Metavirome Analysis Of Wild Birds And Study Their Coronaviruses In Shanghai,2019-2021

In recent years,global climate change,pollution,and loss of wildlife habitats have led to increasing conflicts between wildlife habitats and human activity areas.This has resulted in the outbreak of new infectious diseases worldwide,posing great threats to human health,social stability,economic development,and national security.As one of the most biodiversity vertebrates,wild birds have a large population and wide distribution,they are also natural hosts of some zoonoses.Moreover,due to the clustering and migratory behavior of most birds,they have the potential to spread the viruses across regions or continents,and the potential risk of spillover of pathogens carry to humans,domesticated animals,and other wildlife.However,little is known about the virome of wild birds,which hinders the establishment of warning systems for wild bird-derived zoonoses and effective conservation of wild birds population.With the development of next-generation sequencing technology,there has been a greater understanding of wild bird virome,leading to the discovery and identification of new pathogens.Shanghai is located on the East Asia-Australasia migratory route and has abundant wetland resources,making it an important transit point and wintering site for a variety of migratory birds.To gain insight into the virome of wild birds in Shanghai,this study performed a meta-virome of wild bird samples collected in the region during2019-2021,and the whole-genome amplification and analysis of wild bird coronaviruses,The study’s main findings are as follows:(1)A total of 9,956,825,792 high-quality reads were obtained from 214 swabs and fecal samples of wild birds from 18 species in 4 orders in Shanghai,and these reads were assembled into 34,569,319 contigs,covering 4 viral domains,7 kingdoms,12 phyla,30 orders,43 orders and 83 families.The abundance of DNA viral families was75.15%,RNA viral families was 10.71%,and unclassified viral families was 14.13%.The most abundant DNA viral families were phage viral families,adenoviridae,parvoviridae and circoviridae.The most abundant RNA viral families were picornaviridae,coronaviridae,astroviridae and paramyxoviridae.(2)Abundant phage viruses were identified in wild bird samples from three different sampling sites,Chongming,Nanhui and Jiuduansha.The virus α-diversity of wild birds at different sampling sites were not statistically significant.Both the Charadriiformes and the Anseriformes,which are two major migratory bird taxa,carried abundant phage viruses and picornaviridae,and the virus α-diversity between them is no significant significant.However,the virus α-diversity of different species of Anseriformes were significantly significant.The virus α-diversity of Eurasian Wigeon,Spot-billed Duck and Common Teal was relatively high,while the virus α-diversity of Northern Shoveler and Common Pochard was the relatively lowest.(3)Phylogenetic analysis of adenoviridae,parvoviridae,avian paramyxoviridae,astroviridae,and picornaviridae identified with complete genome sequences,and coronaviruses with high carriage rates,showed that these viruses are more closely related to those found in wild birds and poultry along the migratory routes.This suggests that wild birds can play a potentially important role in the spread and transmission of zoonoses.(4)The whole genome sequences of five coronaviruses,including three Gammacoronaviruses and two Deltacoronaviruses were successfully obtained using rapid amplification of c DNA ends technique.The whole-genome nucleotide homology was 73.9 ～ 93.9% for three Gammacoronaviruses and 65.9% for two Deltacoronaviruses.Phylogeny analysis showed that the three Gammacoronaviruses belonged to the Duck 2714 branch and were closely related to the Australian wild bird coronavirus MK204393 and MK204411.One strain of Deltacoronavirus belonged to the Wigeon HKU20 branch and clustered with the Deltacoronavirus isolated from the Australian Ruddy Turnstone;the other strain clustered with the Deltacoronavirus from the Australian waterfowl in the new wild bird Deltacoronavirus branch.In summary,this study enriches the understanding of the viral diversity of wild birds in Shanghai,emphasizes the importance of strengthening the monitor of wild birds,and provides a theoretical basis for research on early warning,traceability and prevention and control of emerging infectious diseases of wild birds.