云南野鸟源H9N2亚型AIV的遗传进化分析

Genetic Evolution Analysis of H9N2 Subtype Avian Influenza Viruses in Wild Birds from Yunnan Province

【目的】分析云南纳帕海国际重要湿地H9N2亚型禽流感病毒(Avian influenza virus,AIV)的遗传进化和分子特征,掌握H9N2亚型AIV在野禽中的流行特点,为禽流感疫情防控提供数据支持。【方法】采集2022―2023年云南省香格里拉纳帕海国际重要湿地野禽粪样及其周边家禽喉头、泄殖腔和环境水样,经尿囊腔接种10日龄SPF鸡胚分离病毒,结合HA试验和RT-PCR扩增鉴定AIV亚型。利用MBTuni-12/13引物同时扩增H9N2亚型AIV毒株的8个基因(HA、NA、M、PB2、NS、NP、PA及PB1)节段,利用二代测序获取全基因组序列,并分析遗传进化关系及关键氨基酸突变位点。【结果】分离到的4株野鸟源H9N2亚型AIV的HA、NA基因源自Y280-like亚系,M、PB 2基因为G1-like亚系,NS、NP、PA、PB 1基因为F/98-like亚系,表明4株分离株为多种亚型的重组毒株,重组模式与2013年以后在中国流行的G57基因型一致。序列相似性分析结果显示,4株H9N2亚型AIV分离株内部基因与H7N9、H5N6、H5N2等亚型AIV相似性较高,遗传多样性特征突出。HA蛋白裂解位点均符合低致病性禽流感特征,获得了多个可增加跨种感染风险及与哺乳动物致病力增强相关的氨基酸突变位点,包括A198T、Q234L、N166D(HA),I292V、A558V(PB2),I368V、L13P(PB1),N30D、T215A(M1),以及P42S(NS1)等。4株野鸟源H9N2亚型AIV毒株具有家禽源同亚型毒株特征,野禽源AIV可能源于家禽。【结论】从云南纳帕海国际重要湿地分离到的4株野鸟源H9N2亚型AIV可能是由家禽传播的多源重配病毒,属于G57基因型,且具有多个适应哺乳动物宿主相关的氨基酸突变位点。

【Objective】This study was aimed to analyze the genetic evolution and molecular characteristics of H9N2 subtype Avian influenza virus(AIV)originating from Napahai,an international important wetland in Shangri-La,Yunnan province,and clarify the epidemic characteristics of H9N2 subtype AIV in wild birds,so as to provide data for the prevention and control of avian influenza epidemics.【Method】Fecal samples of wild birds,and the oropharyngeal,cloacal swab and and environmental water samples of surrounding poultry from Napahai international important wetland of Yunnan province during 2022-2023 were collected.Allantoic cavity inoculation in 10-day-old SPF chicken embryo was used for isolating virus,and AIV subtype was identified through hemagglutination(HA)assay and RT-PCR amplification.Firstly,all the eight gene(HA,NA,M,PB2,NS,NP,PA and PB1)segments of H9N2 subtype AIV isolates were simultaneously amplify using MBTuni-12/13 primers,followed by the second-generation sequencing to obtain full genome sequences.Finally,the genetic evolutionary relationship and amino acid mutation analysis were performed.【Result】Four H9N2 subtype AIV isolates were identified,which exhibited G57 genotype characteristics that had been prevalent in China since 2013,with HA and NA genes from Y280-like subtype,M and PB 2 genes from G1-like subtype,and NS,NP,PA and PB 1 genes from F/98-like subtype.Four H9N2 subtype AIV might be the recombinant strains from multiple AIV subtypes.The results of sequence similarity analysis showed that the internal genes of four H9N2 subtype AIV isolates had the higher similarity with H7N9,H5N6 and H5N2 subtype AIV isolates,the genetic diversity characteristic was prominent.The HA cleavage sites of four strains were consistent with characteristic of cleavage of low-pathogenic avian influenza.Multiple amino acid mutations associating with increasing the risk of cross-species transmission and enhancing virulence in mammals were acquired by the isolates,including A198T,Q234L and N166D(HA),I292V and A558V(PB2),I368V and L13P(PB1),N30D and T215A(M1),P42S(NS1),etc.Four wild bird-derived H9N2 subtype AIV strains exhibited characteristics similar to those of current Chinese chicken-derived AIVs,indicated the potential for transmission route from poultry to wild birds.【Conclusion】Four H9N2 subtype AIV from wild birds in Napahai international important wetland of Yunnan province belonging to G57 genotype,were likely to be reassortants and may originate from poultry,and had the molecular characteristics related to the adaptation to mammalian hosts.