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Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

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Journal of Integrative Agriculture 2017年第9期16卷 2055-2061页

作者： WANG Xiu-rong GU Lin-lin SHI Jian-zhong XU Hai-feng ZHANG Ying ZENG Xian-ying DENG Guo-hua LI cheng-jun chen hua-lan State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin（HA） cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

关键词： H7N9 highly pathogenic influenza virus real-time RT-PCR

Transcriptomic analyses reveal new genes and networks response to H5N1 influenza viruses in duck(Anas platyrhynchos)

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Journal of Integrative Agriculture 2019年第7期18卷 1460-1472页

作者： huaNG Yin-hua FENG hua-peng huaNG Li-ren YI Kang RONG En-guang chen Xiao-yun LI Jian-wen WANG Zeng ZHU Peng-yang LIU Xiao-juan WANG Xiao-xue HU Jia-xiang LIU Xin chen hua-lan WANG Jun LI Ning State Key Laboratory for Agrobiotechnology China Agricultural UniversityBeijing 100193P.R. China State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China BGI-Shenzhen Shenzhen 518083P.R.China China Veterinary Culture Collection Center China Institute of Veterinary Drug ControlBeijing 100081P.R.China

H5N1 influenza represents one of the great challenges to public *** H5N1 viruses(i.e.,A/goose/Hubei/65/05,GS/65) are weakly pathogenic,while the others(i.e.,A/duck/Hubei/49/05,DK/49) are highly pathogenic to their natural ***,we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 *** demonstrated that,compared to the GS/65 virus,the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways(i.e.,RIG-I and MDA5) in *** found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes,while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 *** also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis(WGCNA).Moreover,we first demonstrated that the duck BCL2 L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication *** analyses,in combination with our comprehensive transcriptomic data,provided global view of the molecular architecture for the interaction between host and H5N1 viruses.

关键词： duck innate immune genes H5N1 influenza viruses transcriptomes

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

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畜牧兽医学报 2010年第S1期41卷 76-81页

作者： WU Yun-pu QIAO Chuan-ling YANG huan-liang chen Yan ZHAN Xiao-guo XIN Xiao-guang chen hua-lan Animal Influenza Laboratory of the Ministry of Agriculture State Key Laboratory of Veterinary BiotechnologyHarbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150001China Liaoning Center for Animal Disease Control and Prevention Shenyang 110164China

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 *** recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot *** results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological *** TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and *** of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

关键词： swine influenza virus H5N1 subtype HA gene recombinant adenovirus

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

Protective efficacy of an H5/H7 trivalent inactivated vaccine(H5-Re13,H5-Re14, and H7-Re4 strains) in chickens, ducks, and geese against newly detected H5N1, H5N6, H5N8, and H7N9 viruses

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Journal of Integrative Agriculture 2022年第7期21卷 2086-2094页

作者： ZENG Xian-ying HE Xin-wen MENG Fei MA Qi WANG Yan BAO Hong-mei LIU Yan-jing DENG Guo-hua SHI Jian-zhong LI Yan-bing TIAN Guo-bin chen hua-lan State Key Laboratory of Veterinary Biotechnology National Poultry Laboratory Animal Resource CenterHarbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

Some H5 viruses isolated in poultry or wild birds between 2020 and 2021 were found to be antigenically different from the vaccine strains(H5-Re11 and H5-Re12) used in China. In this study, we generated three new recombinant vaccine seed viruses by using reverse genetics and used them for vaccine production. The vaccine strain H5-Re13 contains the hemagglutinin(HA) and neuraminidase(NA) genes of an H5 N6 virus that bears the clade 2.***.4.4 h HA gene, H5-Re14 contains the HA and NA genes of an H5 N8 virus that bears the clade 2.***.4.4 b HA gene, and H7-Re4 contains the HA and NA genes of H7 N9 virus detected in 2021. We evaluated the protective efficacy of the novel H5/H7 trivalent inactivated vaccine in chickens, ducks, and geese. The inactivated vaccine was immunogenic and induced substantial antibody responses in the birds tested. Three weeks after vaccination, chickens were challenged with five different viruses detected in 2020 and 2021: three viruses(an H5 N1 virus, an H5 N6 virus, and an H5 N8 virus) bearing the clade 2.***.4.4 b HA gene, an H5 N6 virus bearing the clade 2.***.4.4 h HA gene, and an H7 N9 virus. All of the control birds shed high titers of virus and died within 4 days post-challenge, whereas the vaccinated chickens were completely protected from these viruses. Similar protective efficacy against H5 viruses bearing the clade 2.***.4.4 h or 2.***.4.4 b HA gene was observed in ducks and geese. Our study indicates that the newly updated H5/H7 vaccine can provide solid protection against the H5 and H7 N9 viruses that are currently circulating in nature.

