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Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

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Biomedical and Environmental Sciences 2016年第1期29卷 41-46页

作者： Zou Xiao Hui Chen Wen Bing Zhao Xiang Zhu Wen Fei yang Lei wang da yan Shu Yue Long chinese national influenza center national institute for viral disease control and preventionchina cdckey laboratory for medical virologyministry of healthBeijing 102206China

Objective To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR （M-RTPCR）, for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing （NGS） platform. Methods Virus genome copy was quantified and seria（iy diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. Results The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing Iow-titer virus. Conclusion The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

关键词： Influenza A virus Whole genome sequencing NGS

Development and Assessment of Two Highly Pathogenic Avian Influenza(HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness

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Biomedical and Environmental Sciences 2020年第9期33卷 670-679页

作者： LIU Li Qi LI Zi JIAO Ming LU Jian ZHOU Jian Fang LI Xi yan LIU Jia GUO Jun Feng XIAO Ning ZHAO Xiang wang da yan Chinese National Influenza Center National Institute for Viral Disease Control and PreventionChina CDCBeijing 102206China

Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.

关键词： Highly pathogenic avian influenza H5N6 virus Genetic stability Candidate vaccine virus Reverse genetic technology

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China

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Biomedical and Environmental Sciences 2014年第10期27卷 763-769页

作者： BO Hong DONG Li Bo ZHANG Ye DONG Jie ZOU Shu Mei GAO Rong Bao wang da yan SHU Yue Long National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Key Laboratory for Medical Virology National Health and Family Planning Commission

Objective To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China Methods Recombinant viruses were rescued using reverse genetics. Neuraminidase （NA） and hemagglutinin （HA） segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpoll vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embnjonate survival and antigenicity were compared with those of the respective wild-type viruses. Results The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. Conclusion The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

关键词： Influenza H5N1 Pre-pandemic Vaccine strains

Interferon-induced Transmembrane Protein 3 Prevents Acute Influenza Pathogenesis in Mice

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Biomedical and Environmental Sciences 2020年第5期33卷 295-305页

作者： SUN Qiang LEI Na LU Jian GAO Rong Bao LI Zi LIU Li Qi SUN Ying GUO Jun Feng wang da yan SHU Yue Long School of Public Health(Shenzhen) Sun Yat-sen UniversityShenzhen 510275GuangdongChina National Institute for Viral Disease Control and Prevention Chinese Center for Disease Prevention and ControlBeijing 102206China Chaoyang District Center for Disease Prevention and Control Beijing 100021China

Objective Interferon-induced transmembrane protein 3(IFITM3)is an important member of the IFITM family.However,the molecular mechanisms underlying its antiviral action have not been completely elucidated.Recent studies on IFITM3,particularly those focused on innate antiviral defense mechanisms,have shown that IFITM3 affects the body’s adaptive immune response.The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection in vivo.Methods We performed proteomics,flow cytometry,and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer(NK)cell numbers,their activation,and their immune function in the lungs of Ifitm3-/-and wild-type mice.Results Ifitm3-/-mice developed more severe inflammation and apoptotic responses compared to wild-type mice.Moreover,the NK cell activation was higher in the lungs of Ifitm3-/-mice during acute influenza infection.Conclusions Based on our results,we speculate that the NK cells are more readily activated in the absence of IFITM3,increasing mortality in Ifitm3-/-mice.

关键词： IFITM3 Influenza Immune response NK cells

Full-Length Genome Sequencing of SARS-CoV-2 Directly from Clinical and Environmental Samples Based on the Multiplex Polymerase Chain Reaction Method

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Biomedical and Environmental Sciences 2021年第9期34卷 725-728页

作者： NIU Pei Hua ZHAO Xiang LU Rou Jian ZHAO Li HUANG Bao Ying YE Fei wang da yan TAN Wen Jie National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206China

The new coronavirus,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),is spreading worldwide with the number of confirmed cases increasing dramatically[1,2].Coronaviruses have the largest genome among RNA vi... 详细信息

关键词： acute respiratory Clinical

An uncontrolled open-label, multicenter study to monitor the antiviral activity and safety of inhaled zanamivir （as Rotadisk via Diskhaler device） among Chinese adolescents and adults with influenza-like illness

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Chinese Medical Journal 2012年第17期125卷 3002-3007页

作者： CAO Bin wang da-yan YU Xiao-min WEI Lu-qing PU Zeng-hui GAO yan wang Jing DONG Jian-ping LI Xiao-ling XU Qian HU Ke CHEN Bai-yi YU Yun-song SONG Shu-fan SHU Yue-long wang Chen Beijing Chao-yang Hospital Beijing Institute of Respiratory Medicine Capital Medical University Beijing 100020 China Chinese National Influenza-Center National Institute for Viral Disease Control and Prevention China CDC Beijing 102206 China Pingjing Hospital Logistics College of Chinese People's Armed Police Forces Tianjin 300162 China yantai Yuhuangding Hospital Yantai Shandong 264000 China Peking University People's Hospital Beijing 100044 China Beijing Xuanwu Hospital Capital Medical University Beijing 100053 China Beijng Hai-Dian Hospital Beijing 100080 China Chengdu Infectious Disease Hospital Chengdu Sichuan 610061 China China-Japan Friendship Hospital Beijing 100029 China Renmin Hospital of Wuhan University Wuhan Hubei 430060 China First Hospital of China Medical University Shenyang Liaoning 110001 China Sir Run Run Shaw Hospital Hangzhou Zhejiang 310020 China Pijing Hospital China Ministry of Health Beijing 100730 China

