Molecular Biology Characteristics Of Newly Discovered Mosquito-Borne Viruses And Investigation Of Pathogens On The Wild Rodents In Yunnan Province

Arbovirus is a kind of virus that can be transmitted among humans and animals when blood-sucking arthropods bite sensitive vertebrate hosts,and can cause human and animal diseases.Life history of arboviruses is complex,and most of them have host animals and vector insects that can carry them.Arboviruses can stably circulate in the natural environment.Yunnan Province is located in the southwestern border of China.Species diversity of mosquitoes and small mammals here is owing to the special geographical environment and climatic conditions.Appropriate environmental conditions is conducive to the reproduction and spread of arbovirus.We obtained a total of 82 virus isolates from mosquitoes collected from Yunnan Province during 2007 and 2010.When RT-PCR was used,70 isolates of 8 species belonging to 6 virus families were identified,and there were 12 isolates unknown.On the other hand,wild rodents,as animal host of many pathogens,including mosquito-borne viruses,play an important role in the transmission of diseases.Therefore,the purpose of this study was to identify the unknown isolates and clarify their taxonomy and molecular biological characteristics.We also conducted survey on the wild small mammals and rodent borne pathogens loaded on them in Yunnan Province to understand the distribution of natural focus diseases and the potential epidemic risk.High-throughput sequencing method was used to identify the 12 unknown virusisolates.The taxonomy of 10 isolates were confirmed,including 1 orbivirus-like virus temporarily named as Banna orbivirus(BAOV),6 Aedes-related flavivirus named as Yunnan Aedes-related flavivirus(YNAeFV)and 3 Culex-related flavivirus named as Yunnan Culex-related flavivirus(YNCxFV);Since a variety of arboviruses can coexist in one virus isolate,based on the identification of 10 virus isolates,3 rhabdovirus-like virus were identified simultaneously and named as Menghai rhabdovirus(MRV).Among which,MRV and YNAeFV were simultaneously detected in 2 isolates,and MRV and YNCxFV were detected in one isolate.BAOV was derived from Culex.tritaeniorhynchus in Menghai County.Its genome consisted of 10 segments(S1-S10).S1,S7 and S10 had the highest identities with Tibet orbivirus-XZ0906,while S2,S4,S6 and S8 had the highest identities with Fengkai orbivirus.In S3,BAOV shared the same identity of 80% with Tibet orbivirus-XZ0906 and Fengkai orbivirus.It is considered that the BAOV is a novel orbivirus formed by gene recombination of these 2 orbiviruses.The three MRV isolates were isolated from Aedes.albopictus and Culex.tritaeniorhynchus in Menghai County.Blast analysis showed that its genome had a coverage of 14% and the identity of 66% with Arboretum virus.Phylogenetic analysis showed MRV was a new species of Rhabdoviridae family.The 6 newly identified flaviviruses were all derived from Aedes.albopictus.Complete nucleotide sequence analysis showed that they shared 71%-99% identities and belonged to different species of Aedes-related flavivirus,temporarily named as Xishuangbanna flavivirus(XFV)and Menghai flavivirus(MFV),respectively.Both of the two flaviviruses showed highest identities with Parramatta River virus,but only 65%-68%,which suggested that these two flaviviruses are new species of the genus Flavivirus.A total of 759 wild small mammals were captured using the method of trap-night and catching in Gongshan area of Yunnan Province,including 35 species,belonging to14 genera.Apodemus(29.1%)and Eothenomy(17.4%)were the dominant genera in this region.Nested PCR was used to detect viral and bacterial pathogens from 541 wild small mammals.Twelve were detected positive as Candidatus Neoehrlichia mikurensis from Apodemus,Eothenomys,Sorex and Niviventer;two as Wolbachia from Sorex and Niviventer eha;three as Bartonella from Eothenomys and Rattus nitidus.Besides,seven hantaviruses and 4 Orientia tsutsugamushi were detected.Of four positive samples identified for O.tsutsugamushi,two were found to have the high nucleotide identities with Boryong isolate 2014-H30(97.0%);one had the highest identity of86.0% with Karp sero-type(isolate 2014-JN08);and the other one had the highest identity of 86.0% with Kato-related strain.Our findings indicated that these two samples may be the variants of Karp and Kato-related sero-type.In the present study,several new mosquito-borne viruses were identified.Our findings enriched the types of mosquito-borne viruses.The newly discovered mosquito-borne virus can provide new evidence for etiological diagnosis of patients with unknown fever.Further research on the pathogenicity of newly discovered viruses to humans and animals is necessary.The present investigation further confirmed the species diversity of wild small mammals and rodent borne pathogens loaded on them,which was of great guiding significance for the prevention and control of related natural focus diseases in Yunnan Province.