| BackgroundArboviruses are a class of viruses transmitted by blood sucking insects including mosquitoes,ticks,sandflies,midges and so on,which can cause human and animal diseases by bitting,and also can result in natural focus-based diseases and zoonosis.The arboviruses are RNA viruses generally,whose structure is simple and outer layer has an envelope structure.Arboviruses enter the body of the blood-sucking insect and reproduct in the body,but do not cause disease.Then arboviruses transfer to the host by insect bite,and reproduct in the body,and further cause the relevant clinical symptoms.In the 1950 s,only 35 species were found.In 1974,359 species were registered at the International Arbovirus Center.As of 1989,532 species were developed,of which 125 species were pathogenic to humans and animals,and certain viruses can cause serious diseases such as encephalitis,blindness,etc.Arboviruses are distributed globally,mainly in the mid-temperate,subtropical and tropical zone.The research on arbovirus in China started late.The viruses that have been isolated include Japanese encephalitis virus(JEV),tick-borne encephalitis virus(TBEV),Xinjiang hemorrhagic fever(XHF),Dengue virus(DV),West Nile virus(WNV),Getah virus(GETV),Banna virus(BAV),etc.There are a variety of arboviruses in China,but China has a vast territory,complex environment and numerous species,which poses challenges for further investigation and research of arboviruses.There are many kinds of mosquitoes and mites with wide distribution in China,which are very important groups.Since the 1950 s,research on midges has been carried out in China until now.With the further research,the discovery of new species of midges has been reported frequently.It is a blood-sucking midges that are closely related to humans and animals,and some midges are important media for zoonotic diseases.On the one hand,they can cause animal diseases,which bring serious economic losses to the livestock and poultry industry,and on the other hand,they cause a huge threat to human health.Studies show that most mosquito-borne infectious diseases are transmitted by mosquitoes in three genera: Aedes,Anopheles,and Culex.Species in the genus Aedes,particularly Ae.Albopictus(Asian tiger mosquito)and Ae.aegypti are vectors for anumber of zoonotic diseases,including dengue virus,chikungunya virus,Zika virus,and yellow fever.Anopheles mosquitoes are responsible for transmitting malaria and a few other pathogens,such as canine heartworm(Dirofilaria immitis)and species that cause filariasis(e.g.,Wuchereria bancrofti).Culex mosquitoes,predominantly C.pipiens,C.tarsalis,and C.quinquefasciatus,carry and transmit West Nile virus and Saint Louis encephalitis virus.Mosquitoes in all three genera have experienced range expansions.Aedes aegypti is the most widespread,with an almost global distribution through repeated invasions.As mosquito species expand their geographic distributions,the diseases they harbor are expanding and are predicted to continue to expand,representing new challenges for places previously not affected.Hainan Province(east longitude: 108°37'-117°50';northern latitude: 3°58'-20°20')faces the Leizhou Peninsula in Guangdong Province in the north seperated by Qiongzhou Strait;is adjacent to Beibu Gulf in the west;reaches to Zengmu Reef in the south,is adjacent to Malaysia;is on the verge of the Pacific Ocean in the southeast,facing the Philippines,Indonesia,etc..It belongs to the tropical monsoon climate.There are natural conditions for the propagation and spread of arboviruses.Hainan Province is an epidemic area of Japanese encephalitis and dengue fever.In 1998,there were outbreaks of Japanese encephalitis in Sanya City and Ledong County.And four JEV were isolated as early as 1985;Pandemic of Dengue fever in Hainan province occurred in 1979 and1980-1991,and a DV2 strain was isolated in 1989.In addition,various viruses such as Alphavirus,Flavivirus and so on were isolated.With the rapid development of tourism in Hainan Province,the flow of personnel has increased,which also provided conditions for the input and diversity of arboviruses.However,in the past 30 years,systematic arbovirus research has not been carried out in Hainan Province.In order to understand the species and distribution of mosquitoes in Hainan Province and to understand the types and epidemic characteristics of the virus in the local blood-sucking insects,this study conducted systematic collection of mosquitoes and midges and its arboviruses in six cities and counties in Hainan Province for two years from 2017 to 2018.It is of great significance to prevent and control local arbovirus disease,to formulate corresponding prevention and control measures in the future and to avoid the spread of epidemic.ObjectiveA systematic survey was conducted on the types and densities of mosquitoes and midges in summer in six cities and counties in Hainan Province to understand thearboviruses carried by mosquitoes and midges in the local blood-sucking insects.The basic data will be provided to prevent and control local arboviruses and related human and animal diseases.MethodsBlood-sucking insects were collected from houses and feeding pens(such as cowsheds,sheepfold,pigsties,and so on)in the villages of 6 cities and counties in Hainan Province in august in 2017 