2019-2021年鸭坦布苏病毒广西流行毒株遗传多样性分析

【目的】研究鸭坦布苏病毒(Duck Tembusu virus,DTMUV)广西毒株分子遗传特征,以期了解、掌握DTMUV广西毒株流行新特点,为及时调整、制定有效防控措施提供数据支持。【方法】采集2019-2021年广西各地的鸭组织病料,采用已建立的实时荧光定量RT-PCR方法检测DTMUV,根据毒株检测年限和来源地选取部分阳性样品进行DTMUV全序列扩增及测序,并进行序列相似性、重要蛋白关键氨基酸位点、系统发育、全序列重组及遗传进化速率分析。【结果】共获得8株DTMUV全序列,基因组全长为10 992 bp,为广西毒株。广西毒株之间开放阅读框(ORF)及E、NS5基因的核苷酸序列相似性分别为97.9%~99.8%、97.0%~99.9%和97.8%~99.9%,氨基酸序列相似性分别为99.1%~99.9%、99.0%~100%和99.3%~99.9%;与国内外参考毒株的核苷酸序列相似性分别为86.4%~99.3%、85.8%~99.5%和87.1%~99.2%,氨基酸序列相似性分别为96.0%~99.8%、94.8%~100%和97.5%~99.9%,且与水禽源TMUV相似性高于其他宿主源。与疫苗株FX2010相比,DTMUV广西毒株E蛋白共有11个氨基酸位点发生变异,其中第43、150、153、326、403、464及487位为广西毒株独特的氨基酸突变。ORF遗传进化分析结果显示,广西毒株均分布于2.1亚群,而源自广西的GX2011和GX2015株隶属于2.2亚群,表明DTMUV广西流行毒株进化趋势不完全一致;E、NS5基因系统发育趋势与ORF相似。重组分析结果表明,GXBH01-2019、GXZS02-2020、ziYY150901及HB2016株检测有重组信号。E、NS5基因的遗传进化速率估算分别为1.31×10^-3和1.30×10^-3替换/(位点·年),表明二者进化较为同步。【结论】当前DTMUV广西流行毒株表现为与主要流行毒株亲缘性较近,E蛋白发生特有氨基酸突变,进化趋势不一致,存在重组现象等分子特征,结果为制定有效防控措施提供了基础数据。

Genetic Diversity Analysis of Duck Tembusu Virus in Guangxi During 2019-2021

【Objective】 The aim of this study was to analyze molecular genetics and prevalent characteristics of Duck Tembusu virus (DTMUV) in Guangxi,so as to understand and master the new epidemic characteristics of DTMUV Guangxi strain,and provide data support for opportunely adjustment and formulation of effective prevention and control measures.【Method】 The tissue samples of duck collected in farms of Guangxi during 2019-2021 were detected for DTMUV by Real-time quantitative RT-PCR,the complete genome sequences of DTMUV were acquired by amplification and sequencing from partial positive samples according to detection date and origin area of DTMUV,and the sequence similarity,predominant amino acid,phylogenetic,recombination detection and estimation rate of evolution were analyzed.【Result】 The genome length of 8 DTMUV strains in Guangxi was 10 992 bp.The similarity of nucleotide sequence of open reading frame (ORF), E and NS5 genes among 8 DTMUV strains in Guangxi were 97.9%-99.8%,97.0%-99.9% and 97.8%-99.9%, and the similarity of amino acid sequence were 99.1%-99.9%,99.0%-100% and 99.3%-99.9%,respectively.The similarity of nucleotide sequence of ORF, E and NS5 genes between 8 DTMUV strains in Guangxi and reference strains were 86.4%-99.3%,85.8%-99.5% and 87.1%-99.2%,and the similarity of amino acid sequence were 96.0%-99.8%,94.8%-100% and 97.5%-99.9%,respectively.The similarity of TMUV with waterfowl source was higher than that of other host sources.Compared with vaccine strain FX2010,there were 11 amino acid mutations in DTMUV Guangxi strains,with the amino acids at 43,150,153,326,403,464 and 487 sites mutation were unique for DTMUV in Guangxi.Phylogenetic tree was constructed on ORF indicated that 8 DTMUV strains acquired in this study were belonged to 2.1 subgroup,while GX2011 and GX2015 strains also from Guangxi were formed to 2.2 subgroup,suggesting that the evolutionary trend of DTMUV epidemic strains in Guangxi was divergence.The phylogenetic trees of E and NS5 genes were similar with that of ORF.Recombination analysis results indicated that GXBH01-2019,GXZS02-2020,ziYY150901 and HB2016 strains existed recombination signal.Estimation rate of evolution performed on E and NS5 genes of DTMUV was 1.31×10^-3and 1.30×10^-3 substitution/(sites·year),which suggested that two genes kept similar evolutionary steps.【Conclusion】 At present,the epidemic strains of DTMUV in Guangxi were closely related to predominant prevalence strains,the E protein had unique amino acid mutation,the evolutionary trend was inconsistent,and there were molecular characteristics such as recombination,which provided basic data for formulating effective prevention and control measures.