Research On Molecular Epidemiology, Diagnosis And Recombinant Vaccine Of Japanese Encephalitis Virus

Japanese encephalitis virus （JEV） is transmitted by Culex tritaeniorhynchus, and theswine is reservoir of JEV which play an important role in the epidemic of encephalitis. JE wasprevalent in the Asia, about3-5million people infected annually and1-1.5million people died.JEV caused huge economic losses to the pig industry, and the pig miscarriage after infection,piglets dysplasia or mummified symptoms.Jinghong city of Yunnan province is located in the China-Burma border, belongs to theinsect-borne virus disease in major countries and regions in arbovirus survey in the regionespecially the research on molecular epidemiology of Japanese encephalitis virus is significant.This research carried on the JEV epidemiological survey of Yunnan Jinghong city mosquito andpig brain tissue samples.430mosquito and108pig brain tissue samples was suveillanted with theconventional PCR method amplification to NS1gene of JEV I and JEV III. The results showedthat JEV positive rate of mosquito C. tritaeniorhynchus is13.2%（57/430）, A. sinensis2.7%（3/430），A. subalbatus0.7%（3/430）. And piglet brain tissue samples positive rate was18.5%（20/108）. Positive samples the amplification JEV structural protein E gene from C.tritaeniorhynchus, A. sinensis and pig samples were obtained8,5and3positive resultsrespectively. The sequence analysis of16samples showed the gene homology of99.7%with typeIII JEV. It can be speculated that the type III of JEV is popular in Yunnan province. The instituteof Yunnan province parasite control provided the E gene sequence of human and pig of JEV carryon further contrast the analysis.The results showed in the Yunnan province mosquito source （C.tritaeniorhynchus6, A. sinensis4） the JEV sequence the same to human of JEV sequence, butpig of JEV and the human of JEV sequence homology is96.2%-98.6%.The reasearch of the epidemiological investigation successfully isolated from pig brain tissueand mosquito of JEV Yunnan0901and Yunnan0902strain （GenBank accession numbersJQ086762and JQ086763）. The complete of genome sequence of JEV Ⅲ, comparative analysisisolated in other regions and time of JEV genome.5’-NCR which Yunnan0901strains andYunnan0902strains have a gene replacement rate about1.05%, while the amino acids were notchanged. Structural protein gene capsid genes were2and1nucleotide substitution, replacementrates were0.52%and0.26%respectively. Yunnan0901strain amino acid did not change whileYunnan0902strains have one amino acid change. Membrane gene were3and1nucleotide substitution, the substitution rate of0.59and0.20%, respectively.Yunnan0902strain amino aciddid not change Yunnan0901strains have an amino acid change. Envelope genes were10and4nucleotide substitution, replacement rates were0.67%and0.27%Yunnan0901strains have9andYunnan0902strains have3amino acid changes. NS5genes were25and16nucleotidesubstitution,replacement rate of0.91%and0.59%Yunnan0901strains have18and Yunnan0902strains have9amino acid changes.3’-NCR were not nucleotide substitution. The pig is a reservoirhost of the virus and likely to cause the virus to mutate. The E gene of Yunnan0901and Yunnan0902were compared with93JEV E genes, to provide the basis for the genetic evolution of JEV.Japanese encephalitis virus isolation in the period of Viremia in peripheral blood. ELISAmethod were used to pig JEV diagnostic. However, JEV checked will have a larger cross-reactivewith other viruses in serological methods. Porcine Japanese encephalitis virus detection havemore sensitive using RT-PCR or real-time PCR method, these two methods need expensiveequipment and experienced operators. In recent years Lamp detection methods havecharacteristics of high sensitivity, accuracy, simplicity is widely used in basic testing organizations.Therefore, we