2022年第7期21卷 2095-2105页

作者： ZHAO Yu-hui WEN Xia LI Qi-bing JIANG Li WANG Guang-wen LIANG Li-bin WANG Xiu-rong CHEN Hua-lan LI Cheng-jun State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

关键词： influenza A virus nucleoprotein monoclonal antibody application

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

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Journal of Integrative Agriculture 2022年第3期21卷 819-825页

作者： WANG Peng-fei WANG Ming SHI Zhi-bin SUN Zhen-zhao WEI Li-li LIU Zai-si WANG Shi-da HE Xi-jun WANG Jing-fei State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

关键词： African swine fever virus pB602L recombinant protein indirect ELISA assay pig

TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2

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Protein & Cell 2020年第12期11卷 894-914页

作者： Nan Sun Li Jiang Miaomiao Ye Yihan Wang Guangwen Wang Xiaopeng Wan Yuhui Zhao Xia Wen Libin Liang Shujie Ma Liling Liu Zhigao Bu Hualan Chen Chengjun Li State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China

Tripartite motif(TRIM)family proteins are important effectors of innate immunity against viral infections.Here we identified TRIM35 as a regulator of TRAF3 activation.Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon(IFN)in response to viral infection.777m35-deficient mice were more susceptible to influenza A virus(IAV)infection than were wild-type mice.TRIM35 promoted the RIG-Imediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1.IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3.TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2,thereby antagonizing its suppression of TRAF3 activation.Our in vitro and in vivo findings thus reveal novel roles of TRIM35,through catalyzing Lys63-or Lys48-linked polyubiquitination,in RIG-I antiviral immunity and mechanism of defense against IAV infection.

关键词： influenza A virus PB2 TRIM35 TRAF3 ubiquitination antiviral immunity

Next-generation sequencing and single-cell RT-PCR reveal a distinct variable gene usage of porcine antibody repertoire following PEDV vaccination

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Science China(Life Sciences) 2020年第8期63卷 1240-1250页

作者： Ren Li Fang Fu Li Feng PingHuang Liu State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150001China

Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.

关键词： porcine epidemic diarrhea virus(PEDV) next-generation sequencing(NGS) single-cell RT-PCR antibody repertoire

Generation and application of replication-competent Venus-expressing H5N1,H7N9,and H9N2 influenza A viruses

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Science Bulletin 2018年第3期63卷 176-186页

作者： guangwen wang jie zhang fandi kong qibing li jinliang wang shujie ma yuhui zhao libin liang junping li nan sun lizheng guan yuan zhou chenchen zhou shanyu huang zhigao bu li jiang hualan chen chengjun li State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences Harbin 150069 China

The generation and application of replication-competent influenza A virus(IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase(NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses,used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.

关键词：流行性感冒病毒 H9N2 维纳斯复制 H5N1 tial 种子类型联合使用

Vaccination of poultry successfully eliminated human infection with H7N9 virus in China

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Science China(Life Sciences) 2018年第12期61卷 1465-1473页

作者： Xianying Zeng Guobin Tian Jianzhong Shi Guohua Deng Chengjun Li Hualan Chen State Key Laboratory of veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China

The H7 N9 viruses that emerged in China in 2013 were nonpathogenic in chickens but mutated to a highly pathogenic form in early 2017 and caused severe disease outbreaks in chickens. The H7 N9 influenza viruses have caused five waves of human infection, with almost half of the total number of human cases(766 of 1,567) being reported in the fifth wave, raising concerns that even more human infections could occur in the sixth wave. In September 2017, an H5/H7 bivalent inactivated vaccine for chickens was introduced, and the H7 N9 virus isolation rate in poultry dropped by 93.3% after vaccination. More importantly,only three H7 N9 human cases were reported between October 1, 2017 and September 30, 2018, indicating that vaccination of poultry successfully eliminated human infection with H7 N9 virus. These facts emphasize that active control of animal disease is extremely important for zoonosis control and human health protection.

关键词： H7N9 influenza virus evolution vaccination human infection elimination