| Objective: Rodents are the natural hosts of more than 100 viruses,and their living areas broadly overlap with human activity.Through direct or indirect contact,rodent viruses can infect people and cause infectious disease outbreaks.Due to its tropical area,hot and humid climate,and frequent trade,the China(Hainan)Pilot Free Trade Zone sees an increased danger of the spread of different tropical viral infectious illnesses.Huangjingjiaoling,Bangxi,and Yingge Ling Nature Reserves are the most inaccessible areas,but there are abundant rodent species.A virology study on the Parvoviridae,Coronaviridae,Flaviviridae,Astroviridae,Parvoviridae,and Papillomaviridae carried by representative rodents in the surrounding urban areas and natural reserves of Hainan Island was conducted using next-generation sequencing technology.We identified viral genera or species,described the viral spectrum,genetic diversity,phylogenetic relationships,and host geographic distribution characteristics of the viruses carried by rodents in Hainan,and assessed the possibility of cross-species pathogen transmission to humans or domesticated animals.Methods: The sampling area of this project includes the mountains,plains,and hills of Hainan Island,and the sampling season includes the spring and autumn,when rodents are active most frequently.The collecting area comprises the southern,eastern,northern,and western representative borderlands of cities and nature reserve.A total of 682 throat and anal swab samples of rodents collected from 9 areas in Hainan were grouped according to species,swab type,and collection area,and then mixed into 28pools(15–20 samples in each pool).The pools were filtered and ultracentrifugation to remove non-viral components such as bacteria and fungi,and digested to remove free nucleic acids.The viral nucleic acid was extracted and inverted,then amplified by anchor primers,and purified by magnetic bead sorting to complete the construction of a nucleic acid library.The amplified viral nucleic acid library was analyzed by the Illumina Hi Seq2500 sequencer(Illumina,USA).Each filtered valid read was compared with the non-redundant protein database of the National Center for Biotechnology Information(NCBI)to evaluate the classification of the virus.The virus reads were extracted after bioinformatics quality control of the sequencing results.According to the molecular clues of viral reads,specific primers were designed for nest-PCR amplification of the complete genome or conserved regions of the virus and sanger sequencing,and the infection rate of the virus carried by the host was detected and the phylogenetic characteristics were analyzed.Results: Based on next-generation sequencing(NGS)technology,a total of 682 rodent swabs from different regions in Hainan were studied for virome research.The genome,phylogenetic analysis,and co-evolution analysis with hosts were carried out for six virus families."Filtering-enrichment-nuclease digestion-viral nucleic acid extraction-anchor primer amplification" constituted a strict nucleic acid library construction process,combined with NGS and bioinformatics quality control.A total of 136 GB of data and nearly 86,114,574 effective reads were obtained from 28 nucleic acid libraries.After excluding virus-unrelated sequences such as rodent,bacteria,and fungi,a total of 7,553,938 viral reads were obtained.After concatenating these reads,33,062 contigs were obtained and were spliced into the NR library.The results showed that the reads were related to 67 viral families.After further excluding non-mammalian viruses,a total of 28,999 contigs were associated with 22 mammalian viral families(87.72% of the total viral contigs).The six mammalian viral families,Coronaviridae,Parvoviridae,Flaviviridae,Astroviridae,and Papillomaviridae,which are related to human and domesticated animal viruses and present high abundance,have 3,353 contigs.The nested primers were designed according to the contigs clues to verify the six viral families.The poly A tail should be added to RNA viruses that require amplification of the complete viral genome but do not have the poly A tail.The 5’RACE method was used to amplify the 5’ end of the virus,and the 3’RACE method was used to amplify the 3’end.At present,the complete genome amplification of 8 virus strains from 6 viral families and the genome sequence amplification of 11 viral strains have been completed,among which 7 strains are suggested to be novel viruses.The results showed that the rodent arenavirus was distributed in the Rattus tanezumi from Sanya;the coronavirus was distributed in the Rattus tanezumi from Haikou and Lingao;and the Leopoldamys edwardsi from Baisha;the flavivirus was distributed in the Leopoldamys edwardsi from Baisha and the Rattus tanezumi from Chengmai;and the astrovirus was distributed in the Rattus tanezumi from Chengmai and Lingao.Papillomavirus was distributed in Rattus tanezumi from Haikou,Rattus norvegicus from Tunchang,and Niviventer fulvescens from Baisha,and parvovirus was distributed in Rattus tanezumi from Haikou and Chengmai,and in Rattus andamanensis and Leopoldamys edwardsi from Baisha.We found that the novel arenavirus of rodents in Lingshui shared more than 92.38% identity with the WENV subtype that caused human fever of unknown origin in Southeast Asia,which has the possibility of potential transmission to humans.The identified rodent coronaviruses showed 90.14%–93.91% identities with human coronavirus HKU1 and human coronavirus OC43 isolated from pneumonia patients.Rodent viruses from rural and urban areas showed high identities with known rodent viruses at home and abroad,and were consistent in evolution.However,novel pestiviruses,bocaviruses,and papillomaviruses originating from Huangjingjiaoling,Bangxi,and Yingge of the National Nature Reserve had low identities with known viruses,but Leopoldamys edwardsi and Rattus andamanensis have never been reported as reservoirs of these viruses.Conclusions:(1)Rodent swab samples collected from 16 sampling sites in different regions and ecological environments in Hainan had a high abundance of viruses.The Rattus tanezumi and Rattus norvegicus in Haikou,Sanya,Dongfang,Chengmai,and Lingshui carried high abundances of mammalian viruses,such as coronaviruses,picornaviruses,herpesviruses,astroviruses,and papillomaviruses.There are many unclassified RNA viruses in several sampling sites,such as Dongfang,Haikou,and Sanya.There were pestiviruses,papillomaviruses,and bocaviruses with low identities in rodents from Huangjingjiaoling,Bangxi,and Yinggeling nature reserves,indicating that there were still rich viruses that evolved independently in their unique wild animal hosts in inaccessible areas and waiting to be found.(2)Rattus tanezumi,Rattus losea,and Rattus norvegicus are widely distributed in many areas of Hainan,and the novel virus of pestivirus,coronavirus,and parvovirus carried by rodents have host specificity.Rattus tanezumi and Rattus norvegicus showed a high proportion of viruses in the total data and virus abundance,and the representative rodents in tropical regions,Leopoldamys edwardsi and Rattus andamanensis,carried a high proportion of novel viruses.It was the first time to found that Leopoldamys edwardsi and Rattus tanezumi carried viruses from the genus Pestivirus in the Flaviviridae.It was the first time to found that Rattus andamanensis carried viruses from the genus Bocavirus in the Parvoviridae.Metagenomics,virome research,and molecular biology methods were used to investigate the virus-carrying status of rodents in different ecological environments in Hainan.This study expanded the host species and geographical range of viruses carried by rodents,and the discovery of novel species or genera of viruses expanded the viral classification and greatly increased our understanding of the virus carried by rodents in Hainan Island. |
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