Background It is the first multicenter clinical study in China to investigate zanamivir use among Chinese adolescents and adults with influenza-like illness （ILl） since 2009, when inhaled zanamivir （RELENZA ） was marketed in China. Methods An uncontrolled open-label, multicentre study to evaluate the antiviral activity, and safety of inhaled zanamivir （as Rotadisk via Diskhaler device）; 10 mg administered twice daily for 5 days in subjects≥12 years old with ILl. Patients were enrolled within 48 hours of onset and followed for eight days. Patients were defined as being influenza-positive if the real-time reverse transcriptase-polymerase chain reaction real-time reverse transcriptase-polymerase chain reaction （rRT-PCR） test had positive results. Results A total of 400 patients 〉12 years old were screened from 11 centers in seven provinces from March 2010 to January 2011. Three hundred and ninety-two patients who took at least one dose of zanamivir were entered into the safety analysis. The mean age was 33.8 years and 50% were male. Cardiovascular diseases and diabetes were the most common comorbidities. All the reported adverse events, such as rash, nasal ache, muscle ache, nausea, diarrhea, headache, occurred in less than 1% of subjects. Mild sinus bradycadia or arrhythmia occurred in four subjects （1%）. Most of the adverse events were mild and did not require any change of treatment. No severe adverse events （SAE） or fatal cases were reported. Bronchospasm was found in a 38 years old woman whose symptoms disappeared after stopping zanamivir and without additional treatment. All the 61 influenza virus isolates （43 before enrollment, 18 during treatment） proved to be sensitive to zanamivir. Conclusions Zanamivir is well tolerated by Chinese adolescents and adults with ILls. There is no evidence for the emergence of drug-resistant isolates during treatment with zanamivir. （ChiCTR-TNRC-10000938）

关键词： influenza zanamivir safety susceptibility

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine

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Biomedical and Environmental Sciences 2019年第7期32卷 531-540页

作者： FAN Xue Ting wang Yun Long SU Qiu Dong QIU Feng YI Yao JIA Zhi Yuan wang da yan QIN Kun ZOU Ye Ning BI Sheng Li SHEN Li Ping National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 102206 China Henan Bioengineering Research Center Zhengzhou 4500466 Henan China Sinovac Biotech Co. Ltd. Beijing 100085 China

Objective To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine. Methods Mice were immunized intranasally with PBS(negative control), or H3N2 split vaccine(3 μg/mouse) alone, or CTA1-DD(5 μg/mouse) alone, or H3N2 split vaccine(3 μg/mouse) plus CTA1-DD(5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine(3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured. Results H3N2 split vaccine formulated with CTA1-DD could elicit higher Ig M, Ig G and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory Ig A titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus. Conclusion CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

关键词： Adjuvants H3N2 Influenza Mucosal immunization Split vaccine

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

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Biomedical and Environmental Sciences 2020年第2期33卷 103-113页

作者： SUN Ying CAO yang LI Zi BAI Tian ZHANG Hong HU Shi Xiong LI Fang Cai ZHAO Xiang CHEN Yong Kun LU Jian LIU Li Qi wang da yan SHU Yue Long ZHOU Jian Fang National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and PreventionKey Laboratory for Medical VirologyNational Health and Family Planning CommissionBeijing 102206China Center of Growth Metabolism and AgingKey Laboratory of Bio-Resource and Eco-EnvironmentMinistry of EducationCollege of Life SciencesSichuan UniversityChengdu 610064SichuanChina Hunan Provincial Disease Control and Prevention Changsha 410000HunanChina School of Public Health(Shenzhen) Sun Yatsen UniversityShenzhen 510275GuangdongChina

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

关键词： V^H1-69 D3-9 Avian influenza A(H5N1)virus Cross-neutralizing Antibody

Effect of Mn content on microstructure and properties of AlCrCuFeMnx high-entropy alloy

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China Foundry 2024年第2期21卷 147-158页

作者： Ning wang Kai Ma Qiu-da Li Yu-dong Yuan yan-chun Zhao Li Feng School of Materials Science and Engineering Lanzhou University of TechnologyLanzhou 730050China State Key Laboratory of Advanced Processing and Reuse of Nonferrous Metals Lanzhou 730050China CNNC Nuclear Power Operation and Management Co. Ltd.Haiyan 314300ZhejiangChina

AlCrCuFeMnx(x=0,0.5,1,1.5,and 2)high-entropy alloys were prepared using the vacuum arc melting technology.The microstructure and mechanical properties of AlCrCuFeMnxwere analyzed and tested by XRD,SEM,TEM,nanoindentation,and electronic universal testing.The results indicate that the AlCrCuFeMnxhigh-entropy alloy exhibits a dendritic structure,consisting of dendrites with a BCC structure,interdendrite regions with an FCC structure,and precipitates with an ordered BCC structure that form within the dendrite.Manganese(Mn)has a strong affinity for dendritic,interdendritic,and precipitate structures,allowing it to easily enter these areas.With an increase in Mn content,the size of the precipitated nanoparticles in the dendritic region initially increases and then decreases.Similarly,the area fraction initially decreases and then increases.Additionally,the alloy’s strength and wear resistance decrease,while its plasticity increases.The Al Cr Cu Fe Mn1.5alloy boasts excellent mechanical properties,including a hardness of 360 HV and a wear rate of 2.4×10^(-5)mm^(3)·N^(-1)·mm^(-1).It also exhibits impressive yield strength,compressive strength,and deformation rates of 960 MPa,1,700 MPa,and 27.5%,respectively.

关键词： high-entropy alloys microstructure mechanical properties wear resistance strengthening mechanisms