Genetic Variation Analysis,Isolation And Identificaiton Of Porcine Astrovirus In Hunan Province

Astroviruses are a common cause of gastroenteritis in humans and animals.They are also associated with extraintestinal infections,including hepatitis in ducklings,nephritis in chickens,as well as fatal meningitis and encephalitis in humans and other mammals.Since 2015,outbreaks of disease characterized by visceral gout and swelling of kidneys have been reported in goslings and ducklings in China,with the causative agent revealed to be a novel avian astrovirus designated novel goose astrovirus.In the present study,this novel gout-associated GoAstV was identified in diseased goslings from 2 farms in Hunan province,China.Three genomes were successfully sequenced and analyzed and were shown to have high identities of 99.7to 99.8%between each other,with some specific amino acid alterations revealed in open reading frame 2 when compared with other gout-associated GoAstVs.Two strains were further efficiently isolated in the DF-1 chicken fibroblast cell line with high virus titers of 10¹¹viral genomic copies perm L of culture media.A pilot virus challenge study using GoAstV isolations inchickens demonstrated that this virus can cause clinical visceral gout in chickens,indicating that the virus could infect not only geese,ducks and other waterfowl but also chickens and other land fowl.Based on the phylogenetic analyses of Capsid sequences,the identified GoAstVs were proposed to be classified into 2 genotypes,GoAstV1 and GoAstV2 according to the time of discovery,and the novel gout-associated GoAstVs were all clustered in GoAstV2.Further Bayesian inference analyses indicated a nucleotide substitution rate of 1.46×10^-3substitutions/site/year for avian astrovirus based on open reading frame2sequences,and the time to the most recent common ancestor of GoAstVs was estimated to bearound 2011.This is the first report to confirm GoAstV can infect chickens while also providing an estimation of the evolutionary rates of Avastroviruses,which provides an important basis for the follow-up research on the pathogenesis,epidemiology and replication mechanism of GoAstV in cells.