Characterization Of A New Nephropathogenic Goose Astrovirus And Development Of An Attenuated Vaccine Candidate

Astroviruses(AstVs)are non-enveloped,positive-sense,single-stranded RNA viruses belonging to the Astroviridae family.They are recognized as a leading cause of gastroenteritis in humans and animals.In poultry,AstV infections have been found to be associated with multiple diseases,such as poult enteritis mortality syndrome,runting-stunting syndrome of broilers,white chick syndrome,kidney and visceral gout in broilers and fatal hepatitis of ducklings.Recently,Cases of fatal visceral gout in goslings which was featured with urate deposition on visceral organ and leg muscle have been reported and the disease spread to most goose producing regions in China,leading to significant economic loss.In this study,the pathogen of goose visceral gout was identified,and the research work of pathogen detection and prevention was carried out.It is of great significance for effective prevention and control of goslings visceral gout.Our systematic investigation led to the isolation of an astrovirus via goose embryo inoculate,designated as AAstV/Goose/CHN/2017/SD01.The isolated astrovirus replicated well and resulted in 100%mortality of goose embryos.Experimental infection of healthy goslings showed that the isolate was highly pathogenic in goslings,causing clinical signs,growth repression and high mortality.Histopathological examination indicated that the lesions occurred mainly in the kidneys of infected birds.However,the virus-specific genomic RNA could be detected in all representative tissues,and virus shedding was detected up to 12 days after inoculation.The etiological role of a genetically distinct astrovirus in the fatal infection of goslings was demonstrated for the first time.Sequence analysis revealed that whole genome of the isolate SD01 was 7175nt in length with similar gene organization to other known Avastroviruses,consisting of 5'-UTR.3'-UTR and three sequential ORFs(ORF1a,ORF1b and ORF2).ORFla,ORFlb encode non-structural protein,consisting four transmembrane region,serine protease,nuclear localization signal,ribosomal frameshift signal and RNA dependent RNA polymerase.Phylogenetic analysis of ORF2 sequences showed that SD01 formed a sister clade neighbouring DAstV-1 and TAstV-2,and the mean amino acid genetic distance values analysis based on the complete capsid protein amino acid indicating that GAstV belong to the same species as TAstV-2 and DAstV-1,but with significant variability.Based on the genetics and biological characteristics of the goose astrovirus infection,we expressed the viral spiker protein using the baculovirus expression system and the recombinant protein was purified to immunize mice.Polyclonal antibodies and monoclonal antibodies against astrovirus were generated and used in the indirect immunofluorescence and immunohistochemical methods for the detection of goose astrovirus in the virus infected cell culture or goose tissues.At the same time,the triple RT-PCR and real-time PCR methods were developed for differential and quantitative diagnosis of goose astrovirus infection in waterfowl.In order to develop a live-attenuated vaccine,we passaged the astrovirus isolate in goose embryos sequentially.The passaged virus exhibited pathogenicity attenuation for goslings at the 60th passage(designated as SD01-G60),as to which no virulence reversion was observerd after 5 successive back-passge in susceptible goslings.Administration of a dose of 5 × 107.0 copies of the SD01-G60 protected goslings from the virulent virus challenge on 10 days after immunization.Neutralization antibody detection suggested that vaccination of the SD01-G60 induced significant immune responses,implying the potential for vaccine development.Sequence analysis revealed 8 amino acid substitutions in the SD01-G70 genome in comparing the parental virus.In summary,our work describes the isolation of the astrovirus AAstV/Goose/CHN/2017/SD01 from tissue samples of goslings dying from a disease characterized by visceral urate deposition.The successful reproduction of the disease by experimental infection demonstrates the etiological role of this AAstV.The development of monoclonal antibodies and the goose embryo-adapted attenuated virus provided useful measures for the diagnosis and control of the goose astrovirus infection in the future.