Study On Integrated Multiplex Reverse Transcription-PCR And Microfluidic Electrophoresis Detection System For The Detection Of Mosquito-transmitted Zoonotic Encephalomyelitis Viruses

Mosquito-transmitted encephalomyelitis viruses are pathogens with a wide range of hosts, including birds, rodents, pigs, horses and human beings. West Nile virus(WNV), Saint Louis encephalitis virus(SLEV), Venezuelan equine encephalomyelitis virus(VEEV), Western equine encephalomyelitis virus(WEEV), Eastern equine encephalomyelitis viruses(EEEV), Highlands J virus(HJV), Japanese encephalitis virus(JEV), La Crosse virus(LACV), Sindbis virus(SINV), Chikungunya fever virus(CHKV), Dengue fever virus(DENV) and Ockelbo virus are a threat to public health in certain areas. A common feature of these agents is the seasonal and regional circulation of the virus by infected mosquitoes, which may result in serious infections in susceptible animals or human beings, causing central nervous system inflammation and viraemia. The American continents are the areas that are most affected by these pathogens except of JEV, which is predominantly distributed throughout Asia. However, increased international trade and movement of passengers and cargo have led to some of these agents expanding from old enzootic sites to new epidemic regions. Although no clinical cases of the above mosquito-transmitted encephalomyelitis virus have occurred in China(except JEV), the hosts and competent vectors of EEEV, WEEV, VEEV, WNV, SLEV and HJV have been found. It is also impossible to quarantine these pathogens at the port of entry and, lacking a reliable detection method, particularly a high-throughput method, the epidemiological situation of these agents in China remains unclear. Considering the potential threat to public health if an outbreak of one of these agents occurred, an effective surveillance method is important for detecting an incursion of these pathogens.The aim of this study was to develop a novel PCR-microfluidic combined assay for the simultaneous surveillance of the zoonotic encephalomyelitis viruses listed at the start of the introduction in the mosquito population. The analytical sensitivity and specificity were evaluated separately using synthetic viral RNA standards and reference viral strains from the national veterinary services laboratories. Additionally, it was field tested with mosquitoes captured in local places and compared to real-time RT-PCR using mock field specimens.Results of the current study as follows:(1) Vesicular stomatitis virus(VSV) was used as gene carrier in this paper. Three recombinant viruses, including rVSV-EEEV-E2, rVSV-WEEV-E2 and r VSV-VEEV-E2 were rescued separately by vitro transfection of 4 plasmids into BHK-21 cells. The recombinant viruses were showed good titer in BHK-21 and were verified by RT-PCR, microscope and immunofluorescence staining(IFA). Evidence shown it can be used for the viral surrogate in related diagnostics assay or candidate vaccine.(2) Serial of novel synthetic viral RNAs were generated by tailed PCR strategy and vitro-transcription. The svRNAs were verified by DNA sequencing and RT-PCR. The svRNA was flanked by viral sequences on both ends and imbedded with heterogenetic nucleotides from plasmids in the centre. The viral sequence region contained viral-specific primer binding sites and viral_svr primer binding sites, which functioned for producing the svRNA. The heterogenetic sequence imbedded in the centre was identified for the investigation of false positives. Evidence shown it can be used for the viral surrogate in related diagnostics assay.(3) A Wsp-RT-mPCR diagnostic method was developed by the combining a novle tailed priming PCR and microfluidic electrophoresis to simultaneously detect multiple pagthogens. This work was the first report of an RT-mPCR assay combining a novel primer strategy with Lab-on-Chip electrophoresis to detect EEEV, WEEV, VEEV, WNV, HJV, JEV, LACV, SINV, SLEV, CHKV, DENV and Ockelbo virus in a high-throughput manner. EEEV, WEEV, VEEV, WNV, HJV, SLEV and JEV were verified by reference virus provided by OIE reference lab. Results shown the specificity, sensitivity and reliability of the assay were sufficient for the simultaneous detection of these targets. The analytical sensitivity of the Wsp-RT-mPCR developed here was 102 copy/?L and the reliability of Wsp-RT-mPCR was comparable to that of the singleplex real-time RT-PCR.(4) A software programme called ‘Virus feature interpretation software V1.0' was designed based on the length and concentration of the fragments. This software could automatically interpret the Wsp-RT-mPCR results.(5) Wsp-RT-mPCR was used for the surveillance of above pathogens. More than 2000 wild female mosquitoes were collected from five different locations of the Three Gorges of the Yangtze River Basin. Except of 2/115(1.7%) pools were positive for JEV, all other pools were negative for all other viral targets in these assays.(6) Two SN standards were established in this study. Quarantine protocol for equine encephalitis(eastern and western)(SN/T 2863-2011) and Quarantine protocol for venezuelan equine encephalitis(SN/T 2833-2011) were accredited and published by the General Administration of Quality Supervision, Inspection and Quarantine(AQSIQ).