2株广西猪源H9N2亚型流感病毒遗传进化及生物学特性分析

Genetic evolution and biological characteristics analysis of two strains of H9N2 subtype influenza virus from swine in Guangxi

【目的】明确广西猪源H9N2亚型流感病毒的遗传特征及分子生物学特性(抗原性、耐药性和致病性),为广西猪源H9N2亚型流感病毒的防控提供科学依据。【方法】以分离自广西百色地区的2株猪源H9N2亚型流感病毒(SW/GX/P2/2011株和SW/GX/P3/2011株)为研究对象,运用RT-PCR扩增其全基因组的8个基因片段(HA、NA、NP、M、NS、PB1、PB2和PA基因),经克隆测序后进行核苷酸序列及氨基酸位点分析。【结果】2株广西猪源H9N2亚型流感病毒的核苷酸序列开放阅读框(ORF)分别是PB2:2280 bp、PB1:2274 bp、PA:2151 bp、HA:1683 bp、NP:1497 bp、NA:1410bp、M:982 bp和NS:838 bp。2株广西猪源H9N2亚型流感病毒全基因组仅M基因核苷酸序列与猪源H9N2亚型流感病毒的相似性较高,而HA、NA、NP、NS、PA、PB2和PB1基因核苷酸序列均与禽源H9N2亚型流感病毒的相似性较高,在基因型分类上属于G57基因型,为我国广泛流行的H9N2亚型基因型。2株广西猪源H9N2亚型流感病毒的HA蛋白发生R180Q、T213A、D216E、M224L、N285S和V287T突变,NA蛋白抗原决定簇S331V、W403S和Q431K也发生突变;NA蛋白在N2亚型的耐药性关键位点119E、151D、292R、276E和294N位点未发生突变,但在NA蛋白抗原区存在S331V、K367E和Q432K突变,且M2氨基酸位点发生S31N突变。2株广西猪源H9N2亚型流感病毒HA蛋白连接HA1和HA2的氨基酸均为RSSR↓GLF;HA蛋白存在1个因P315S突变而新增的潜在糖基化位点(NCS);NA蛋白未缺失NA潜在糖基化位点,也未出现NA蛋白颈部杆状结构63~65 aa缺失现象,在NA潜在糖基化位点中69、86、146、200和234aa处非常保守,但发生W402S突变。【结论】从广西百色分离获得的2株猪源H9N2亚型流感病毒(SW/GX/P2/2011株和SW/GX/P3/2011株)虽为低致病力毒株,但在流感病毒基因重组过程中发挥重要作用,且其致病性有增强趋势,已对金刚烷胺类药物产生耐药性。因此,应加强广西地区哺乳动物H9N2亚型流感病毒的监控,并警惕其跨种间传播。

【Objective】To understand the genetic and molecular biological characteristics(antigenicity,drug resistance and pathogenicity)of pig H9 N2 subtype influenza virus from swine in Guangxi and to provide scientific basis for the prevention and control of swine H9 N2 influenza virus in Guangxi.【Method】Two strains of pig H9 N2 subtype influenza virus from swine isolated from Baise in Guangxi(SW/GX/P2/2011 and SW/GX/P3/2011)were used as materials,eight segments(HA,NA,NP,M,NS,PB1,PB2 and PA)of both virus strains whole genome were amplified by RT-PCR.After cloning and sequencing,the nucleotide sequence and amino acid sites were analyzed.【Result】Nucleotide sequence open reading frame(ORF)of two strains of H9 N2 influenza virus isolated from pigs were PB2:2280 bp,PB1:2274 bp,PA:2151 bp,HA:1683 bp,NP:1497 bp,NA:1410 bp,M:982 bp and NS:838 bp,respectively.In genomes of the two isolated strains,only the nucleotide sequence of M gene of the two isolates had high similarity with H9 N2 subtype influenza virus of swine origin,while the nucleotide sequences of HA,NA,NP,NS,PA,PB2 and PB1 genes had high similarity with H9 N2 subtype influenza virus of avian origin,and belonged to G57 genotype in genotype classification,which was the H9 N2 subtype genotype widely prevalent in China.Mutations of R180 Q,T213 A,D216 E,M224 I,N285 S and V287 T occurred in HA protein of the two isolated strains,and the NA protein antigenic determinants S331 V,W403 S and Q431 K were also mutated.In the drug resistance locus analysis,the isolated virus strains did not mutate at the 119 E,151 D,292 R,276 E and 294 N sites of the NA protein in the N2 subtype resistance,but S331 V,K367 E and Q432 K mutations were found in the antigen region of NA protein,and the M2 amino acid site produced S31 N mutations.The amino acids connecting HA1 and HA2 of the two isolates were RSSR↓GLF.A new potential glycosylation site(NCS)existed in HA protein due to P315 S mutation.There was no deletion of NA potential glycosylation site and 63-65 aa deletion in the neck rod structure of NA protein.Among the potential glycosylation sites of NA,69,86,146,200 and 234 aa were very conservative,but W402 S mutation occurred.【Conclusion】The two H9 N2 influenza viruses(SW/GX/P2/2011 and SW/GX/P3/2011)isolated from Baise,Guangxi are low virulence strains,but they play an important role in the gene recombination of influenza viruses,and their pathogenicity tends to increase.They have developed amantadine resistance.Therefore,it is necessary to strengthen the surveillance of H9 N2 subtype influenza virus in mammals in Guangxi,and to guard against its cross species transmission.