一株H1N1亚型猪流感病毒的分离鉴定及全基因序列分析  

Isolation,Identification and Genetic Analysis of an H1N1 Subtype Isolate of Swine Influenza Virus

作　　者：路伟[1] 张秀华[1] 王秀东[2] 武华[1,2] 

机构地区：[1]中国农业科学院特产研究所人兽共患病研究室,吉林132109 [2]北京必威安泰生物科技有限公司,北京100176

出　　处：《病毒学报》2010年第5期396-401,共6页Chinese Journal of Virology

摘　　要：2006年从辽宁省某猪场采集具有流感症状猪鼻拭子共30份,经9~11日龄SPF鸡胚分离,对分离株进行了血凝试验、血凝抑制试验、RT-PCR亚型鉴定,全基因序列比对和接种试验动物试验。结果表明：该毒株具有凝集0.5%鸡红细胞的活性,且与抗猪流感H1标准血清发生血凝抑制反应,RT-PCR分别扩增得到全基因8个片段,利用DNAStar生物学软件进行序列分析,HA基因与GenBank登录的H1~H16中的H1基因序列同源性最高,NA基因与N1~N9中的N1基因序列同源性最高,故该LN株分离毒为猪流感H1N1亚型病毒。全基因序列中,除了M基因不是猪源的,其它基因片段均与国内H1N1亚型猪流感参照株高度同源,推测LN株可能是由类人和类禽谱系的流感病毒与古典猪谱系的流感病毒重排形成的;用该病毒接种试验动物可成功复制出猪流感典型症状。该病毒的分离鉴定及全基因组序列分析为我国进一步调查猪流感的流行规律提供了基础数据。In 2006,a swine influenza virus（SIV） isolate was isolated from 30 nasal swabs samples collected from pigs with clinical syndromes of swine influenza in a pig farm of Liaoning Province.The virus isolate was studied and identified by the growth in 9~11 days old chicken embryo,hemagglutination（HA） assay,hemagglutination inhibition（HI） assay,reverse transcription-polymerase chain reaction assays（RT-PCR） for its genetic subtype,whole gene sequence analysis and animal trial for its virulence.The virus isolate demonstrated the hemagglutination activity.Result of HI test against H1 subtype of SIV was positive,however,the results were negative when the HI studies were conducted using SIV H3 subtype virus and Newcastle Disease Virus（NDV）.Eight gene segments of the virus isolate were amplified by RT-PCR.Phylogenetic analysis of the gene sequence of the virus isolate by using DNAstar software program revealed that the isolate have the H1 HA gene,by comparing to the sequences of H1~H16 in the GenBank.Furthermore,sequencing results also demonstrated that the virus isolate＇s NA gene belongs to N1 subtype.Therefore,the subtype of the SIV isolate is H1N1.The results of sequence analysis indicated that the genome of the SIV-H1N1 LN strain includes 8 fragments,among which only M protein gene is not swine originated.All other 7 fragments have close relationship with the domestic standard swine H1N1 strains.Results suggested that the SIV isolate LN strain might be created by a possible triple reassortants among the classic swine influenza virus,human influenza-like virus,and avian influenza-like virus.Piglets were inoculated with the SIV LN strain virus preparations and the virus caused the typical clinical symptoms of swine influenza in the inoculated piglets.This study,the isolation,identification and genetic analysis of the SIV LN strain provided useful information and basic data for the further investigation of epidemic principles and patterns of swine influenza virus in China.

关 键 词：猪流感病毒 H1N1 

分 类 号：S852.659.5[农业科学—基础兽医学]