Identification Of New Mosquito-borne Viruses And Their Molecular Characteristics And Serological Survey For The Virus Infections In Yunnan Province

Arthropod-borne viruses(Arboviruses)were kinds of viruses which can be transmitted among humans and animals when blood-sucking arthropods bite sensitive vertebrate hosts,and can cause human and animal diseases.To date,there were more than 550 arboviruses identified worldwide,of which more than 130 viruses,including over 100 mosquito-borne viruses have been confirmed to infect humans and/or animals.Because of the species variety and wide distribution of mosquitoes,Yunnan province was an important natural focus of infectious disease caused by mosquito-borne viruses.After over ten years of unremitting efforts,we had obtained more than 20 new mosquito-borne viruses involving six virus families in Yunnan Province,and several viruses have potential pathogenicity to human and animal.In this study,high-throughput sequencing technology was used to identify the unknown isolates obtained in Yunnan Province from 2010 to 2016.The molecular characteristics of newly discovered viruses were analyzed to clarify their taxonomy.Moreover,a serological survey was conducted to understand the potential pathogenicity of newly obtained viruses.A total of 25 viruses involving 8 genera of 7 virus families were identified from 112 unknown isolates through high-throughput sequencing.12 novel or variant viruses were discovered,including one novel orbivirus,one novel negevirus,one variant Banna virus,two variant totiviruses,one variant Omono river virus,three new rhabdovirus and three new unclassified RNA viruses.Three viruses were first identified from mosquitoes collected in Yunnan Province,including Bluetongue virus(BTV),Ngewotan virus(NWTV),and Wenzhou sobemo-like virus 4(Wz SV4).The most virus species(13 viruses)were found in Culex tritaeniorhynchus mosquitoes.The nucleotide sequence of VP2 of the newly isolated orbivirus,tentatively named Lianghe orbivirus(LHOV),had the highest identity(73%)to CHe RI orbivirus isolated from American,then 68% and 69% identity to the Yunnan orbivirus and Peruvian horse sickness virus,respectively.The amino acid sequence of VP2 of the LHOV has 75% identity to the CHe RI orbivirus,so it was identified as a new species of genus orbivirus.The whole genome of the newly isolated negevirus,tentatively named Manglie virus(Ma V),had the highest similarity to Ngewotan virus,with 79% identity of nucleotide sequence,and 89% identity of amino acid sequence.The result of phylogenetic analysis showed that it was genetically related to the viruses of genus Negevirus,suggesting it was a new member of this virus genus.For the first time,two Bluetongue viruses(BTV)were isolated from mosquito samples,and phylogenetic analysis based on VP2 indicated that they were belonged to serotype 2 of BTV.We obtained the full genome sequence of segment 1 of the new Banna virus(BAV).It had 92% nucleotide sequence identity to the previously reported BAV in China,and 83% identity to the virus isolated in Vietnam,suggesting it was variant Banna virus.Three newly identified totiviruses,tentatively named Omono river virus-YJ(ORV-YJ),Yuanmou totivirus(YMTV),Yongshan totivirus(YSTV),had similar genome structure.Their whole genome nucleotide sequence similarities were during 76%-92%,and the similarity with other totiviruses were from 65% to 93%,suggesting they were variant strains of totivirus.To understand the potential pathogenicity of the newly isolated viruses,we collected 1622 human sera in four cities(counties)of Yunnan Province,and detected the Ig G antibodies anti-Banna virus,Nam Dinh virus,and Tibet orbivirus by indirect immunofluorescence assay(IFA).The results showed that the positive rates of Banna virus and Nam Dinh virus antibodies were 3.8%(61/1622)and 3.0%(49/1622),respectively.The antibody titers of the both viruses in the positive sera were 1:80-1:640.No Ig G antibody to Tibet orbivirus was detected.In conclusions,our findings enriched the species of mosquito-borne viruses,The clues of the infection of Banna virus and Nam Dinh virus were found in human population though the serological survey,suggesting the potential pathogenicity of the two viruses.