猪流感病毒广东株分离鉴定及遗传进化分析

doi:10.16431/j.cnki.1671-7236.2020.08.032

摘要/Abstract

摘要： 本研究从广东省某猪场采集37份疑似猪流感症状的猪鼻拭子样品，接种于9日龄SPF鸡胚并收集尿囊液，通过血凝试验、血凝抑制试验和RT-PCR鉴定，分离得到一株猪流感病毒，经RT-PCR分别扩增8个基因片段，进行基因测序及序列分析，与GenBank收录的参考毒株比对并构建进化树。结果显示，分离毒株为H1N1亚型猪流感病毒，将其命名为A/swine/Guangdong/2/2018（H1N1）。遗传进化分析显示，分离株8个片段的核酸序列与A/swine/Guangdong/L3/2009（H1N1）对应序列的同源性均达99%以上，与经典型H1N1亚型猪流感病毒处于同一分支。分离毒株HA的裂解位点为PSIQSR↓GL，符合低致病性流感病毒分子特征。HA基因受体位点为190D、225G和226Q，表明本毒株既可以结合SAα-2，6-Gal型人类流感病毒SA受体，也有结合SAα-2，3-Gal型禽类流感病毒SA受体的可能，在28、40、104、304、498、557位氨基酸处有6个潜在糖基化位点；NA蛋白在50、58、63、68、98、146、235位氨基酸处有6个潜在糖基化位点，NA蛋白氨基酸序列活性中心位点为119E、199D、223I、275H、293R、295N，氨基酸分析位点未出现突变，表明本分离株对神经氨酸酶抑制剂类药物的敏感性较高，但在M2蛋白中，31位氨基酸由敏感型的（S）突变为抗药的（N），提示可能对金刚烷胺类药物产生耐药性。开展猪流感病毒分离鉴定与遗传进化分析将为广东地区的猪流感流行和变异情况提供重要信息。

关键词: 猪流感; H1N1亚型; 分离鉴定; 遗传进化

Abstract: In this study,37 swabs were collected from a pig farm with suspected swine influenza in Guangdong province,inoculated into 9-day-old SPF chicken embryos and collected alluvial fluid,and a strain of swine influenza virus was isolated by hemagglutination (HA) test and hemagglutination inhibition (HI) test and RT-PCR identification.Eight gene fragments were amplified for sequencing and sequence analysis,and then compared with the reference strains in GenBank to construct the evolutionary tree.The results showed that the virus strain was H1N1 swine influenza virus.The isolate was designated as A/swine/Guangdong/2/2018 (H1N1).The complete genes were sequenced and the genetic analysis showed that the nucleotide sequences of the 8 segments of the isolate were similar to the corresponding sequences of A/swine/Guangdong/L3/2009(H1N1) by more than 99%,and were in the same branch as the classical swine influenza virus of the H1N1 subtype.The cleavage site of HA gene of the isolate was PSIQS↓GL,which was consistent with the molecular characteristics of low pathogenic influenza virus.HA gene receptor sites were 190D,225G and 226Q,which indicated that the strain could bind to both SA receptor of SAα-2,6-Gal human influenza virus and SA receptor of SAα-2,3-Gal avian influenza virus.There were six potential glycosylation sites at 28,40,104,304,498 and 557 amino acids.There were six potential glycosylation sites in amino acids of 50,58,63,68,98,146 and 235 of NA protein.The active sites of amino acid sequence of NA protein were 119E,199D,223I,275H,293R and 295N.There was no mutation in amino acid analysis site,indicating that the isolate was highly sensitive to neuraminidase inhibitors,but in M2 protein,31 amino acids changed from sensitive (S) to resistant (N),which might be resistant to amantadine drugs.The evolution analysis of swine influenza virus would provide important information for the prevalence and variation of swine influenza in Guangdong province.

Key words: swine influenza; H1N1 subtype; isolation and identification; genetic evolution

中图分类号:

S855.3

黄良宗, 颜广智, 邓汝森, 陈盛楠, 张海龙, 顾万军. 猪流感病毒广东株分离鉴定及遗传进化分析[J]. 中国畜牧兽医, 2020, 47(8): 2625-2633.

HUANG Liangzong, YAN Guangzhi, DENG Rusen, CHEN Shengnan, ZHANG Hailong, GU Wanjun. Isolation,Identification and Genetic Evolution Analysis of Swine Influenza Virus from Guangdong Province[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2625-2633.

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