H3N2禽流感病毒反向遗传操作平台的建立  

Development of a Reverse Genetics System for the Rescue of Reassortant H3N2 Avian Infl uenza Virus

作　　者：石晓娜 闫婉婉 陈新武 潘学 李雪松[1] 刘芹防[1] 杨健美[1] 李泽君[1] 滕巧泱[1] SHI Xiaona;YAN Wanwan;CHEN Xinwu;PAN Xue;LI Xuesong;LIU Qinfang;YANG Jianmei;Li Zejun;TENG Qiaoyang(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2022年第5期150-156,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发项目(2018YFD0501500);上海市自然科学基金(18ZR1449000)。

摘　　要：近几年,禽流感病毒(AIV)跨越禽-犬屏障产生新型H3N2犬流感病毒,后者在亚洲多国的犬中广泛流行,造成潜在的公共卫生安全威胁。尽管如此,H3N2 AIV如何跨宿主传播至犬的分子机制尚不清楚。为此,本研究利用早期筛选到的一株不感染犬和鼠,但感染禽的H3N2禽流感病毒A/Duck/Shanghai/01/2009(H3N2)(简称为SH01),通过RT-PCR技术扩增了病毒的8个全基因片段,并分别克隆至PLLB双向表达载体上。将构建成功的8个质粒纯化后共转染293T细胞,转染48 h后加入TPCK胰酶作用2 h,将上清液和细胞一同接种9~11日龄SPF鸡胚,检测其血凝效价。经测序分析,确定获救病毒的8个基因片段序列与亲本毒株的序列完全一致,表明该病毒拯救成功。H3N2禽流感病毒反向遗传操作平台的成功建立为探索该病毒跨宿主传播犬的分子机制提供了工具。Recently,Avian infl uenza virus(AIV)has evolved a new H3N2 canine infl uenza virus in dogs across the avian-dog barrier,and the latter has been widely prevalent in dogs in many Asian countries.However,the molecular mechanism of this new H3N2 AIV transmitting across host barrier to dogs remains unclear.In order to discover this mechanism,an Avian influenza virus A/duck/shanghai/01/2009(H3N2)(SH01)was used in the present study.The SH01 virus did not infect dogs and mice but infected avian species.Eight fragments of the SH01 virus were amplifi ed by RT-PCR and separately cloned into the vector of PLLB.Then recombinant plasmids were purifi ed and co-transfected into 293T cells.After treatment with TPCK trypsin for 2 h at 48 h post-transfection,the supernatant and cells were co-inoculated into 9-day-old SPF embryonated chicken eggs and then the rescued viruses were confi rmed by hemagglutination assay(HA).Sequence analysis demonstrated that eight genes of the rescued virus were all identical to those of the parent virus,indicating that the virus was rescued successfully.The successful utilization of the reverse genetic system for the rescue of reassortant H3N2 avian infl uenza virus provided a tool for exploring the molecular mechanism of the viral transmission across animal species.

关 键 词：H3N2 禽流感病毒 反向遗传操作系统 

分 类 号：S852.65[农业科学—基础兽医学]