广东省活禽市场外环境重配H3N2亚型禽流感病毒污染调查

Reassortant H3N2 avian influenza virus isolated outside live poultry markets in Guangdong Province

目的了解2017-2018年广东省活禽市场外环境中H3N2亚型禽流感病毒的污染和变异情况。方法于2017-2018年在广东省禽流感监测点采集环境样品进行核酸检测,阳性样品接种鸡胚分离病毒并进行全基因组测序,利用相关软件进行生物信息学分析。结果成功获得7株H3N2亚型禽流感病毒,其HA蛋白裂解位点序列均为PEKQTR↓GL,与典型低致病性禽流感病毒的序列特征相符合。其中一分离株NS基因发生D92E干扰素抗性突变。HA蛋白受体结合位点、神经氨酸酶耐药位点、M基因上的金刚烷胺类药物耐药位点、PB2基因上与哺乳动物适应性相关位点等均未发生突变。系统进化分析显示,7株分离株的8个基因片段均处在欧亚禽源分支中,但相互间存在差距。结论 7株分离株中存在5种组合的重配H3N2亚型禽流感病毒,其中1株NS基因发生D92E干扰素抗性突变。因此应加强活禽市场及环境监测,防止产生能在人群中流行的重配H3N2病毒。

Objective To ascertain contamination with and mutations of H3 N2 subtype avian influenza virus in live poultry markets in Guangdong Province from 2017 to 2018. Methods Environmental specimens for nucleic acid detection were collected at monitoring points in 21 cities in Guangdong Province by sampling poultry droppings and stall waste liquid and by wiping cage surfaces and cutting boards. Positive specimens were isolated using chicken embryo inoculation, sequenced using an Ion PGM system and CLC Genomics Workbench, and analyzed using MUSCLE and IQtree. Results Seven H3 N2 subtype avian influenza virus isolates were successfully obtained. The nucleotide sequence similarity of the HA gene among the seven isolates was 86.4 to 99.5%, and that of the NA gene was 92.2 to 99.8%. The nucleotide sequence similarity of the internal gene PB2 was 84.7% to 98.6%, that of PB1 was 88.5% to 97.6%, that of PA was 88.3% to 99.3%, that of NS was 91.5% to 99.8%, that of NP was 88.7% to 99.7%, and that of M was 92.2% to 99.8%. Phylogenetic analysis indicated that eight gene fragments of seven isolates were in the branch of Eurasian lineage but were not clustered in each phylogenetic tree. The HA protein cleavage site sequences were all PEKQTR↓GL, consistent with the sequence characteristics of typical low pathogenic avian influenza viruses. A D92 E interferon resistance mutation was detected in the NS gene of one isolate. No mutations were detected in the HA protein receptor binding site, the neuraminidase resistance site, the amantadine resistance site in the M gene, or mammalian adaptation-related sites in the PB2 gene. Conclusion Five combinations of reassortant H3 N2 subtype avian influenza viruses were noted in the seven isolates. Monitoring of live poultry markets and the environment needs to be enhanced to prevent the production of reassortant viruses that can spread among the population.