and 2018.The samples were classified and identified according to their morphology under ice bath conditions.The basic information were registered according to the collection information,collection date,breeding environment,mosquito species,quantity and other basic information.Then these samples were immediately placed in liquid nitrogen and transferred to the laboratory for isolation and identification.The collected specimens were ground,centrifuged and dispensed in the laboratory.And the supernatant was inoculated in parallel with BHK-21 cells(Mesocricetus auratus kidney cells)and C6/36 cells(Aedes albopictus cells)for virus isolation.Meanwhile,a cDNA library of mosquito specimens from Hainan Province was prepared,and specific nucleic acid screening was performed by RT-PCR.When cytopathic effect(CPE)occurred in the cell,the virus suspension was harvested,and further identification are used for further molecular biological identification.The results of nucleotide of the viral genome were analyzed by using various softwares to obtain the data homology analysis and molecular genetic analysis.ResultsA total of 15097 mosquitoes belonging to seven species of four genera and 11360 midges were collected from Danzhou City,Chengmai County,Wenchang City,Changjiang Li Autonomous County,Dongfang City and Ledong Li Autonomous County in Hainan Province in august in 2017 and 2018.The collected mosquitoes included13,990 Culex tritaeniorhynchus,accounting for 92.67% of the total number of collected mosquitoes(13990/15097).In addition,780 Armigeres,37 Aedes albopictus and 37 Anopheles were collected.It can be seen that Culex tritaeniorhynchus is the dominant mosquito species at the above collection points.From August 18 th to August 21 st,2017,19,604 identified mideges were collected in three areas of Danzhou City,Chengmai County,Wenchang City,Hainan Province,which include 22 species of 1 genera(C.arakawai?C.peregrinus?C.oxystoma and so on).Among them,the species collected from Danzhou City are mainly C.oxystoma and C.flavescens,which are 3,593 and 1,640 respectively,accounting for 40.5%(3593/8872)and 18.55%(1640/ 8872)of the total number of collected midges in Danzhou City.It can be seen that the C.oxystoma is the local dominant species.The species collected from Chengmai City are mainly C.flavescens and C.arakawai,which are 887 and 529 respectively,accounting for 33.2%(887/2673)and 19.8%(529/ 2673)of the total number of collected midges in Chengmai City.It can be seen that the C.flavescens is the local dominant species.The species collected from Wenchang City are mainly C.peregrinus and C.arakawai,which are 3102 and 2102 respectively,accounting for 38.5%(3102/8059)and 26.1%(2102/8059)of the total number of collected midges in Wenchang City.It can be seen that the C.peregrinus is the local dominant species.In general,the number of C.oxystoma and C.arakawai accounted for a relatively large proportion,accounting for 28.1%(5499/19604)and 18.3%(3589/19604)respectively of the total number of Culicoides.There were a total of 199 pools of all mosquitoes(162 pools)and not identified midges(37 pools).75?63?30?9?8?14 pools respectively in Danzhou City,Chengmai County,Wenchang City,Changjiang Li Autonomous County,Dongfang City and Ledong Li Autonomous County.After routine treatment in the laboratory,the supernatant was inoculated in BHK-21 and C6/36 cells.And 4 strains of virus isolates were obtained that could be stably passaged.Three strains of viruses were detected by molecular biology,and all of them belonged to JEV I(all isolated from Culex tritaeniorhynchus),1 GETV(isolated from Culex tritaeniorhynchus),and no virus isolates were obtained from the midges specimens.Viral gene amplification of 199 pools of specimens was carried out by using 12 virus-specific and species-specific viral gene amplification primers.The results showed that 8 pools in JEV gene amplification and 1 pool in GETV gene amplification of mosquito specimens were positive,and the others were negative;while in the midges specimens,5 pools of OYA virus gene amplification were positive,all collected in pigsties.4 of them are from Danzhou City and 1 is from Chengmai County.ConclusionIn 2017 and 2018,six cities and counties in Hainan Province in China were selected for the investigation of arboviruses in this study in the summer.A total of 15097 mosquitoes belonging to seven species of four genera and 11360 midges were collected,of which Culex tritaeniorhynchus is the dominant mosquito species at the above collection points.All mosquitoes and midges specimens were groud 199 pools.And 4strains of virus isolates that could be stably passaged were isolated,which isolated from Culex tritaeniorhynchus and coming from Danzhou City,three of which were JEV whose genotype was I.Another strain is GETV.The results suggest that there is still acirculation of Japanese encephalitis virus in nature.In addition,positive results of the AKAV gene amplification was detected in a large number of midges specimens collected in Hainan Province.This is the first time that the presence of the AKAV has been found in the blood-sucking insect samples collected in the natural world of Hainan Province,which pose new challenges to prevent and control local animal diseases. |
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