一株H5N1亚型禽流感病毒全基因组克隆及HA基因分子进化分析

Cloning and sequencing full-length genome of H5N1 avian influenza A virus and molecular evolution analysis of HA gene

目的对禽流感H5N1亚型病毒株A/Chicken/Hubei/489/2004的全基因组进行克隆和测序,并分析血凝素基因HA的遗传突变特点及其与1996年以来其他病毒株的亲缘关系。方法通过RT-PCR扩增病毒株A/Chicken/Hubei/489/2004的8个基因,并将其克隆到测序载体;在对病毒株全基因组序列测定基础上,利用生物信息学软件对HA基因进行遗传进化分析。结果病毒株A/Chicken/Hubei/489/2004的全基因组克隆到PMD18-T;遗传进化分析显示该毒株HA蛋白具有与致病性有关的切割位点"PQRERRRKKR",并且与2000～2006年在香港从人和禽体内分离的H5N1亲缘关系相近,也与2003～2004年在东南亚从人和禽体内分离的H5N1极其相关。结论 A/Chicken/Hubei/489/2004病毒分离株具有与A/Chicken/HongKong/YU777/2002更相似的遗传特性,极可能是2002年禽流感病毒家系的再次出现。

Objective To clone and sequence full-length genome of the avian influenza A/Chicken/Hubei/489/2004（H5N1） virus,in order to analyze genetic mutation patterns of HA gene and polygenetic relationship between A/Chicken/Hubei/489/2004（H5N1）virus and other strains since 1996.Methods 8 genes of the avian influenza A/Chicken/Hubei/489/2004（H5N1）virus were amplified and cloned,and then genetic mutation analysis and phylogenetic trees were made by bioinformatics software.Results Full-length genome of the avian influenza A/Chicken/Hubei/489/2004（H5N1）virus were cloned into the vector of PMD18-T.Genetic evolution analysis showed that there is a specific cleavage site of ＂PQRERRRKKR＂,which was proved be related with virulence.In addition,molecular phylogenetic trees of HA gene revealed that A/Chicken/Hubei/ 489/2004 virus were closely related to H5N1 viruses of 2000-2006 isolated in Hong Kong and in Southeast Asia.Conclusion the influenza A/Chicken/Hubei/489/2004（H5N1） virus was closest genetic relatives to the influenza A/Chicken/HongKong/YU777/2002（H5N1） virus,and it was most possible that the avian influenza outbreak was caused by the 2002 lineage of Hong Kong.