Screening And Genetic Characterization Of Viruses Carried By Birds In Yunnan Province

In recent years,the frequent emergence of new and sudden infectious diseases has had a great negative impact on the world’s economic development and the management of people’s life and health safety.Birds have received increasing attention as natural hosts of many human and avian commensal viruses such as avian influenza,West Nile fever,and Japanese encephalitis.The unique immune system of wild birds and their long-distance migratory behavior allow viruses to be transmitted over long distances.Compared to other wildlife,wild birds are extremely abundant in human settlements,res μlting in high frequency contact with people and food.Yunnan is located in the overlapping region of the East Asia-Australasia and Central Asia migratory areas of the global migratory bird route,while there are more than 50,000 birds passing through Yunnan every year,and is one of the important migratory corridors for migratory birds.Therefore,the identification of virus carriage in birds,especially migratory birds,and the discovery of novel viruses and exploration their genetic characteristics in Yunnan are of great importance for the prevention and control of human and avian infectious diseases.The aim of this study is to establish the pathogenic spectrum of wild birds in Yunnan,explore potential novel viruses,and explore their molec μlar characteristics and genetic evolutionary relationships.In this study,1259 bird samples,including 1114 wild bird samples and 145 poμltry samples,were collected from November 2021 to January 2022 in seven migratory bird habitats in Zhaotong,Kunming,Honghe,Qujing,Wenshan,Pu’er,and Dali.The bird samples were processed and macro-transcriptome sequenced by our established sample pre-treatment method;and analyzed to explore the virus carriage of birds;and validated and screened for routinely known viruses using conventional PCR.A total of 13 sequencing libraries were constructed based on bird species and sample types,and data analysis revealed that: all libraries were annotated to a total of76 viruses from 50 virus families,including 16 species from 16 families of vertebrate-associated viruses,including 3.08% of Picornaviridae,1.08% of Paramyxoviridae,Circoviridae 0.08%,Parvoviridae 0.31%,Orthomyxoviridae 9.15%and Flaviviridae 0.69%.Astrovirus were found in the feces of Anser indicus and Larus ridibundus.Parvovirus are unique to Larus ridibundus.The feces of Anser indicus and Grus nigricollis included different species of Picornavirus and various arboviruses,while avian influenza viruses were carried in the tissues of Anser indicus.the tissues of Grus nigricollis were mostly of Picornavirus;The hepatitis viruses were detected only in pigeons,peacocks,egrets,white pheasants,and geese,while in farmed birds such as pigeons,peacocks,egrets,white pheasants,and geese,they consisted of a large number of phages and low levels of DNA viruses such as circovirus and herpesvirus.The analysis revealed that retroviruses were detected in all species;virus species were more abundant in bird fecal samples than in dead bird tissues,and small amounts of plant viruses were present in the lungs of some dead birds,probably because plant particles entered the lungs through the respiratory tract.The composition of digestive tract viruses in different bird species showed diversity;meanwhile,astroviruses and picornavirus were detected in Anser indicus and Larus ridibundus.Based on the migration habits of birds,more virus species were annotated in migratory birds,except for unclassified viruses,Picornaviridae had the widest host range,while Anelloviridae were only found in the lungs of egrets.Eight common viruses,avian influenza virus,Newcastle disease virus,coronavirus,astrovirus,circovirus,rotavirus,avian pox virus,and infectious bursal virus,were screened by conventional PCR methods based on viral macrogenomics resμlts.In this study,four viruses,avian influenza virus 0.64%(8/1259),coronavirus 4.1%(52/1259),astrovirus 1.99%(25/1259),and circovirus 1.27%(16/1259)were identified.Coronavirus hosts were most abundantly sourced.In addition to three species of migratory birds,including Larus ridibundus,Anser indicus and Grus nigricollis,there are also Gallin μla chloropus,mallard ducks,Streptopelia and other resident birds.Further genetic analysis of key genes revealed abundant coronavirus and astrovirus subtypes,which indicates its potential interspecific transmission.Circovirus was more host specific and often co-infected with other viruses,which enhanced the replication of other viruses.Avian influenza viruses were subtyped and f μll-length amplification was completed,and the whole genome sequences of five H5N1 strains were obtained,all the isolated strains were highly pathogenic avian influenza viruses.The genetic evolutionary tree showed that all five viruses were subtype 2.3.4.4b of the GS/GD spectrum and divided into two genotypes;they were genetically related to the H5 Nx of Eurasia 2021-2022 and belonged to the large Eurasian branch.In contrast,the NS and NP gene fragments of DSBBTY5 have a closer genetic relationship with Asian low pathogenic avian influenza H4N6.The mutations at S128 P,S137A,T160 A and S227 R of HA protein may enhance the ability of the virus to bind to mammalian receptors;the mutations at L473 V of PB1 protein,N383 D,N409S and S515 T of PA protein,N30 D,I43M and T215 A of M protein and A/P42 S of NS protein may alter the pathogenic potential of H5N1 HPAI viruses,and no other amino acid variants associated with viral pathogenicity and drug resistance were identified.In conclusion,this study applied viral transcriptomic techniques and PCR assays to investigate the pathogens carried by birds in Yunnan,enriching the pathogenic spectrum of birds in Yunnan.We identified m μltiple subtypes of avian influenza viruses and astrovirus with potential cross-species transmission,and a large number of avian coronaviruses in a variety of bird species.There were significant differences in enterovirus communities and diversity among host types,and migratory birds carry a richer variety of viruses.The large number of unclassified viruses suggested that the viruses we examined were only the tip of the iceberg of viruses carried by birds,and the molec μlar characterization of more unknown viruses in birds still needs to be continuously explored and analyzed.