Virome Analysis And Epidemiological Investigation Of Coronavirus In Bat Guano From Four Counties Of Dali Prefecture,Yunnan Province,China

ObjectivesThe study aimed to gain insight into the viral spectrum carried by bats and prevalence of coronaviruses in bats in Dali Prefecture,Yunnan Province,with a view to providing a basis for the prevention and control of local zoonoses.MethodsIt was selected for the survey and bat guano samples were collected in the four Counties of Heqing,Binchuan,Yangbi and Nanjian in Dali Prefecture,Yunnan Province.It was carried out that viral metagenomics by next generation sequencing(NGS)and coronavirus infection in bats by nested RT-PCR.Subsequently,analysis of coronavirus positivity and mapping of geographical distribution were used by SPSS 23 and Arc GIS10.2 statistical software.The whole coronavirus genomes were obtained using Sanger sequencing and NGS.The sequences were then analysed using bioinformatics software such as BLAST,ORF Finder,MAFFT 7,MEGA X,Geneious Prime,RDP4 and Simplot to determine the viral genotype,annotate the genome,construct phylogenetic trees and analyse gene recombination events.ResultsFrom June to July 2022,519 samples were collected at six sampling sites in the four counties of Dali Prefecture,including Heqing,Binchuan,Yangbi and Nanjian counties.A total of 1,654,519 virus-associated reads were matched by NGS.The highest number of reads was Parvoviridae with 75.99%,followed by Totiviridae and Coronaviridae with 7.11% and 6.83%,respectively.143 positive samples were investigated for coronavirus with an overall positive rate of 27.55%(143/519).The positivity rate in the cave of Banpo Village,Bixi Township,Nanjian County was98.41%(124/126),and the positivity rate at this sampling site was statistically higher than that at other sampling sites(P<0.001).23 full-length coronavirus sequences were obtained in this study.A number of 21 coronaviruses belonged to the subgenus Sarbecovirus of the genus Betacoronavirus,of which 19 sequences were from Rhinolophus osgoodi and the remaining 2 sequences were from Rhinolophus affinis.In addition,two coronaviruses belonged to the subgenus Decacovirus and Rhinacovirus of the genus Alphacoronavirus,with hosts from R.affinis and Rhinolophus ferrumequinum,respectively.Although the main structure of the coronavirus genomes was consistent between the three subgenera,there were few differences in their accessory protein genes.Fifteen recombination events were identified by RDP4 software and the 15 potential recombination events were re-validated using Sim Plot software,which revealed the presence of five significant recombination events in the subgenus Sarbecovirus.The results showed that recombination events were common in the subgenus Sarbecovirus across regions,hosts and years.ConclusionViral metagenomics discovered 17 families of viruses and successfully obtained the basic characteristics of the viral spectrum from bat guano in Dali Prefecture.Bats from Heqing,Binchuan and Nanjian counties carried the genera Alphacoronavirus and genus Betacoronavirus,with rich genetic diversity.In addition,Whole genome sequences of 23 coronaviruses were obtained,all from the genus Rhinolophus.Furthermore,this study revealed a frequent,complicated and long-term recombination history of the subgenus Sarbecovirus.