新型鹅源星状病毒广西流行株GoAstV-GXHZ01-2022基因组分子特征分析

Molecular characteristics of complete genome of novel goose-origin astrovirus strain GoAstV-GXHZ01-2022 in Guangxi

为研究新型鹅源星状病毒(GoAstV)广西流行株基因组的分子特征,本研究采集广西3只疑似GoAstV感染发病雏鹅的3份组织病料样品(每份样品包括肾脏、肝脏和脾脏),采用荧光定量RT-PCR(RT-qPCR)检测GoAstV,以GoAstV阳性样品的核酸为模板,经RT-PCR分段扩增、测序及拼接获得GoAstV广西株的全基因组序列,采用BioEdit软件分析上述获得的GoAstV全基因组序列与该病毒参考株各功能基因(ORF1a、ORF1b及ORF2)的相似性,采用Bepipred及NetNGlyc软件预测该病毒Cap蛋白的抗原表位与N-糖基化位点,通过Mega 7.0软件绘制各功能基因的进化树,通过RDP 5软件分析ORF基因序列的重组事件。结果显示,RT-qPCR检出3份GoAstV阳性样品,并经测序获得1条GoAstV广西流行株GoAstV-GXHZ01-2022全基因组序列,该基因组全长为7 149 bp,其中5'非编码区(5'UTR)长143 bp,ORF全长6 929 bp,3'UTR长77 bp;GoAstV-GXHZ01-2022株与经典GoAstV代表株的ORF1a、ORF1b及ORF2基因序列的相似性分别为53.0%~53.4%、63.2%~63.5%及48.2%~48.4%,与新型GoAstV参考株相应基因序列的相似性分别为97.0%~99.6%、98.1%~99.8%及97.5%~99.8%;Cap蛋白抗原表位预测结果为aa6~aa16、aa20~aa66及aa80~aa86等共29个区域存在抗原表位,N-糖基化位点预测结果为^(81)NSTD^(84)、^(401)NTTG^(404)、^(464)NITF^(467)及^(531)NTST^(534);功能基因的进化树结果显示,GoAstV-GXHZ01-2022株与新型GoAstV参考株均处于同一进化分支,与禽星状病毒3型(AAstV-3)流行株遗传距离较近,与AAstV-1次之,与AAstV-2最远;基因重组分析结果显示,GoAstV-GXHZ01-2022株的ORF1a和ORF2检测到重组信号,主要亲本株为新型GoAstV安徽株AstV/AH01/Goose/0512/18,二者相似性为98.6%,次要亲本株为新型GoAstV河南株AstV/HB02/Goose/0310/19,二者相似性为99.1%。表明GoAstV-GXHZ01-2022株与新型GoAstV参考株相似性最高,进化趋势也一致,并检测到重组现象。本研究为丰富GoAstV基因库中广西流行株全基因组序列信息提供基础数据。

In order to understand the molecular characteristics of complete genome of a novel goose-origin astrovirus (Go Ast V)prevalent in Guangxi,three tissue samples (kidney,liver and spleen) from goose suspected of Go Ast V infection were collected and detected using the fluorescence quantification RT-PCR assay (RT-qPCR).Positive Go Ast V samples were used as template for the amplification and sequencing of the complete genome sequences.The similarity analysis was conducted using Bio Edit software,while epitope sites and N-glycosylation sites prediction of Cap protein were predicted using Bepipred and Net NGlyc 1.0 online software.Phylogenetic and recombination analysis were performed with Mega 7.0 and RDP 5.Three Go Ast V positive samples were identified and the genome sequence of Go Ast V-GXHZ01-2022 strain was obtained.The genome length was 7149bp,consisting of a 143bp 5'untranslated region(UTR),a 6929bp open reading frame (ORF) and a 77bp 3'UTR.Similarity analysis based on the nucleotide sequences of ORF1a,ORF1b and ORF2 of Go Ast V-GXHZ01-2022 showed 53.0%-53.4%,63.2%-63.5%,and 48.2%-48.4%similarity to classical Go Ast V strains,and 97.0%-99.6%,98.1%-99.8%,and 97.5%-99.8%similarity to novel Go Ast V reference strains.Epitope sites prediction of Cap protein of Go Ast V-GXHZ01-2022 identified sites at aa6-aa16,aa20-aa66,aa80-aa86.N-glycosylation sites prediction were identified at^(81)NSTD^(84),^(401)NTTG^(404),^(464)NITF^(467)and^(531)NTST^(534).Phylogenetic analysis based on nucleotide sequences of ORF,ORF1a,ORF1b and ORF2 genes indicated that Go Ast V-GXHZ01-2022 and novel Go Ast V reference strains belonged to the same branch,most closely related to AAst V-3,followed by AAst V-1 and AAst V-2 strains.Recombination signal was detected in the ORF1a and ORF2 genes of Go Ast V-GXHZ01-2022 strain,with the major parent being the Ast V/AH01/Goose/0512/18 strain from Anhui Province (98.6%similarity),and the minor parent being Ast V/HB02/Goose/0310/19 strain from Henan Province (99.1%similarity).The findings suggested that the Go Ast V-GXHZ01-2022 strain shares the highest similarity to novel Go Ast V reference strains,exhibits an identical genetic trend,and shows evidence of recombination.This study enriches the genomic database and provides valuable information on Go Ast V strains prevalent in Guangxi.