一株H1N1猪流感病毒的进化分析与分子特征

【目的】了解猪流感病毒的分子流行病学,为动物流感的防控提供科学依据。【方法】将猪的鼻拭子接种鸡胚进行病毒分离,对血凝试验阳性的样品在SPF鸡胚上进一步纯化、增殖,采用RT-PCR对分离毒株的全基因组进行扩增,使用Applied Biosystems 3500x L Genetic Analyzer进行序列测定,利用DNASTAR软件对测序基因片段进行整个阅读框的核苷酸序列同源性比对分析,并用MEGA 6.0绘制遗传进化树及分析氨基酸位点。【结果】分离毒株A/swine/Zhejiang/245/2013(SW/ZJ/245/13)为H1N1亚型病毒,核苷酸的分析结果发现该分离毒株的8个基因片段均与中国近期分离的H1N1亚型流感病毒高度同源,属于类禽型猪H1N1的进化分支,没有出现不同基因型流感病毒片段之间的重组。分离株HA蛋白的裂解位点序列为IPSIQSR↓G,具有典型低致病力流感病毒的分子特征;HA蛋白5个抗原位点区域与其同源性最高毒株完全一致,表明抗原性未发生改变;HA蛋白受体结合位点中190 D以及225 E,表明SW/ZJ/245/13与SA-α-2,6-Gal受体有较强的结合能力,而SA-α-2,6-Gal受体普遍存在于哺乳动物宿主(猪和人)上呼吸道中;HA共有6个潜在糖基化位点,其中4个(N27、N40、N212和N291)位于HAl区,2个(N498和N557)位于HA2区;NA有7个潜在糖基化位点,其中4个(N50、N58、N63和N68)在链接区,其他3个(N88、N146和N235)在结构域;PB2蛋白具有271T、627E和701N的氨基酸组合;M2蛋白发生抗药位点S31N的突变。【结论】类禽型H1N1猪流感病毒的持续存在和不断变异,提示应加强猪流感的监测,为动物流感的防控提供重要的科学依据。

Phylogenetic Analysis and Molecular Characteristics of an H1N1 Subtype Swine Influenza Virus

【Objective】 The objective of this study is to understand the molecular epidemiology of swine influenza virus, to provide scientific references for prevention of animal influenza in China. 【Method】Nasal swab samples collected from pigs were inoculated into SPF chicken embryos for virus isolation. The HA positive samples were further purifiedand proliferated in SPF chicken embryos. Eight gene fragments of the isolated virus were amplified by RT-PCR and then sequenced by Applied Biosystems 3500xL Genetic Analyzer. Nucleotide homology was analyzed by using DNASTAR. The phylogenetic trees of the genes were constructed using MEGA 6.0. 【Result】 The isolated virus was identified as an H1N1 subtype influenza virus, designated as A/swine/Zhejiang/245/2013(H1N1). Nucleotide homology analysis showed that eight gene fragments of this strain were highly homologous with the respective gene of the viruses recently isolated from China. Phylogenetic analysis revealed that the strain belonged to the avian-like swine H1N1 lineage, no gene reassortment occurred. Amino acid sequence at the HA cleavage site was IPSIQSR↓G, which had characteristics of low-pathogenic influenza viruses. And its receptor-binding sites contained 190D and 225E, which prefer sialic acid (SA)-2,6-Gal-terminated saccharides that are abundant in mammal upper respiratory epithelium. Among the six glycosylation sites, four sites were located in HA1 section and the rest in HA2. The isolates contained 271T, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker which may confer resistance to amantadine and rimantadine antivirals.【Conclusion】The sustained existence and continual mutation of avian-like swine H1N1 influenza virus suggested that intensive swine influenza surveillance should be carried out in the future.