Isolation,Identification,Whole Genome Sequencing and Genetic Evolution Analysis of Goose Astrovirus in Some Areas of Guangdong Province
[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 7 131 bp were obtained,including 18 bp of 5'-UTR and 184 bp of 3'-UTR,as well as three reading open frames(ORF1a,ORF1b and ORF2 genes),Among them,the ORF1a,ORF1b and ORF2 genes were 3 255,1 551 and 2 115 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-Ⅱ type.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 ORF1a gene and 14 mutations in the ORF2 gene.[Conclusion]The four isolated strains of GAstV were GAstV-Ⅱ type 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.
Goose astrovirusisolation and identificationwhole genome sequencinggenetic evolution analysismutations analysis
万方数据
维普