关键词： avian influenza H5/H7 trivalent vaccine H5-Re13 H5-Re14 H7-Re4 strains protective efficacy chickens ducks and geese

Establishing an ELISA,Based on the HA1 Protein,for Detecting Antibodies against H3N2 Subtype Swine Influenza Virus

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畜牧兽医学报 2009年第S1期40卷 57-61页

作者： DING Xuan-ya QIAO Chuan-ling chen Yan YANG huan-liang XIN Xiao-guang HAN Qing-gong chen hua-lan Animal Influenza Laboratory of the Ministry of Agriculture State Key Laboratory of Veterinary BiotechnologyHarbin Veterinary Research InstituteChinese Academy of Agricultural Sciences

The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named *** was transformed into *** BL21(DE3),and expressed by induction with *** expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was *** purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of *** assay has excellent specificity,sensitivity and *** 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.

关键词： swine influenza virus HA1 protein subtype specific ELISA

过氧化氢氧化二氯荧光素动力学荧光法测定痕量镍

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冶金分析 2007年第11期27卷 41-43页

作者：陈兰化严波淮北煤炭师范学院化学系安徽淮北235000 淮北煤炭师范学院化学系

在近中性介质中,痕量镍(Ⅱ)对过氧化氢氧化二氯荧光素的反应具有很强的催化作用,当加入痕量镍(Ⅱ)时,可使反应体系的荧光强度减小,据此建立了催化动力学荧光法测定痕量镍(Ⅱ)的新方法。反应体系的激发波长和发射波长分别为491 nm和517 ... 详细信息

在近中性介质中,痕量镍(Ⅱ)对过氧化氢氧化二氯荧光素的反应具有很强的催化作用,当加入痕量镍(Ⅱ)时,可使反应体系的荧光强度减小,据此建立了催化动力学荧光法测定痕量镍(Ⅱ)的新方法。反应体系的激发波长和发射波长分别为491 nm和517 nm,测定镍(Ⅱ)的线性范围为4.0~80.0μg/L,检出限为2.4μg/L。该法用于废水中痕量镍(Ⅱ)的测定,加标回收率为103.6%。

关键词：动力学荧光法二氯荧光素镍

Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens

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Journal of Integrative Agriculture 2020年第9期19卷 2294-2300页

作者： ZENG Xian-ying chen Xiao-han MA Shu-jie WU Jiao-jiao BAO Hong-mei PAN Shu-xin LIU Yan-jing DENG Guo-hua SHI Jian-zhong chen Pu-cheng JIANG Yong-ping LI Yan-bing HU Jing-lei LU Tong MAO Sheng-gang GUO Xing-fu LIU Jing-li TIAN Guo-bin chen hua-lan State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China Harbin Weike Biotechnology Development Company Harbin 150069P.R.China

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5N6) (DK/GZ/S4184/17) (a clade 2.***.4.4d virus), A/chicken/Liaoning/SD007/2017(H5N1) (CK/LN/SD007/17) (a clade 2.***.2.1d virus), and A/chicken/ Guangxi/SD098/2017(H7N9) (CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.

关键词： avian influenza H5/H7 trivalent vaccine Re-11 Re-12 and H7-Re2 strains protective efficacy

Subtype Identification of Avian Influenza Virus on DNA Microarray

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Agricultural Sciences in China 2005年第9期4卷 700-706页

作者： WANG Xiu-rong YU Kang-zhen DENG Guo-hua SHI Rui LIU Li-ling QIAO Chuan-ling BAO Hong-mei KONG Xian-gang chen hua-lan Animal Influenza Laboratory of the Ministry of Agriculture and National Key Laboratory of Veterinary Biotechnology of Harbin VeterinaryResearch Institute of Chinese Academy of Agricultural Sciences Harbin 150001 P.R.China

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

关键词： Avian influenza virus DNA microarray Subtype identification