H3N2亚型猪流感病毒重庆株的分离、鉴定、基因组测定及序列分析  

Isolation,chararcteristics and sequence analysis of the genes of swine influenza viruses H3N2 subtype Chongqing strain

作　　者：杨柳[1,2] 翟少钦[1,2] 杨睿[1,2] 张素辉[1,2] 郑华[1] 张邑帆[1] 付利芝[1,2] 

机构地区：[1]重庆市畜牧科学院,重庆荣昌402460 [2]农业部养猪科学重点实验室,重庆荣昌402460

出　　处：《中国兽医学报》2015年第3期359-366,共8页Chinese Journal of Veterinary Science

基　　金：国家科技支撑计划资助项目(2010BAD4B01-8)

摘　　要：从猪流感疑似病例中分离鉴定猪流感病毒,并对病毒进行全基因组测序及其序列的分析。采用鼻棉拭子法从猪流感疑似病猪的鼻中收集病样,制成病毒悬液,经SPF鸡胚增殖培养和纯化。采用红细胞凝集、血凝抑制和神经氨酸酶抑制试验进行病毒亚型的判定,并作电镜观察。运用RT-PCR法扩增病毒的8个基因片段,分别克隆到pMD18-T载体,进行全基因组测序及序列分析。结果表明,获得1株猪流感病毒,经血凝、血凝抑制和神经氨酸酶抑制试验,判定为H3N2亚型,命名为A/swine/Chongqing/CQ/3/2011(H3N2),简称SIV CQ3。基因组序列比较分析显示,SIV CQ3 8个节段与不同时间的猪流感广东分离株(H3N2亚型)相应基因的核苷酸相似性最高,可能来源于H3N2亚型SIV。然而NP、HA和NA基因编码氨基酸序列与参考毒株A/New York/392/2004(H3N2)的相似性比较低,分别达95%、89%和90%;而血凝素(HA)裂解位点处的氨基酸序列为P-E-K-Q-T-R↓G,与参考毒株的一致,不具有高致病分子特征。此外,HA和NA的糖基化位点、受体结合位点处氨基酸存在一定程度的差异。最后,系统进化树分析显示NP、HA基因位于SIV群,与A/swine/Guangdong/2006/(H3N2)处于同一分支;而NA基因与禽流感(A/duck/Ontario/2000/AJ697881)和猪流感位于同一个进化单元,表明这株H3N2亚型流感病毒在不断的发生演变。The aim of the trial was isolation,chararcteristics and sequence analysis of the genes of swine influenza virus(SIV)from some SIV-suspected pigs in 2011.The viral suspensions were collected from nasal swab material from some SIV-suspected cases,and inoculated into the allantoic cavity of 10-day-old chicken embryos for virus proliferation.The subtype of colleted SIVs can be preliminarily ascertained by using hemagglutinin test(HA),hemagglutination inhibition test(HI)and nueraminidase inhibition test(NI).All the eight genes of the SIV were amplified by RTPCR,cloned into pMD18-T vector and sequenced,respectively.A SIV H3 subtype was isolated from SIV-suspected cases according to the results of HI.DNA sequence comparison revealed the eight genes from the SIV had shared the highest degree of similarity to that of the corresponding genes from the SIV H3N2 subtype from Guangdong isolates at different time,suggested that SIV may be from SIV H3N2 subtype.Comparisons of deduced amino acid sequences of NP,HA and NA of the isolates with that of the reference viruses A/New York/392/2004(H3N2)shared95%,89% and 90% amino acid similarities,respectively.While the amino acids sequence of cleavage site of HA is P-E-K-Q-T-R↓G,suggesting that SIV did not has the molecular characteristics of high pathogen.In addition,amino acid substitution detection at previously defined glycosylation sites,and receptor binding sites of the HA or NA proteins has some difference.Finally,analysis of NP,HA,NA phylogenetic trees showed the NP,HA might originate from swine influenza virus H3N2 subtype,while NA might originate from aive influenza virus,all that showed this SIV is evolution during the course of its life.

关 键 词：猪流感病毒 H3NA亚型 HA基因 NA基因 序列分析 

分 类 号：S852.65[农业科学—基础兽医学]