野鸭源H3N8亚型禽流感病毒的系统进化和分子遗传特征分析

Genome sequencing and genetic analysis of H3N8 subtype avian influenza virus isolated from mallard

为了解在江苏某湿地野生水禽中分离到的1株禽流感病毒的来源、流行特点、进化和分子遗传特征,将采集的野生水禽粪便经处理后,使用A型禽流感通用荧光RT-PCR检测,阳性样品接种10日龄SPF鸡胚,收取接种后第24~96小时的尿囊液,进行血凝试验,随后对其进行全基因测序和遗传进化分析。结果显示,该毒株为H3N8亚型禽流感病毒,命名为A/Mallard/Xuyi/14/2015（H3N8）。HA蛋白的裂解位点氨基酸序列为PEKQTR↓GLF,无连续碱性氨基酸插入,符合典型低致病性禽流感病毒的特征;HA与NA的糖基化位点没有发生突变;NA耐药性氨基酸位点出现突变位点;PB2蛋白第627和701位氨基酸分别为谷氨酸（Glu）和天冬氨酸（Asn）,是禽源流感病毒的特征性氨基酸。在Gen Bank中对该毒株进行序列比对分析,结果显示,与所测基因片段同源性最高的10株毒株基本位于我国东部候鸟迁徙路线上。结果表明,A/Mallard/Xuyi/14/2015（H3N8）株为从江苏分离的低致病性禽流感病毒,基因主要来源于我国东部候鸟迁徙路线上的禽流感病毒。

An avian influenza virus（AIV） has been isolated from a mallard in Jiangsu wetland.To investigate the origin,molecular evolution and epidemic characteristics of the strain,the entire of the isolate was subjected to genome sequencing and genetic evolution analysis.Fecal samples from wild waterfowl were texted by A common type of avian influenza fluorescent RT-PCR reagents.The positive ones were inoculated with the 10-day-old SPF chicken embryos. Allantoic fluid was collected within 24 h to96 h. Hemagglutination test was done and the sequence was analyzed. The results showed that the virus belongs to the H3N8 subtype AIV,named as A/Mallard/Xuyi/14/2015（H3N8）.It indicated that the amino acid motif of the HA gene cleavage site was PEKQTR ↓GLF, which is the typical characterization of the low pathogenic AIV.Glycosylation sites of HA and NA did not have mutations.NA resistant amino acid sites appeared mutation.The 627 and 701 amino acids of PB2 protein is glutamate（Glu） and aspartic acid（Asn）,which are AIV signature amino acids.In Gen Bank,the strain sequence alignment analysis showed that 10 strains with the highest affinity for each gene located in the China Eastern migratory area. Inconclusion,the strain is low pathogenic AIV that genes were derived from different regions of the viral genes which mainly from China Eastern migratory area.