Establishment Of Rapid Detection Methods For Important Arboviruses Have Not Found In China

Arboviruses comprise a group of viruses which cause natural foci of disease and zoonosis by blood-sucking arthropads bite sensitive vertebrate. They are mainly include genus Alphavirus in Togoviridae, genus Flavivirus in Flaviridae,genus Orthobunyavirus in Bunyavirudae,and Reoviridae,such as Barmah Forest virus(BFV),Chikungunya virus(CHIKV), Sindbis virus(SINV), Eastern equine encephal omyelitis virus(EEEV), Western equine encephal omyelitis virus(WEEV), Ross river virus(RRV), Venzuelan Equine Encephal omyelitis virus(VEEV) in genus Alphavirus. Yellow fever virus(YFV), Kunjin virus(KUNV),Dengue virus(DENV), West Nile Virus(WNV)and Japanese encephalitis virus(JEV) in genus Flavivirus. La Crosse virus(LACV), Snowshoe virus, California encephalitis virus(CEV) and Tahyna virus(TAHV) in genus Orthobunyavirus. The viral transsion to human and livestock through insects causes human disease and death, it also causes disease of a large number of livestock, and even death, resulting in tremendous economic loss and had been regarded as a public health problem of every country. At present our country has not yet been isolated KUNV and BFV, but there are mosquito related media, Ecology and have WNV epidemic conditions, KUNV as a subspecies of WNV into China is very risky. Also there is no YFV current popular or confirmed reports, But due to the parts of our country’s geography, climate, media- mosquitoes, the source of infection-monkey conditions such as similar to Africa, middle and South America, And with the development of global economy, the integration of global trade and trend of urban life, Each year about more than 3 million travelers from YFV popular area, this makes the virus and media form more easily spread between different countries. Arboviruses distribute widely, but mainly distributes in temperate, subtropical and tropical zones. Most arboviruses are only distributed in a region or a continent, but can be spread to different places with crowd, migration of host and vectors. Therefore, arboviruses carried by medical vectors shoud be strengthen surveillant.At present, detection of arboviruses depend on virus isolation, ELISA and/or molecular biology techniques. But these methods owned some disadvantages such as taking long time, low sensitivity, lacking of specific antibodies, and ELISA were used for antibody detection of a fatal flaw is due to the same genus virus similar antigenicity, affect the accuracy of the results, In actual work hard to get promotion and application. At present can be used to test the commercialization of arboviruses reagent quantity is less, for has not been introduced into china arboviruses then there is no corresponding whole virus antibody to detect virus antigen. In recent years, with the popularization and application of PCR technology, real-time fluorescent PCR and conventional reverse transcription PCR amplification(RT-PCR) detection aspects of virus in mosquitoes media is widely used. Rt-PCR method is rapid, sensitive, and can be detected in 5-8 hour virus, suitable for the port. This research use double RT-PCR and real-time fluorescent rt-pcr technology, establish a rapid, sensitive, high specificity testing method of arboviruses applied in frontier port arboviruses monitoring and testing.Objective:This study intends to carry out important arboviruses have not found in China, especially the entry medical media arboviruses rapid detection technology research, to establish detect duplex RT-PCR of KUNV and BFV method and KUNV, BFV,YFV fluorescent RT-PCR detection methods,applied to the frontier port vector mosquitoes carry arboviruses detection.Methods:The complete genome sequences of 34 strains KUNV,26 strains BFV, 30 strains YFV were searched from GenBank. The conserved sequences of the viruses were aligned and blasted with DNAstar software.Choose KUNV and BFV highly conservative E gene, YFV conservative E gene, for the purpose.The conserved sequences of the viruses were aligned and blasted with primer5.0 and DNAstar software.E gene sequences of KUNV and BFV commissioned Shanghai Ying Wei Jie-based Trade Co., Ltd. synthesis, then follow the instructions in vitro transcription kit extracted RNA KUNV and BFV, and then reverse transcribed into cDNA. QIAGEN of RNA extraction kit was used to extract YFV RNA, establish KUNV and BFV duplex RT-PCR detection method, establish KUNV, BFV and YFV fluorescence RT-PCR detection methods.Results:①Set up KUNV and BFV duplex RT-PCR method, sensitivity of KUNV: 1.83 x 106 copies/μL, BFV: 2.02 x 106 copies/μL and LACV, SSH, MAYV, YFV, JEV no cross reaction②Established KUNV fluorescent RT-PCR method, has the very good specificity and sensitivity, the sensitivity of 1.83 x 102 copies/μL, and LACV, SSH, BFV, YFV, MAYV, JEV, and Banna virus(BAV) no cross reaction.③Established BFV fluorescent RT-PCR method, has the very good specificity and sensitivity, the sensitivity of 2.02 x 103 copies/μL, and LACV, SSH, KUNV, YFV, MAYV, JEV, and Banna virus(BAV) no cross reaction.④Established YFV fluorescent RT-PCR method, has the very good specificity and sensitivity, the sensitivity of 1.4 x 103 copies/μL, and LACV, SSH, KUNV, BFV, MAYV, JEV, and Banna virus(BAV) no cross reaction.Conclusion:Established KUNV and BFV doplex RT-PCR method, KUNV, BFV and YFV fluorescenceRT-PCR method, for port mosquito-borne insect-borne virus detection has extensive application value.