Identification And Genetic Variation Of Novel Astrovirus And H5N8 Subtype Highly Pathogenic Avian Influenza Virus From Wild Birds

Astrovirus infection in poultry can lead to severe fatal hepatitis and nephritis.Avian influenza is caused by avian influenza virus(AIV),H5N8 highly pathogenic avian influenza virus(HPAIV-H5N8)causes a systemic disease with a mortality rate of up to 100%.Astrovirus and HPAIV-H5N8 can infect wild birds,poultry and humans,which cause huge economic loss to poultry industry and pose a threat to human health.Carry out the epidemiological invesigation and the genetic variation of these two viruses,will broaden the genetic evolution of these two viruses in wild birds.In December of 2017,more than 300 crows died suddenly in a province of China.No obvious histological change was observed,and no bacterium was detected in the tiusses of the dead birds.So this disease outbreak was suspected as a virus infection.We firstly used RT-PCR or q RT-PCR methods to test sevral viruses including influenza A virus,West Nile virus,Newcastle disease virus,Japanese encephalitis virus,and forest encephalitis virus.However,all the detected viruses were negative.Then we further used Next Generation Sequencing(NGS)method to screen the potential pathogens.The results showed that there were three astroviruses with whole genomes in the detected RNAs from tissues of the dead crows.To confirm the NGS results,we also used PCR and Sanger sequcing method to amplify and sequence the whole genome of the identified astroviruses.The PCR and NGS results are generally identical.Further genetic analysis showed that the whole genomes of these three astrovirus strains presented low homology with other avian atroviruses.(47.8-52.3%,46.1-51%,and45.7-50.5%,respectively).Interestingly,these three strains also presented low genomic identities with each other as 65.6-82.9%.Phylogenetic analysis for the whole genome and each gene showed that these crow source astroviruses(Crow Astroviruses,Cr Ast V)formed an independent clade in avian astrovirus.In December of 2020,a number of whooper swans died suddenly in a province of China,the reason is unknown.The Oral and cloacal swabs from a diseased swan(a total of 2 samples)and 23 samples of fresh environmental feces were collected for pathogen detection.Only avian influenza virus was detected in the samples,and four whole genome sequences of HPAIV-H5N8 were obtained.Genetic analysis showed that the cleavage site on HA contained several basic amino acids(REKRKR?GLF),suggesting the H5N8 virus was a highly pathogenic influenza virus.These H5N8 viruses contain glutamine(Q)at 226 and glycine(G)at 228 of HA(H3-number)suggesting these viruses prefer avian receptors(?-2,3 sialic acid receptor).However,these strains possess a threonine(T)to alanine(A)mutation at position 160(T160A),which may enhance the ability to bind human receptor(?-2,6 sialic acid receptor).Genetic analysis showed that our H5N8 strians possessed 92.13-97.86% genomic identity to the wild bird source H5N8 vrisues circulating in Asia during 2020-2021 season,while possessed99.70-99.95% identity to the European isolates.Importantly,these new identified H5N8 viruses presented a high homology(99.23%-99.80%)to the H5N8 isolates identified in humans,Russia,in 2020.Phylogenetic analysis showed that the eight gene fragments of our isolates were all clustered together with the European isolates from wild birds.In this study,these new identified astroviruses,formed a new clade in avian astrovirus lineage,were firstly identified in crows,which enriches the ecological map and genetic diversity of astroviruses.The identification and genome sequence analysis of the European-like HPAIV-H5N8 from whooper swans suggested that whooper swan play an important role for the transmission of HPAIVs.The results of the present study provide an evidence for strengthening the surveillance of unkown pathogens in wild birds/animals.