Molecular Epidemiological Study Of Some Mosquito-borne Arbovirus In Longgang District, Shenzhen City

ObjectiveTo explore the infection of arboviruses in mosquitoes and investigate the molecularepidemiological characteristics of epidemic strains in Longgang district of Shenzhen cityfor providing the evidence of arbovirus diseases control and prevention.MethodsMosquito samples were collected in the monitoring site belong to10towns ofLonggang District and21Universiade venues by the program of the vector biologicalmonitoring scheme during2009and2011. Arbovirus include Japanese encephalitis virus(Japanese encephalitis virus，JEV)，dengue viruses (Dengue virus，DV) and Nam Dinh virus(Nam Dinh virus，NDiV) were detected by using the method of SYBR GreenⅡreal-timefluorescent RT-PCR，and the arbovirus infection rate were calculated；The C6/36andBHK-21cells were used to isolate viruses.TaqMan-MGB real-time PCR and Geneamplification technology were used for virus identification.The specific gene segment wasamplificated and sequenced.The homology of nucleotide or amino acid sequence andphylogenetic analysis were carried out by using ClustalX、Mega4、DNASTAR softwate.ResultsTotal25，556mosquitoes were collected and sorted into220pools.These samples werebelong to3genera and total7species，including Genus Anophelinae Meigen， Anophelessinensis Wiedemann，Anopheles minimus Theobald，Anopheles anthropophagus Xu etFeng， Genus Culex Linnnaeus， Culex tritaeniorhynchus Giles， Culex pipiensquinquefasciatus Say，Genus Aedes Meigen，Aedes togoi，Aedes albopictus. Culex pipiens quinquefasciatus Say was the dominate species in Longgang District and its constituentratio was23962（93.8%）.The constituent ratio of Aedes togoi、Aedes albopictus、Culextritaeniorhynchus Giles and the remain were637（2.5%），408（1.6%），385（1.5%），102（0.6%）.The SYBR GreenⅡ real-time RT-PCR assay result showed that totally2Culex pipiensquinquefasciatus Say and1Culex tritaeniorhynchus Giles samples were detected positivefor JEV RNA，the mosquito infection rates were1.4%（3/220）.1Aedes togoi and1Aedesalbopictus were detected positive for DV RNA respectively and the mosquito infectionrates were0.9%（2/220）.4Culex pipiens quinquefasciatus Say were detected positive forNDiV RNA and the mosquito infection rates were1.8%（4/220）.The cytopathic effect （CPE）were caused by4positive NDiV sample in C6/36cells，but not in BHK-21.The positive isolates were not obtained by inoculated the positive JEVand DV RNA samples in either C6/36cells or BHK-21.The NDiV proliferation changing with time were detected by TaqMan-MGB real-timeRT-PCR.The test results showed that Ct value of isolates gradient reduce as the time goeson.These indicated the viral nucleic acid was increased with increasing of the incubationtime.Four target gene of NDiV strains in Shenzhen according to the RdRp gene and partialfragment of ZmHel1gene were observed using agarose gel electrophoresis.Four strains ofNDiV of nucleotide sequences showed a high homology which between99.8~100.0%，andthe homology of its amino acid sequences showed between99.7~100.0%.The homology ofamino acid sequences and nucleotide sequences between four strains of NDiV of Shenzhenand the02VN178strains of Vietnam reached more than99.0%.The strains of Shenzhenwere showed a close phylgenetic relationship with NDiV strains isolated from mosquitoesin Vietnam，and They belong to the same evolutionary branch.ConclusionsThis is the first time systematic and comprehensive investigation of mosquito inLonggang District，Shenzhen city. Culex were the dominate species in local area.Thenucleic acid of DV and JEV detected in mosquitoes were suggesting that the viruscirculated in natural environment in low level.It can be the tendency that the local areabecome the natural epidemic foci of Japanese encephalitis and dengue fever. After the first time discovery of insect nidovirus-NDiV in Vietnam，the re-discovery of these virus in ourstudy which is the first time in China found NDiV. Phylogenetic analysis showed that therewas a link in genetics between the NDiV strains of Shenzhen and Vietnam.