临床细菌与H3N2流感病毒共感染特征及病毒血凝素裂解位点的变异研究

Characteristics of clinical bacteria and H3N2 influenza virus co-infection and variations in viral hemagglutinin cleavage site

目的了解流感样病例中临床细菌与H3N2流感病毒共感染及病毒血凝素(HA)裂解位点的变异特征。方法收集2013年1月至2018年12月的流感样病例样本12250份,实时荧光PCR检测H3N2流感病毒。选取部分流感阳性病例样本,采用实时荧光PCR的方法进行金黄色葡萄球菌、肺炎链球菌和B型流感嗜血杆菌的检测,分析其临床感染特征。分离流感病毒,RT-PCR扩增病毒HA,测序,构建进化树,并分析裂解位点氨基酸变异特征。结果2013年以来,每年都有H3N2流感病毒的流行,H3N2在流感阳性病例中的占比为44.69%。随机选取2013—2018年间流感流行高峰的H3N2阳性病例样本295份,29.2%的病例存在临床细菌感染。对分离到的210株H3N2流感病毒进行HA裂解位点测序,68株存在变异,其中63株为K342R单氨基酸位点变异,裂解位点由PEKQTR转变为PERQTR。裂解位点未变异样本的细菌共感染率为31.25%(45/144),裂解位点变异样本的细菌感染率为23.53%(16/68),二者的差异没有统计学意义(χ2=1.34,P>0.05)。杭州地区流行的H3N2流感病毒主要属于3C.3a和3C.2a两个进化簇,裂解位点发生K342R变异的病毒属于3C.3a进化簇。结论H3N2流感病毒在杭州地区的流感病毒流行中占据重要地位。流感阳性病例存在着一定程度的细菌共感染。裂解位点变异具有一定的地域流行特征,但与细菌共感染没有显著相关性。

Objective To investigate the characteristics of clinical bacteria and H3N2 influenza A virus coinfection and variations in the hemagglutinin(HA)cleavage site of H3N2 among influenza-like cases.Methods A total of 12250 samples were collected from influenza-like cases for real-time PCR detection of H3N2 influenza virus from January 2013 to December 2018.To analyze the characteristics of co-infection,some H3N2-positive samples were selected and analyzed by real-time PCR to detect Staphylococcus aureus,Streptococcus pneumoniae and haemophilus influenzae type B.HA genes of H3N2 isolates were amplified by RT-PCR and sequenced.A phylogenetic tree was constructed based on HA gene sequences.Amino acid variations in cleavage sites were analyzed.Results H3N2 influenza viruses had been detected every year since 2013,causing 44.69%influenza-positive cases.There were 295 randomly selected H3N2-positive samples,of which 29.2%had clinical bacterial infection.The HA cleavage sites of 210 H3N2 isolates were sequenced and 68 had variations,including 63 carrying K342R(PEKQTR to PERQTR)single-amino acid site variation.The co-infection rate was 31.25%(45/144)in unmutated samples and 23.53%(16/68)in mutated samples(χ2=1.34,P>0.05).The H3N2 influenza viruses circulating in Hangzhou mainly belonged to two evolutionary clusters of 3c.3a and 3c.2a,and the viruses with K342R mutation at the cleavage site belonged to the evolutionary cluster of 3c.3a.Conclusions H3N2 influenza virus played an important role in the epidemic of influenza virus in Hangzhou.There were some bacterial co-infections in influenza-positive cases.Cleavage site variations showed regional epidemic characteristics,but had no significant correlation with bacterial co-infection.