Isolation,Identification And Biological Characterization Of Goose Astrovirus

Astrovirus(Ast V)is a non-enveloped,single stand positive sense RNA virus,which belongs to the Astroviridae family.The virus has been detected in a variety of mammals and birds such as chickens,turkeys,ducks,geese and pigeons,and it is related to diarrhea diseases in many animals.Since 2017,goose-derived astrovirus has been reported in Shandong,Anhui,Guangdong,Hunan and Jiangsu province,with a fatality rate of more than 30%,resulting in serious economic losses in the goose industry.In this study,RT-PCR was used to detect goose-derived astrovirus.Visceral grinding supernatants of dead geese with positive results were collected for virus isolation and two stable breeding astrovirus strains were isolated: GAstV/Goose/2019/HLJPF and GAstV/Goose/2018/HLJ01,of which the ELD50 of GAstV/Goose/2019/HLJPF was 10-2.6/0.2 m L.The results of isolation and passage of goose embryos showed that the virus inoculated goose embryos could cause systemic bleeding,inhibition of growth and development or death of goose embryos.A group of primers were designed and synthesized.The virus genome was amplified by RT-PCR,and the whole genome of the virus was sequenced.The structural composition,antigenic epitopes,phylogeny,recombination and mutation of the virus were analyzed.The results showed that the full length of the genomes of GAstV/Goose/2019/HLJPF and GAstV/Goose/2018/HLJ01 were 7 233 bp and 7 166 bp respectively,encoding three open reading frames(ORFs),namely ORF1 a,ORF1b and ORF2.Phylogenetic analysis of the sequences of two GAstV viruses showed that they belonged to Avastrovirus,and were closely related to Avastrovirus 3 and TURKEY Astrovirus.In addition,the mutation rates of ORF1 a and ORF2 were higher.The tertiary structure of the mutant ORF2 protein was smooth and its antigenic epitope was highly mutated,which may be related to the pathogenicity of the virus and was caused by the antibody pressure of the host.Based on genetics and biological characteristics of GAstV,the prokaryotic protein expression system was employed to express the capsid protein of HLJ01 strain,and the purified recombinant protein was employed to immunize mice to prepare polyclonal antibodies against virus capsid protein.At the same time,a pair of specific primers were designed for theconservative region,and a Taq Man probe Real-time quantitative PCR method for the detection of GAstV was established.To sum up,in this study,we isolated and identified GAstV,systematically studied the biological characteristics of GAstV,analyzed its genome sequence,predicted its protein structure and function,established a Taq Man probe Real-time quantitative PCR method for detecting GAstV,and prepared specific polyclonal antibodies against its capsid protein.It has laid a certain theoretical foundation and practical support for further deepening the understanding of GAstV.