Genetic Variability And Antigenicity Of Duck Astrovirus (DAstV) And Transcriptome Analysis On Livers Of DAstV-infected Ducklings

Duck viral hepatitis(DVH)is an important disease affecting ducks.Duck astrovirus 1(DAstV-1)and 2(DAstV-2)are two of the causative agents of DVH,and DAstV-1 has been documented in several regions of China.To understand the molecular epidemiology of DAstV in China,we performed molecular detection of DAstV in field samples,genomic characterization of newly discovered DAstVs,and investigation of the pathogenicity and the transmission route of a novel DAstV.A total of 678 samples were collected between 2012 and 2014.DAstV was detected in 94(13.9%)of the samples.Phylogenetic analysis of the partial ORF1b sequence revealed that the 94 astroviruses were identified as DAstV-1(51.1%),DAstV-2(4.3%),a novel DAstV(designated DAstV-3)(35.1%),and chicken astrovirus-like(2.1%)and Northern Pintail astrovirus-like(7.4%)viruses,respectively.The results demonstrated that multiple astroviruses were circulating in duck populations in China.Four DAstV-2 and one DAstV-3 strains were subjected to genomic characterization.The 7319-nt-long DAstV-2 and 7463-nt-long DAstV-3 were shown to possess a typical avastrovirus genome organization.Genetic analysis of the full-length capsid sequence revealed that DAstV-2 and DAstV-3 shared high levels of mean amino acid genetic distance with each other(0.612)and official avastrovirus species(0.579-0.721 and 0.577-0.744,respectively),suggesting that they may represent two additional avastrovirus species.Phylogenetic analyses of three ORFs provide evidence that recombination events may have occurred during evolution of DAstV-2,which may influence the taxonomy of avastroviruses.DAstV-3 was detected in newly hatched ducklings which were used in experimental infection of DAstV-2.Using a DAstV-3-specific RT-PCR developed in this study,DAstV-3 was detected in 46%fresh feces of breeder ducks,60%breeding eggs,46%dead embryos,and 60%newly hatched ducklings,which were taken from a DAstV-3 positive farm.The findings,and the detection of DAstV-3 in 36/130 dead-in-shell duck embryo samples collected from different hatcheries located in six provinces,suggest that the virus may be horizontally and vertically transmitted.Inoculation and repeated passages in embryonating duck eggs resulted in isolation of DAstV-3.The virus caused severe chorioallantoic membrane lesions as well as growth retardation and embryo mortality.8/10 duck embryos inoculated with DAstV-3 were unable to hatch,with most embryos succumbing in the final stage of incubation.The use of RT-PCR on the hatched ducklings provided evidence that the embryos could develop into infected ducklings.It is concluded that DAstV-3 may play a role in hatchability problems.Among duck astrovirueses,DAstV-1 is the most prevalent in ducks.It is therefore important to develop the vaccine for the control of DAstV-1 infection.However,DAstV-1 cannot be propagated efficiently in vitro,making it difficult to develop conventional vaccine.Thus,we evaluate the immunogenicity of recombinant capsid protein which was expressed in the baculovirus system.High levels of antibody were detected in sera collected from Pekin ducklings after immunization with the recombinant capsid protein at 1 and 14 days of age,respectively.Challenge with DAstV-1 D17 at 24 days of age resulted in liver hemorrhagic lesions in 30%of ducks in the immunized group and 80%of ducks in the control group.This suggests that immunization with the recombinant capsid protein can provide partial protection against DAstV-1 infection.In addition,we synthesized 12 peptides based on prediction of linear B cell epitopes of the capsid protein.In indirect enzyme linked immunosorbent and indirect immunofluorescence assays,antisera against DAstV-1 recognized all of the 12 peptides,whereas antisera against recombinant capsid proteins only reacted with peptides A1,A5,and A6.Additionally,reactions were observed between the recombinant capsid proteins and antisera against peptides A1,A4,A5,and A6.From these investigations,we identified three linear immunodominant epitopes,which were found in fragments covering amino acid residues 7-22,166-179,and 199-213.Our results may contribute to the understanding of the antigenic properties of DAstV-1.So far,little is known about the pathogenesis of DAstV-1.Therefore,we screened the differentially expressed genes from livers of the DAstV-1-infected ducklings using RNA-seq.A total of 2377 differentially expressed genes resulted.Among them,38.75%belonged to up-regulated genes,which were associated with immune response,and 61.25%belonged to down-regulated genes which are related with coagulation factors and growth and metabolism genes.GO analysis suggested that the differentially expressed genes may participate in 36 biological processes,18 molecular functions,and 5 cellular component GO terms.Using the KEGG searches,43 signaling pathways were significantly enriched,including signaling pathways associated with metabolism and immune response.These results may provide clues for further research on the pathogenesis of DAstV-1.