广东部分地区鹅星状病毒的分离鉴定、全基因组测序及遗传进化分析

Isolation,Identification,Whole Genome Sequencing and Genetic Evolution Analysis of Goose Astrovirus in Some Areas of Guangdong Province

【目的】了解鹅星状病毒(Goose astrovirus, GAstV)在广东部分地区的流行及遗传变异情况,阐明其遗传进化与全基因组的特征,为GAstV的防控提供理论基础。【方法】使用GAstV特异性引物对2021―2022年广东地区养鹅场内出现疑似痛风的36份鹅病料进行PCR检测,选取4份具有代表性的GAstV强阳性样品,利用鹅细小病毒、坦布苏病毒、禽流感病毒、新城疫病毒的特异性引物进行检测,以排除病料中存在其他常见的外源性病毒;在此基础上利用鹅胚及鸡肝癌细胞系(leghorn male hepatoma, LMH)对4份GAstV样品进行病毒的分离和纯化,使用透射电镜对病毒粒子的形态进行观察;设计7对特异性引物对4株GAstV进行全基因组扩增和测序,运用DNAStar软件对序列进行整理与拼接以获取GAstV的全基因组序列,利用MegAlign软件进行核苷酸相似性比对,并对关键氨基酸位点的变异情况进行分析,应用Mega-X软件对全基因及单个ORF基因(ORF1a、ORF1b、ORF2)进行系统进化树构建。【结果】经PCR鉴定发现被检样品中共有30份是GAstV阳性,选取的4份代表性GAstV阳性样品均不存在其他外源性病毒感染,利用鹅胚及LMH细胞成功分离出了该4株GAstV病毒,并获得了4株全长均为7 131 bp的GAstV基因组序列,包括18 bp的5′-UTR和184 bp的3′-UTR、3个阅读开放框(ORF1a、ORF1b和ORF2基因),其中ORF1a、ORF1b和ORF2基因分别长3 255、1 551和2 115 bp,与报道的GAstV全基因组特征基本一致。相似性分析显示,4株GAstV之间的核苷酸相似性在99.4%~99.8%之间,与GenBank收录的其他19株GAstV的核苷酸相似在57.6%~99.6%之间。全基因组及3个ORF基因的遗传进化分析显示,获得的4株GAstV均属于GAstV-Ⅱ型。氨基酸序列分析显示,4株GAstV的氨基酸存在22处位置上的突变,其中ORF1a基因有8处突变,ORF2基因有14处突变。【结论】分离的4株GAstV均为GAstV-Ⅱ型毒株,与中国其他地区流行的基因型基本一致,该4株病毒的氨基酸突变主要发生在ORF1a和ORF2区域,为后续疫苗研发、流行病学调查及致病机理等方面提供参考依据。

【Objective】The aim of this study was to understand the prevalence and genetic variation of Goose astrovirus(GAstV)in some areas of Guangdong,elucidate its genetic evolution and genome-wide characteristics,and provide a theoretical basis for the prevention and control of GAstV.【Method】Using GAstV specific primers,PCR detection was performed on 36 goose samples suspected of gout in goose farms in Guangdong province from 2021 to 2022.Four representative samples with strong positive GAstV were selected,and specific primers for GPV,TMUV,AIV and NDV were used for detection to exclude the presence of other common exogenous viruses in the samples.On this basis,the four GAstV samples were isolated and purified using goose embryos and chicken liver cancer cell lines(LMH),and the morphology of the virus particles was observed using transmission electron microscopy.7 pairs of specific primers for whole genome amplification and sequencing of 4 strains of GAstV were designed.The sequences were organized and spliced to obtain the whole genome sequence of GAstV by DNAStar software.The nucleotide similarity was compared and the variation of key amino acid sites were analyzed by MegAlign software.The phylogenetic tree for the entire gene and individual ORF genes(ORF1a,ORF1b and ORF2)were constructed by Mega-X software.【Result】Through PCR identification,it was found that a total of 30 tested samples were GAstV positive.The four representative GAstV positive samples selected did not contain any other exogenous virus infections.The four strains of GAstV virus were successfully isolated using goose embryos and LMH cells,and four GAstV genome sequences with a total length of 7131 bp were obtained,including 18 bp of 5′-UTR and 184 bp of 3′-UTR,as well as three reading open frames(ORF 1 a,ORF 1 b and ORF 2 genes),Among them,the ORF 1a,ORF 1 b and ORF 2 genes were 3255,1551 and 2115 bp long,respectively,which was basically consistent with the reported whole genome characteristics of the GAstV.Similarity analysis showed that the nucleotide similarity between the four strains of GAstV was between 99.4%and 99.8%,and the nucleotide similarity with the other 19 strains of GAstV included in GenBank was between 57.6%and 99.6%.The genetic evolution analysis of the entire genome and three ORF genes showed that all four strains of GAstV obtained belonged to GAstV-Ⅱtype.Amino acid sequence analysis showed that there were 22 mutations in the amino acids of the 4 strains of GAstV,including 8 mutations in the ORF 1 a gene and 14 mutations in the ORF 2 gene.【Conclusion】The four isolated strains of GAstV were GAstV-Ⅱtype strains,which were basically consistent with the genotypes prevalent in other regions of China.The amino acid mutations of these four strains mainly occur in the ORF1a and ORF2 regions,providing reference for subsequent vaccine development,epidemiological investigations,and pathogenic mechanisms.