Arbovirus Isolation And Identification Of Mosquitoes And Midges Collected In Guizhou And Sichuan Province In China

Objective To understand the types and densities of mosquitoes and midges in summer in 5counties in Guizhou and Sichuan provinces find the insect-borne viruses carried by mosquitoes and aphids in the main blood-sucking insects in the area.The results of this study provide basic data for the prevention and control of local humans and livestock diseases transmitted by mosquitoes and midges.Methods Blood-sucking insects were collected from houses and feeding pens,such as pigsties,and cowsheds,in Sichuan,Guizhou Province in 2016 and 2017.The mosquito samples were classified according to their morphology under ice bath conditions(50 insects per pool).midges were not classified according to their morphology(about 400 insects per pool).The samples were stored in liquid nitrogen and transferred to the laboratory.BHK-21cells(Mesocricetus auratus kidney cells)and C6/36 cells(Aedes albopictus cells)used in this study were isolated the viruses.The cells were cultured in incubators for three generations.The CPE of each well was observed each 12 h.The virus suspension was harvested when CPE occurred and stored at–80°C until further identification.Molecular biology methods was used to identification of viruses.The analyses of the nucleotide and amino acid sequences were analyzed using software.Results1.Isolation and identification of arbovirus in Guizhou province A total of 16175 mosquitoes belonging to five species of three genera and 1300 midgeswere collected from Dejiang,Jiangkou in Guizhou Province in 2016 and 2017.The mosquitoes in consist of Anopheles sinensis,Culex quinquefasciatus,Culex tritaeniorhynchus,Armigeres subalbatus and Culex fuscanus.43.7%(7072/16175)were Armigeres subalbatus and 34.1%(5518/16175)were Anopheles sinensis that were the dominant mosquito.The samples of mosquitoes and midges were divided into 270 pools before processing.The BHK-21 and C6/36 cells were incubated with the sample supernatants and observed for CPE.eleven virus isolates were obtained.PCR products indicated that two ZIKV strains,four JEVs,two banna viruses(BAVs),two GETV strainsand one Densovirus(DNV).No isolates from the sample of midges.No viral positive results were obtained from the genomic amplification of 270 pools using 12 virus-specific and species-specific viral gene amplification primers.2.Isolation and identification of arbovirus in Sichuan province17019 mosquitoes belonging to three species of three genera and 12700 midgeswere collected from Hejiang,Nanxi and Gulin,Sichuan Province in 2016.Culex tritaeniorhynchus is the dominant species.The percentage is79.4%(13519/17019).The samples were divided into 275 pools before processing.The BHK-21 and C6/36 cells were incubated with the sample supernatants and observed daily for CPE.Three positive(JEVs)isolated were obtained in Hejiang in 2016.four positive results were obtained from the genomic amplification of 270 pools using 12 virus-specific and species-specific viral gene amplification primers.The results showed that the 3virus were positive for PCR amplification using the Akabane virus(AKV)primers.However,Akabane virus not isolated from midges.Conclusion This is the study for arbovirus investigating in Guizhou and Sichuan Province.A number of mosquitoes belonging to five species of three genera and many midgeswere collected from 5 county.Anopheles sinensis were the dominant mosquito in Guizhou,while Culex tritaeniorhynchus were the dominant mosquito in Sichuan province.14 strain virus were isolated from 5 species mosquito samples(two ZIKV,eight JEV,two BAV,one DNA,two GETV).The ZIKVs isolated from mosquitoes represent the first ZIKV isolates in mainland China.Multiple strains of JEVs were isolated from mosquitoes in the above two places,suggesting that there is still a natural circulation of JEV.This research result provides etiological basis for explaining the persistence of JEV for many years.In addition,AKV gene amplification was detected in a large number of midges collected in Sichuan Province.This is the first time that AKV was found in blood-sucking insect specimens collected in the natural world of Sichuan Province.It provide the new challenges in the prevention and control of animal diseases.