泉州地区2014-2017年活禽相关外环境禽流感病毒监测及遗传进化特征

Surveillance and phylogenetic characteristics of H7N9 avian influenza virus in the live poultry related external environment of Quanzhou city during 2014-2017

目的分析泉州地区2014—2017年活禽相关外环境中H5、H7、H9亚型禽流感病毒的分布情况和分子生物学特点,为本地区人感染禽流感的防控和预警提供参考依据。方法采集泉州地区活禽监测场所标本,采用实时荧光定量PCR方法检测甲型流感病毒以及H5、H7、H9亚型禽流感病毒,然后对检测结果进行统计学分析;并对4株代表性H7N9禽流感病毒毒株的HA和NA基因进行核苷酸序列测定和同源性分析,用DNAstar和MEGA7.0软件进行序列分析。结果外环境标本甲型流感病毒核酸阳性率为29.04%(377/1289),其中H5、H7、H9亚型的阳性率分别为3.80%、13.34%、12.02%。各监测年份中,H7N9阳性率均高于其它亚型,其中2017年最高(21.88%)。不同类型标本中,案板的H7N9阳性率最高(65.4%)、污水次之(59.3%)、禽类饮水最低(29.6%)。采集场所中,最高的是农贸市场(61.7%)和活禽交易市场(52.8%)。对4株H7N9病毒的HA和NA基因序列分析显示,外环境和人源病毒分别属于珠江三角洲和长江三角洲谱系。HA蛋白的裂解位点均为PKGR/G,没有发生高致病性突变;NA蛋白292位氨基酸都为R,没有出现奥司他韦耐药突变;PB2蛋白中,627位氨基酸从E突变为了K,526位和701位氨基酸没有出现突变。结论泉州地区活禽相关外环境存在H7N9禽流感病毒污染,特别是农贸市场和活禽交易市场是人感染H7N9亚型的高风险场所,需要加强监测。

Objective To analyze the distribution and the molecular biological characteristics of variant subtypes (H5, H7 and H9) of avian influenza virus (AIV) in the live poultry related external environment of Quanzhou form 2014 to 2017, and provide regional references for the prevention, control and early-warning of human infections. Methods Samples from monitoring sites of live poultry were collected in Quanzhou from 2014 to 2017. Influenza A and variant subtypes of AIV (H5, H7 and H9) were detected by real time RT-PCR, and the detection results were further analyzed statistically. Furthermore, the HA and NA genes of four representative H7N9 strains were sequenced, and the results were further analyzed with DNAstar and MEGA7.0. Results Among the samples from external environment, the positive rate of nucleic acid of influenza A was 29.04% (377/1 289), of which the positive rates of H5, H7 and H9 subtypes were 3.80%, 13.34% and 12.02%, respectively. The positive rate of H7N9 was higher than those of the other subtypes in all monitored years, of which the highest rate was found in 2017 (21.88%). As to the different types of samples, chopping board possessed the highest positive rate of influenza A (65.4%), followed by waste water (59.3%) and drinking water for the poultry (29.6%). Among the different monitoring sites, the positive rate of poultry farm is 6.94%, far lower than that in the open air (61.7%) and the live poultry trading market (52.8%). Sequencing of the HA and NA genes of four strains of H7N9 showed that the strains from external environment and the strains from H7N9 patients belonged to Pearl River Delta and Yangtze River Delta lineage, respectively. The cleavage sites of HA proteins of these four strains were all PKGR/G without highly pathogenic mutation. Meanwhile, they were low pathogenic H7N9 without oseltamivir resistant mutation (R292 K in NA), while they all possessed the E627 K mutation in the PB2 genes associated with virulence. Conclusions H7N9 AIV existed in the live poultry related external environment of Quanzhou, especially the farmers’ and the live poultry trading market, so that more persistent surveillance could be needed in the future.