| Avian astrovirus(AAstV)belongs to the family of astroviridae,which is an important pathogen in poultry industry.AAstV includes avian nephritis virus(ANV),duck astrovirus(DAstV),chicken astrovirus(CAstV),goose astrovirus(Go AstV),etc,which can infect a variety of birds and cause chicken nephritis,duck hepatitis,diarrhea,growth retardation,gout and other symptoms.In recent years,AAstV is widely infected in the world,and it occured occasionally in China,such as Gosling visceral Gout Caused by novel goose astrovirus in Shandong,Jiangsu and other places in 2017.In addition,the emergence of new epidemic strains,such as CAstV and DAstV,also brings new pressure to the prevention and control of the disease.To understand the prevalence of avian astroviruses in China,the molecular epidemiological investigation was carried out on poultry samples collected from 8 Provences.The whole genome sequencing and pathogenicity analysis of the representative strains of avian astroviruses were carried out to provide scientific basis for the prevention and control.1.Molecular epidemiological investigation of avian astrovirusesIn this study,we designed universal primers for the RNA-dependent RNA polymerase(RdRp)gene of avian astrovirus,and a total of 1210 throat and anal swab samples were collected from wholesale markets,retail markets,farms and slaughterhouses in 8 provinces.The avian astrovirus was sequenced by reverse transcription polymerase chain reaction(RT-PCR)and phylogenetic tree was constructed according to the sequencing results.A total of 17 AAstVs were detected,including 7CAstVs,5 ANVs,2 DAstVs,2 Go AstVs,and one novel avian astrovirus(AAstV-3),and the overall positive rate was 1.4%.The detected AAstVs were isolated by inoculating chicken embryo(duck embryo or goose embryo),Notably,the existence of AAstVs has been confirmed from breeding,slaughtering to marketing.The results showed that the pollution of AAstV was serious in the whole poultry industry chain,and the circulation of products between each link promoted the spread,which also suggested that we should strengthen the monitoring of AAstV.This study confirmed the genetic diversity of AAstV in China and further enriched the epidemiological data of AAstV in China.2.Avian astrovirus isolation and characterizationVirus isolation was performed on the detected avian astroviruses by inoculation with chicken embryos(duck embryos or goose embryos).The results showed that ANVs and CAstVs could be isolated from chicken samples using chicken embryos,DAstV could be isolated from duck samples using duck embryos,and Go AstV could be isolated from chicken embryos.In addition,two novel goose astroviruses could also be isolated from duck embryos.This suggests that the isolated novel goose astrovirus could be transmitted across hosts.The strain isolated were selected for detection of the exogenous virus,and the strain of chicken astrovirus N-2P which can proliferate stably was quantitated as purified and inoculated into 1-day-age SPF chickens for pathogenicity testing,which showed that the chickens in the challenge group were in stable condition,without die,and had no obvious lesions at necropsy within 15 d of observation.Three of seven chicks in the experimental group tested positive for chicken astrovirus nucleic acid in the liver,spleen,and kidney(42.86%).The results showed that this chicken astrovirus isolate could infect SPF chickens,but did not cause obvious lesions,which illustrated that the chicken astrovirus isolated in this study was less pathogenic.It can be concluded that there are avian astroviruses with weak pathogenicity in China,which are not very harmful,and this is also the main reason that they have not received enough attention.Mixed or secondary infection with avian astroviruses and other pathogens has been shown to cause stronger pathogenicity,and therefore,enhanced prevention and control of avian astrovirus disease is warranted.3.Analysis of the genomic properties of avian astrovirusesThe 17 avian astroviruses isolated were subjected to whole genome sequencing by next-generation sequencing to identify homologies and evolutionary trees with Gen Bank reference sequences and to predict epitope,transmembrane region structure,and hydrophobicity.Genetic evolution analysis revealed that the 17 avian astroviruses were distributed in six clades,consisting of seven chicken astroviruses,two novel goose astroviruses,five avian nephronophthisis viruses,one novel astrovirus,one duck viral hepatitis 3 virus,and one duck astrovirus type 1.Of these,the isolate G1272 had only the ORF2 region that could be matched to a highly homologous reference strain sequence.However,the ORF1 a and ORF1 b regions share low sequence homology with known sequences and have less than 50% nucleotide and amino acid identity compared with the reference strains such as DAstV-1,CAstV and DAstV-2.Nucleotide and amino acid homology analysis of the remaining isolates in the whole genome sequence and in the three ORF regions showed high homology to their corresponding reference strains,including Go AstV,DAstV-1,CAstV,DAstV-2 and ANV.Hydrophobicity,epitope and transmembrane region prediction results showed that the proteins encoded by the three ORF regions in all isolates were well antigenic and all were hydrophilic,with only the ORF1 a region predicted to have 4-6 transmembrane region structures and no related structures predicted in ORF1 b and ORF2.In this study,whole genome sequencing analysis and prediction analysis of structures such as hydrophilicity of the isolated avian astroviruses not only confirm that the avian astroviruses circulating in domestic poultry in China are genetically diverse,but also enrich the epidemiological repertoire of avian astroviruses and provide technical support for follow-up studies of avian astroviruses. |
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