Establishment Of The Methods Of Flavivirus Universal Primers-based RT-PCR, Real-Time PCR And Application In The Detecting Of Field-collected Mosquitoes

The Genus Flavivirus belongs to the Family Flaviviridae,comprises more than 70 viruses. Most of them are arbovirus transmitted by either mosquito or tick vectors, include Japanese encephalitis virus(JEV), Dengue virus(DEN), yellow fever virus(YFV), West Nile virus(WNV), St.Louis encephalitis virus(SLE), etc.. JEV prevalenced in most areas in Asia, and Dengue fever/dengue hemorrhagic fever (DF/DHF) are also in an upward epidemic trend obviously in America, Asian and Pacific regions. St.Louis encephalitis is endemic mainly in South and North America areas, and West Nile virus is found to be widely distributed throughout Algeria, Russia, Italy, America, etc. In September 2000, Rift Valley fever was found for the first time outside Africa in the Kingdom of Saudi Arabia and Yemen since the disease was identified in 1930. Half of the viruses in flavivirus can lead to high rate of mortality and lower rate of curing for both mankind and animals, leaving some serious sequelaes among those survivors and pose a large threat to the human's health.The Flavivirus are transmitted by mosquito and tick vector, most of these diseases are hard to be effective treated, prevented and controlled. The transmission of the viruses usually lead to serious public health problems and large challenges to the prevention and control of these diseases. The expansion of epidemic areas of the flavivirus represented by JEV, DEN and WNV had aroused extensive concerns and worries of all over the world. In addition, among the vectors of WNV reported by overseas, some species are found in our country. For example, Culex bitaeniorhynchus, Cx. gelidus, species of Cx. pipiens complex, Cx. tarsalis, Aedes albopictus, Ae. Aegypti, Ae. vexans and Ochlerotatus aponicus are effective and medium effective vectors. To meet the requirement of strengthen the investigation and epidemiology survey of flavivirus, and to develop the early warning system of mosquito-borne diseases, it is necessitated for the establishment of a rapid, convenient and precise methods for the detection of flavivirus in mosquitoes.Take WNV to illustrate, ,flavivirus is a positive strand RNA virus and the genome size is about 11 kb, which serves as the only viral mRNA. The length of 5′-termini of each of the different flavivirus genome RNAs were similar, and the structure, in each case consisting of a stem with a small top loop and a larger side loop(m7GpppAmp). The members of the Flaviviridae family are the only known animal positive-strand RNA viruses that do not have a poly(A) tract at the 3′end of their genomes, end with CU-OH. The single open reading frame encoded by the viral RNA produces a polyprotein that is cleaved by viral and cellular proteases into three structure and seven nonstructure proteins. Within the virus genenome, these protein-encoding sequence are as follows: C --PrM --M --E --NS1 --NS2A --NS2B --NS3 --NS4A --NS4B --NS5. Among them, NS5 is one of the conserve structural domain of all flavivirus genome. We developed a routine PT-PCR method with genus-universal primers within NS5 region, further species identification was conducted by using species specific primers.Suckling mice was infected with JEV SA14-14-2 strain( attenuated vaccine), mice brain were dissectted .and homogenated to make virus suspension, which was served as positive control. JEV cDNA and DEN cDNA were RT-PCR amplified with the universal primers. The amplified fragment coincidence with the target DNA fragment. The sensitivity of RT-PCR methods was 0.568 PFU/ml. Another SYBR GreenⅠdye Real-Time PCR method using the same primer was established simultaneously to detect Flavivirus or JEV complex viruses infection in field-collected mosquitoes.West Nile virus has been epidemic in many countries all over the world (including those countries neighboring China). It's dissemination was mainly through the migration of fling birds. The health authorities are concerned about the fact that the virus could be imported by the fling birds or through other routes. The Department of Health of our country had listd it as the potential emerging infectious agent. Considerating this, we set up a detection method for identification WNV. According to the reported protein E sequence of WNV, we synthetized protein E gene fragment, connected with pMD18-T and pMD19-T vector, transform E coli, then identify positive plasmid. These plasmid was amplified and used as positive control in TaqMan probe PCR method. To test the sensitivity of Real-Time RT-PCR method, Stock solution of PCR product was diluted at geometric series, the maximus dilution which had positive amplification was 1:107 for routine RT-PCR, and 1:108 for Real-Time PCR, respectively. OD value shown that the lowest consentration wich RT-PCR could detect was 0.01 pg, while Real-Time PCR could detect 1 fg of plasmid containning WNV E fragment. Sensitivity for Real-Time PCR method was 10 fold higher than that of the conventional RT-PCR. Also, Real-Time RT-PCR could detect virus in mosquitos in a semi-quantitative mode.Based on the above methods, we detected field-collected mosquites which were trapped from 5 province (Jiangsu, Fujian,Yunnan, Shanghai, Xinjiang). Chosing the trapping place was taken into account of the route of bird migration and wetland natural prodection area, Wild mice also trapped in Xinjiang Autonomous Region. All samples were identified and allocated into pooled groups, and detected by RT-PCR. Each group of mosquitoes contained 30/pool or less for engorged mosquitoes and 50-60/pool for non-engorged mosquitoes, respectively. Tissues (brain,liver, spleen) from mice were cut at size of 0.5 cm3/pool. Altogether 261 pools of 12539 mosquitoes and 8 pools of mouse tissues were detected with the Flavivirus-universal primer-based RT-PCR. There was a positive amplicaiton in 1 mosquito pool which were captured from the wetland natural protection area of Jiangsu province. Further amplification using species-specific primers, the positive result for JEV was found. Further sequencing the amplified product showed the sequence was similar to the JEV sequence deposited in GenBank(Accessing No.NC 001437). All the 269 pools were deteced further by SYBR GreenⅠdye Real-Time PCR.There were 22 mosquito pools showing positive amplification curve. These samples were re-identified by WNV-specific TaqMan probe Real-Time PCR, no amplification curve appeared.Monitoring flavivirus infection in mosquito is an earlier detection and surveillance approach in forecast the epidemiology of arbovirus infections. The methods we established were targeted directly to flavivirus, especially Japanese encephalitis virus complex in mosquitoes. The methods are simple, effective, and rapid for detection flavivirus in mosquitoes, and are superior over virus culture method in its high-throughput detection. From this study, we found JEV in mosquitoes in wetland natural protection area of Jiangsu province. Health workers in each level of CDC should pay more attention to this result and proceed further surveillance to this region and other similar regions. The results also provide experimental documents for arbovirus surveillance, for dignosis and precaution of newly vector-borne disease agents which might have potential to be imported to our country.