应用焦磷酸测序技术快速检测猪流感病毒金刚烷胺耐药性的分子标签

【目的】本研究旨在通过焦磷酸测序技术对我国分离的H1N1、H3N2、H9N2等3种基因型的10株猪流感病毒分离株进行金刚烷胺耐药性鉴定。【方法】流感病毒M2蛋白5个关键位点氨基酸残基(第26、27、30、31和34位)中的任何一个发生突变会导致抗流感病毒药物中金刚烷胺抗药性的产生。本研究利用焦磷酸测序技术对2004-2008年国内分离的10株猪流感病毒M基因金刚烷胺耐药性分子决定区进行了鉴定,并进行抗药性分析。【结果】基于M2蛋白基因保守区序列建立的焦磷酸测序技术能用于国内猪流感病毒的快速检测,且具有较好的特异性和重复性。抗药性分析表明10株猪流感病毒国内分离株中5株H1N1分离株全部耐药,主要存在M2蛋白的V27T、V27I或S31N位点的突变,而4株H3N2和1株H9N2猪流感病毒分离株在M2蛋白5个关键位点上均未出现变异,表明其对金刚烷胺敏感。【结论】基于M基因的焦磷酸测序技术可以用于我国猪流感病毒金刚烷胺耐药性快速鉴定。

Detection of molecular markers of amantadine resistance in swine influenza viruses by pyrosequencing

Abstract: Objective] To evaluate the resistance against the adamantine of multi-genotype (H1N1, H3N2 and H9N2) swine influenza viruses isolated from China in recent years by pyrosequencing. Methods] Mutation in one of five key amino acid residues (positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses, leading to resistance against the adamantine class of anti- influenza drugs. The residues L26, V27, A30, S31,and G34 in the M2 protein were targeted for pyrosequencing, and 10 swine influenza viruses obtained from China during 2004 to 2008 were used to perform the amantadine resistance analysis. Results] All 5 H1N1 swine influenza viruses were adamantine resistance, three mutations were founded in these isolates, namely V27T,V27I and S31N. Other five isolates, including four H3N2 and one H9N2 swine influenza virus, were proved to be sensitive to amantadine. Conclusion] Pyrosequencing technology based on the M2 gene can be used to determine the amantadine resistance for multi-genotype swine influenza viruses.