develop a simple, fast and accurate Porcine Japanese encephalitis diagnosticmethod for grassroots department. lamp amplification primers were designed in JEV Ⅰ（K94PO5） and JEV Ⅲ （SA14-14-2Strains）. The Optimization of Lamp reaction systemsensitivity and accuracy were similar to real-time PCR, but the Lamp is more time-saving and didnot need equipment requirements.It only need a constant temperature water bath. LAMP andconventional PCR compare its superiority more obvious, not only time-saving but also itssensitivity is conventional PCR100times. The LAMP test result is completely consistent withvirus isolation method.At present, there is no porcine Japanese encephalitis virus vaccine, instead of humanattenuated vaccine SA14-14-2strain. The SA14strain may exists cause animal disease, so weneed to develop a veterinary new Japanese encephalitis virus vaccine. The E protein antigen caninduce virus neutralizing antibodies and CTLs effect.The vaccinia virus genome can be inserted into a large number of exogenous gene, and non-essential gene deletion does not affect viral replication and function. This study was constructedJE recombinant vaccinia virus vaccine containing the Japanese encephalitis virus E antigen andknock out TK and E3L of gene of the vaccinia virus, and its enables to purpose of virus attenuated.E protein was successfully expressed through western blot analysis. Moreover, the the E proteinproduction biological functions. Recombinant vaccinia virus can induce JEV specific the Tlymphocyte reaction, muscle immunized mice and pig experiments Induced antibody levels higherthan the attenuated vaccine and other immunization groups （p <0.05）. The Neutralizingantibody of pig also proves this result. The mouse Lymphopoiesis proves the JEV specificitystimulation the SI index, rVVTK/E3LΔ–E+rVVTK/E3LΔ–E （1.86）>SA14-14-2 （1.59）>rVVTK/E3LΔ–E （1.37）>VVE3LΔ（1.21）>PBS （1.01）. The pig lymphocyteproliferation experiments rVVTK/E3LΔ–E+rVVTK/E3LΔ–E （1.65）>SA14-14-2（1.45）>rVVTK/E3LΔ–E （1.29）>VVE3LΔ（1.16）>PBS （1.02）. The mice and pig JEVof stimulus their results indicate that the recombinant vaccinia virus immunohistochemistrystimulation index （si） significantly higher than the other groups （p <0.05） to prove that iteffectively stimulate the cell-mediated immunity.The cellular immune response is an important protective immunity against Japaneseencephalitis virus infection. IL-4and IL-10is an important indicator of evaluation Th2celldifferentiation. IFN-γ and IL-2, generally are considered as the Th1of cell factors, similarly mayinduce the production of Th2of cell factors. We from the mouse immunity experiment IFN-γresult, rVVTK/E3LΔ–E+rVVTK/E3LΔ–E （1207）>SA14-14-2（870）>rVVTK/E3LΔ–E（688）>VVE3LΔ（367）>PBS （197）. IL-4result: rVVTK/E3LΔ–E+rVVTK/E3LΔ–E（728）>SA14-14-2（603）>rVVTK/E3LΔ–E （460）>VVE3LΔ（230）>PBS （117）.Recombinant vaccinia virus cytokine levels significantly higher than the control group of PBS（p<0.05）.Pig immune test IFN-γ results: rVVTK/E3LΔ–E+rVVTK/E3LΔ–E（1078）>SA14-14-2（866）>rVVTK/E3LΔ–E（778）>VVE3LΔ（474）>PBS（329）. IL-4result: rVVTK/E3LΔ–E+rVVTK/E3LΔ–E （469）>SA14-14-2（393）>rVVTK/E3LΔ–E （320）>VVE3LΔ（221）>PBS （107）. Recombinant vaccinia virus cytokine levels significantly higher than thecontrol group of PBS（p <0.05）.The results of the recombinant vaccinia virus vaccine in mice and pigs can stimulate the bodyto produce humoral and cellular immune responses. Mouse and pig challenge protection testproved that recombinant vaccinia virus vaccine against this result was significantly higher than theattenuated vaccine group and control group of mice （5/5）, and pigs have very good protectioneffect. Compared to the other group significantly reduced swine virus in patients with clinicalsymptoms, so we developed a new recombinant vaccinia virus is safe and effective. It is expectedto instead of JE attenuated vaccine.