Surveillance And Diversity Of Wild Bird Coronaviruses In Shanghai,2019-2021

Coronaviruses have a wide host range and can cause pandemics in humans.Wild birds are thought to be the natural hosts of gammacoronaviruses and deltacoronaviruses,and these coronaviruses can be widely distributed among different regions and species of wild birds.Migratory birds as links and messengers,may play an important role in inter-species and intra-species transmission of coronaviruses across the globe.However,due to the lack of systematic monitoring of coronaviruses in wild birds worldwide,research on coronaviruses detection methods,virus ecology,virus diversity and the relationship between coronaviruses and hosts still needs to be further studied.Shanghai located in the estuary of Yangtze River,has abundant wetland resources which have provided a quality habitat environment for many wild birds.In addition,Shanghai is a crucial stopover and wintering site for a variety of migratory birds on the East Asia-Australia migratory route,and has a high diversity of wild bird species.In view of the importance of wild birds in the ecological and epidemiological studies of coronaviruses,this study established a sensitive and specific molecular biology method that can be directly used to screen wild bird for coronavirus,and then the method was applied to detect coronaviruses in wild bird in Shanghai from 2019-2021,and analyzed the ecological characteristics of the virus and its prevalence.The main results were as follows:(1)In this study,by screening the molecular detection methods published on literature,referring to the gene sequences in the database,and optimizing the amplification primers for avian-derived coronaviruses,we established a specific and sensitive nested RT-PCR assay that can be directly applied to detect both gammacoronaviruses and deltacoronaviruses of clinical samples of wild birds.(2)A total of 3344 wild birds with 5284 samples were collected in 2019-2021.The number of birds tested positive for coronavirus was 225,with a positive rate of 6.7%.Of which,the positivity rate in swab samples > tissue > environmental samples,and the positive rate of gammacoronaviruses was remarkably higher than that of deltacoronaviruses.Coronaviruses have a wide temporal distribution and are prevalent throughout the year,but the peak of the epidemic is in autumn and winter.Positive species were distributed among 6 species of Charadriiformes,10 species of Anseriformes,2 species of Pelecaniformes and 1 species of Coloumbiformes.The highest positivity ratewas found in Anseriformes(16.1%)and the lowest in Charadriiformes(2.2%).In this study,coronaviruses were detected for the first time in Mandarin Duck,Common Pochard,Falcated Duck,Gadwall,Broad-billed Sandpiper,Common Redshank,Great Knot,Red Collared-Dove.Some species may also have virus subtype preference,for example,both types of viruses were found in the order Anseriformes and Charadriiformes,while deltacoronavirus were only found in the order Pelecaniformes and Coloumbiformes.(3)Homology and phylogenetic analysis of gammacoronaviruses revealed that183 strains were mainly distributed in three different clades,namely Avian coronavirus9203,Goose coronavirus CB17 and DCUK 2714 branches.One strain of gammacoronavirus from common teal had high homology with the poultry IBV strain,and the transmission from poultry to wild birds might have occurred;another strain from mallard differed from IBV and other wild bird-derived coronaviruses all belonged to the Goose CB17 clade;the remaining 181 strains of gammacoronaviruses clustered into the DUCK 2714 clade and distributed in six different subclades,which clustered with a large number of wild bird-derived gammacoronaviruses with no obvious temporal geographic pattern.(4)Molecular evolutionary analysis of deltacoronaviruses revealed that there was a high diversity of deltacoronaviruses,and 36 strains belonged to six different clades of deltacoronavirus.Five strains of coronaviruses from the family Heronidae and one strain from an environmental sample belonged to clade of the Hong Kong night heron coronavirus HKU19 in 2011;six strains of deltacoronaviruses from the order Anseriformes and one strain of deltacoronavirus from Mandarin Duck belonged to clade of Wigeon coronavirus HKU20 and clade of common moorhen coronavirus HKU21,respectively;another 14 strains of Anseriformes formed a small clade of the novel wild bird deltacoronavirus 2.The eight strains from the Scolopacidae family are more closely related to the deltacoronavirus,which also comes from the Scolopacidae family in the United States and Australia;the deltacoronavirus found in one environmental sample belongs to the sparrow and porcine coronavirus clade,but is more similar to the sparrow coronavirus,the possibility of its transmission to mammals should be closely monitored.In summary,the avian-derived coronaviruses are widespread in wild birds in Shanghai,involving a wide range of species,high genetic diversity of viruses,and the potential for inter-species and intra-specific transmission of host species.To better understand the ecological and epidemiological characteristics of coronaviruses in wild birds in Shanghai,and the role of wild birds in the transmission of coronaviruses,the prevalence of coronaviruses carried by wild birds of different species in the region should be continuously monitored.