Molecular Epidemiology And Phylogenetic Analysis Of Japanese Encephalitis Virus In Nature,China

Japanese encephalitis is one of the National Notifiable Infectious Diseases and is classified as B in China.Japanese encephalitis has high mortality and most survivors have severe sequelae of the central nervous system.Since 2007,Japanese encephalitis vaccines have been included in the national Expanded Program of immunization(EPI)and the incidence of Japanese encephalitis annually reported decreased significantly.However,there are still cases occurring each year and causes serious economic burdens for society and family.Therefore,establish rapid,reliable and simple techniques for the detection of Japanese encephalitis virus and differentiation of genotypes is essential for accurately mastering of the epidemic and variation of Japanese encephalitis virus in natural,China.Moreover,the combination of society,environment,economy and many other factors can provide important scientific bases for the further development of vaccination strategies,targeted implementation for focus areas,populations and even animals so as to effectively protect the health of the population,which have significant meaningfulness for public health.1.Establishment of TaqMan real-time RT-PCR assay for Japanese encephalitis virus.In this study,the full genome sequences of Japanese encephalitis virus G1～G5 were downloaded from Genbank database.The analysis software of ClustalX Version 2.0 was used to compare the download genome sequences and identified the most conservative regions NS1,NS2 and M genes.Six sets of primers and probes were manually designed and evaluated by Primer Express 3.0(Applied Biosystems,Foster City,CA,USA)for the physical properties.Broad spectrum and specificity of primers and probes were analyzed online by blasting on NCBI.Method of TaqMan real-time RT-PCR was applied to screen six sets of primers and probes which mostly suitable for JEV detection.At the same time,we established standard curves with 10-fold serial dilution of JEV titers and in vitro-transcribed RNAs to furtherly evaluate the specificity,sensitivity and reproducibility of the detection systems.In this experiment,samples with Ct values under 35 and amplification curves presented typical S were considered as positive.The results showed that the detection system had higher specificity among JEV,other flaviviruses and arboviruses.The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/μL.Each ten-fold dilution was amplified in three replicates and the coefficients of variation were all<2.8%.The amplification efficiency of this method was between 90%and 103%.In addition,Twenty batches of mosquitoes previously collected in Guizhou province in 2004,Shandong province in 2008 and Tibet in 2009 were selected for the comparison of virus isolation in cell culture,semi-nested PCR and TaqMan real-time RT-PCR were used to identified the differences for the positive and negative samples and genotypes.The results showed that only 5 samples had obvious cytopathic effect(CPE).Seven specimens were detected by semi-nested PCR,while,twenty samples were detected by TaqMan qPCR with Ct values<35.The above results indicate that the latter is significantly superior to the first two in terms of sensitivity and accuracy for JEV detection and genotype.Therefore,we established a quantitative method that can quickly,easily,and specifically detect JEV in samples and differentiate all five genotypes with high specificity,sensitivity and reproducibility,providing good support techniques in the field of laboratory testing for the routine detection.Meanwhile,it provides a more effective basis for rapidly diagnosing suspected JE cases,which can ensure the implementation of surveillance and prevention of Japanese encephalitis carried out effectively.2.Analysis of the detection results of Japanese Encephalitis virus of mosquitoes in ChinaConsidering above findings,we extracted RNA manually from the 140ul supernatant of mosquito samples in this study.Based on the established TaqMan Real-time RT-PCR detection system to screen and differentiate JEV of 19 Provinces from 2004 to 2016 in China’s mainland,We amplified the E gene of samples which identified as positive to obtain target sequence information and analyzed the homology of nucleotide and amino acid among the newly sequenced E gene,as well as the attenuated vaccine strain SA14-14-2.So,we can clarify the characteristics of the JEV strains in nature in China and its relationships with foreign JEV strains in the molecular level.The purpose of this study is to master the basic epidemiology of JEV and the genotype variation in the natural of China’s mainland.In this study,a total of 3,996samples were collected from 19 areas in China during 2004-2016,including 3,935 mosquito samples,45 ticks,14 biting midges and 2 phlebotomus.Mosquito specimens included 4 genera,28 species and unclassified mosquitoes with a total of 266,679 mosquitoes.Culex mosquitoes and Aedes mosquitoes accounted for the majority of the collected mosquitoes,especially Culex tritaeniorhynchus and Armigeres subalbeatus.The 19 provinces and autonomous regions including four eastern provinces,five central and nine western provinces or autonomous regions.A total of 278 positive(Ct value<35)mosquito samples detected JEV in twelve provinces or autonomous regions,including G1、G3 and G5.There were no positive JEV in mosquitoes for other provinces in that year.Moreover,Qinghai and Xinjiang provinces remained no JEV in these years.In this study,the detection rate of JEV ranged from 0.89%to 31.11%among the 12 provinces,moreover,the detection rate was closely related to mosquito species.Detection rate of JEV from Culex tritaeniorhynchus is highest.One-step PCR method was used to amplify samples with Ct values less than 35 by three pairs of primers in order to get E gene sequences.At present,a total of 63 samples of the E gene of JEV were sequenced.The homology of nucleotide between SA14-14-2 and newly sequenced E gene ranged from 87.1%to 88.3%.The homology of nucleotide sequence among the newly sequenced sequence information range from 96.1%to 99.9%.The homology of amino acid among the newly sequenced E gene and SA14-14-2 ranged from 96.4%to 97.2%based on the E gene.The homology of amino acid among the sequenced E gene and SA14-14-2 were ranged from 98.4%to 100%.Analysis of amino acid site differences revealed that there were 14 common differences between the newly sequenced samples and SA14-14-2.At the same time,the results of phylogenetic analysis showed that 63 samples belonged to G1,which were consistent with the TaqMan real-time RT-PCR method.This study provides a convenient,rapid,sensitive,and specific detection technique for the rapid detection and differentiation for JEV genotypes.Through furtherly phylogenetic analysis,we found that G1 strains in mosquitoes have an obvious advantage since 2004,while,the G3 strains almost not prevalent.The G5 strains are still limited to detected in mosquitoes in Tibet.This study provides an targeted and planned scientific basis to develop and improve the vaccination strategy and environmental governance for Japanese encephalitis,which can effectively protect the vulnerable populations or general populations and even domestic animals.What’s more,it can actively promote social stability and healthy.