2016-2021年广西H9亚型禽流感病毒HA和NA基因遗传进化动态分析

Genetic Evolutionary Dynamics Analysis of HA and NA Genes of H9 Subtype Avian Influenza Virus in Guangxi from 2016 to 2021

【目的】研究H9亚型禽流感病毒(Avian influenza virus,AIV)广西毒株的遗传进化动态,以期掌握其地域流行新特点,为及时优化调整有效防控措施提供数据支持。【方法】采集2016-2021年广西各地的家禽喉头、泄殖腔及环境拭子样品3600份,接种9~11日龄SPF鸡胚分离病毒,并结合血凝和血凝抑制试验及实时荧光定量RT-PCR方法鉴定AIV亚型,根据毒株检测年限和来源地选取部分H9亚型AIV核酸阳性样品进行HA、NA基因扩增、测序及进化动态分析。【结果】共获得46株H9亚型AIV广西毒株的HA、NA基因序列,其中HA基因全长1683 bp,编码560个氨基酸;4株NA基因全长1410 bp,编码469个氨基酸,42株缺失3个氨基酸,编码466个氨基酸。BLAST分析结果显示,越南及国内多个省份的毒株与本研究获得的广西毒株HA、NA基因相似性最高,分别为97.62%~99.88%和93.13%~99.79%,宿主源也不尽相同,表现遗传多样性特征。广西毒株之间HA基因核苷酸和氨基酸序列相似性分别为93.0%~99.1%和94.6%~99.1%,NA基因核苷酸和氨基酸序列相似性分别为83.9%~98.2%和86.1%~98.2%,表明HA基因较NA基因更为保守;与同期的参考毒株相似性高于早期,表明毒株存在持续进化。遗传进化分析结果显示,广西毒株的HA、NA基因属于当前优势流行的G57基因型分支,与疫苗株遗传关系较远。进化速率估算结果显示,HA、NA基因的平均进化速率分别为3.99×10^(-3)和4.59×10^(-3)替换/位点/年,最近共同祖先估算时间(tMRCA)分别为66.77和58.24年,表明HA基因比NA基因进化缓慢。重组分析结果显示,从鸡、鸭及环境中各分离到的毒株HA基因存在重组现象,主要亲本和次要亲本毒株均为人源的A/Beijing/1/2017株和禽源的A/quail/Hainan/250/2012株。【结论】当前H9亚型AIV广西流行毒株HA、NA基因与越南及国内省份的毒株相似性较高,具有遗传多样性特征,进化趋势以G57为优势基因型,与人源流感毒株发生基因重组,为新型流感产生创造条件。

【Objective】This study was aimed to further understand the evolutionary dynamics and epidemic characteristics of H9 subtype Avian influenza virus(AIV)in Guangxi,which could provide data support for disease prevention strategy constituted and modified.【Method】3600 swabs samples of poultry throat,cloaca and environment were collected in several area markets of Guangxi from 2016 to 2021,then were inoculated in 9-11 days age SPF chicken embryos to isolate virus,the hemagglutinin(HA)and neuraminidase(NA)genes of H9 subtype AIV were amplified,sequenced and genetic evolutionary drynamic analyzed based on hemagglutinin and hemagglutinin-inhibit test,and Real-time quantitative RT-PCR detection assays.【Result】46 HA and NA genes sequence were acquired,the length of HA gene was 1683 bp,coding for 560 amino acids and 4 NA gene was 1410 bp,coding for 469 amino acids except 42 isolates were 466 amino acids with absence of 3 amino acids.BLAST analysis indicated that isolates shared the highest similarity with 97.62%-99.88%and 93.13%-99.79%to HA and NA genes in this study were from Vietnam and several provinces of China,and hosts were not identical,which suggested that the genetic of HA and NA genes was diversity.The nucleotide and amino acid similarity of Guangxi isolates intra were 93.0%-99.1%and 94.6%-99.1%,83.9%-98.2%and 86.1%-98.2%,respectively,which indicated that HA gene was more conserved,the similarity of Guangxi isolates with the reference strains of after 2016 was higher than strains of before,which manifested that the isolates were persisting evolution.The genetic analysis showed that HA and NA genes of Guangxi isolates belonged to G57 branch prevalent in China,the genetic distance of which and vaccine strains was farther.The evolutionary rate estimation of HA and NA genes was 3.99×10-3 and 4.59×10-3 substitution/sites/year,and the most recent common ancestor of which was 66.77 and 58.24 years,which suggested that the rate of HA gene was slower than NA gene.The recombination analysis of HA gene acquired from the chicken,duck and environment was positive signal,respectively.The major and minor parents strains were A/Beijing/1/2017 and A/quail/Hainan/250/2012.【Conclusion】The HA and NA genes of Guangxi isolates with higher similarity to those of Vietnam and provinces of China,the genetic of them was diversity,and evolution progress was close to the dominant gene type G57,the recombination of HA genes was occurred between AIV and human-origin influenza strain,which could provide outbreak condition for novel subtype influence in future.