Investigation Of The Emerging Infectious Virus In Important Animal Reservoir In Hainan

Research purpose:In order to prevent and control the outbreak of emerging and re-emerging infectious diseases in Hainan Island,we provide scientific data and technical support by investigating and monitoring the virome of important natural animal reservoir,including bats,rodents,and avian.Research methods: According to the characteristics of the ecological environment and distribution of important natural animal reservoir in different regions of Hainan Island,we choose the sample collection site with representative ecological environment.High-throughput viral sequencing and bioinformatics analysis were performed to obtain a preliminary view of virome present in important natural animal reservoir and analyze the diversity and difference of virome between different sample.Research results: In the study,740 animal samples were collected from five counties and cities in Hainan Island during the period from 2015 to 2016.The information of species and collection sites and groupings were as follows: 652 bats(210 in the Haikou crater HHB group and 442 in the Tunchang TC group)and 85 rodents(18 in the HKS group of Haikou City,24 in CMS group of Chengmai County,24 in the LGS group of Lingao County,19 in the LMS group of Limu mountain),and 3 samples of poultry droppings from Haikou live poultry market(chicken CH group,duck DU group,and pigeon PI group).Viral metagenomics analyses showed that a total of 34396 and 241206 virus sequencing reads were generated from the HHB group and the TCB group,respectively.After sequence alignment and bioinformatics analysis,the virus community composition of the two groups of bats is basically similar,the virome were related to five virus families: Densovirinae,Herpesviridae,Circoviridae,Baculovirida,and Myoviridae.Mammalian viruses of both two groups are annotated into Gallid herpesvirus,Bat circovirus,and Falconid herpesvirus.In addition,225 reads of the HHB group were annotated to the human herpesvirus,and 880 reads of the TCB group were annotated to the bat circovirus with much higher abundance than in the HHB group.The rest of insect virus reads are annotated to Densovirus and Granulovirus.Viral metagenomics analyses of rodent showed that 7515,5221,and 3,503 virus sequencing reads were generated CMS(Chengmai),LGS(Lingao)and LMS(Limushan),respectively.The most abundant viral family were annotated to mammalian viruses(CMS: 89.15%,6700/7515;LGS: 100%,5221/5221;LMS: 98.40%,3447/3503),and the majority of mammalian viral sequences was parvovirus(CMS: 88.01%,6614/7515 LGS: 97.43%,5087/5221;LMS: 90.43%,3168/3503).However,bovine hokovirus and rat bocavirus were the main constitution among the CMS group,mouse parvovirus and rat minute virus were the main constitution the LGS group,and mouse bocavirus and porcine bocavirus were the main constitution among the LMS group.Moreover,human adenovirus(Human adenovirus)was also present in the CMS group and LMS group,and a arenavirus was found in the HKS group.Viral metagenomics analyses of the poultry faeces showed that 13,063,1,370 and 862 virus reads were generated from group A(chicken),group B(duck)and group C(pigeon)received,respectively.Most of the viral sequences of poultry faeces were annotated to mammalian viruses(A: 99.3%,12975/13063;B: 95.3%,1306/1370;C: 81.1%,706/862),including: Adenoviridae,Herpesviridae,Retroviridae,Reoviridae,Circoviridae,Astroviridae,Caliciviridae,Coronaviridae,Paramyxovirus,Picornaviridae and Picobiraviridae.Among them,the virus composition of group A and group B was similar,and most of the viral reads were annotated to Avian coronavirus.However,circovirus comprised the main constitution of group C.In addition,we found two sequences of influenza virus A in the C group,but the chick embryo isolation culture and PCR assays results were negative.We determine the presence of rotavirus A pigeon faeces by performing a PCR and genomics analysis.Based on metagenomics analysis data,we discovered a HMU virus(Hainan Medical University virus)from a Rattus norvegicus from Haikou,belonged to Wenzhou Virus of Arenaviridea.We successfully obtained the nearly full genome sequence of HMU virus.The homologous and phylogenetic analysis showed that HMU virus was homologous to Wenzhou Virus(WENV)isolated from Rattus norvegicus in Zhejiang(92%)and Cardomon virus isolated from human in Cambodia(82%).The complete genome sequence of HMU virus has been submitted to Gen Bank with accession numbers MF595889.1 and MF595888.1.In addition,we found a Rotavirus A(RVA)in pigeon droppings from the live poultry market in Haikou.The sequences of VP4,VP6,and VP7 gene were obtained and determined as RVA/Pigeon-Haikou/CHN/ 2016/G18P[17] based on the Virus Taxonomy of International Society for the Classification(ICTV).This RVA Haikou strain was homologous to RVA/Fox-tc/ITA/288356/2011/G18 P [17])in foxes from Italian(92%)and PO-13 strains with the infectious ability to mammals(85%).The RVA VP7 and VP4 genome sequences have been submitted to Gen Bank with accession numbers MG518479 and MG518480.Based on the bioinformatics analysis,the two viruses have a high potential ability to infect human.To further investigation of the prevalence and effective prevention and control of the two viruses,the detection methods of Real-time PCR assay were established with good specificity and sensitivity,the lowest detection limit was 1.0×102 copies/?l.Conclusion: Bats,rodents and poultry are the natural hosts of many viruses and have close contact conditions with humans.Therefore,the investigation of the virome of these important natural animal reservoir in Hainan Island has important public health significance for the prevention and control of emerging and re-emerging infectious diseases.In this study,high-throughput sequencing and bioinformatics analysis were performed to obtain the virome metagenomics information of bats,rodents,and poultry in Hainan Island(including Haikou,Chengmai,Tunchang,Lingao,and Limushan).The bats in Haikou and Tunchang are carrying the human herpesvirus and bat circovirus,the results suggesting that the people live near these two sites has the potential to infect and transmit herpesvirus and circoviruses.In addition,an abundantinsect viruses are present among bats from two sites,which certainly reflect the ecological environment of the two places.Parvovirus presents a very high abundance in rodents from Tunchang,Lingao and Limushan areas.The rodents carrying parvoviruses may spread the virus to humans by polluting the water sources,foods and so on.Therefore,we should closely monitor and prevent the occurrence of intestinal diseases in the above regions,especially residential areas.We also identified a HMU virus of arenavirus,which provided evidence for the widespread epidemic of arenavirus in Asia and provided scientific warnings for the prevention and control of arenavirus in Haikou for the future.With the rapid development of Hainan Island's public service of double-creation,the safety of live poultry food become a hot spot of concern.In this study,the mammalian viruses were very abundance in the poultry faeces in Haikou live poultry market,suggesting that more attention should be paid to health and safety issues in poultry breeding and circulation.However,no influenza virus A has been found in this study.In addition,the RVA/Pigeon-Haikou/CHN/2016/G18 P [17] have high homologous with the virus can cause neurological diseases and infect the mammal through the species barrier.Whether the RVA we founded has the ability to infect the central nervous system,or has the ability to cross the species barrier to infect mammals remains to be further explored.In this epidemiological investigation,we have obtained abundant basic background data and conducted in-depth genomics analysis of the virus carried by important natural animal reservoir in Hainan Island.Efficient detection assay for two important new-emerging viruses were established.It provided scientific data support for the prevention and control of the new outbreak and epidemic of new infectious diseases in Hainan Island,and provided scientific direction for the identification of other important virus and the investigation of emerging and re-emerging infectious diseases research in the future.