| Japanese encephalitis virus(JEV)is a member of the flavivirus which cause Japanese encephalitis(JE),it is a serious impact on human and animal health of mosquito borne zoonotic diseases.JEV mainly damage human patients with central nervous system,some of the survivors have neurological sequelae of severe.JEV can cause sow reproductive disorders,boars orchitis and so on.Pigs and waterfowl are intermediate hosts of JEV,all of which are important in the mechanism of JEV circulating transmission.Dongting Lake's special wetland ecosystem provides a good growth and reproduction environment for a variety of water birds and mosquitoes.In addition,there are more than 20 counties around the Dongting Lake area,with a large scale and a large number of pig farms.Therefore,we studied the epidemiological of Japanese encephalitis in three breeding farms in Dongting Lake area,and learned about the infection of JEV in mosquitoes and pig farms.To study the molecular epidemiology of JEV in this region,and it has certain significance to prevent and control local JE disease.Amplified by RT-PCR JEV 3 'UTR region,type I and III JEV prM gene,the mosquitoes,the positive rate was 42.9%(45/105),piglet tissue samples the positive rate was 8.2%(10/123),semen samples were not detected in JEV;The blood samples collected from 24 heads of pigs were detected by JEV antibody,and the mosquitoes captured in the pigpen were tested for pathogen detection,the results showed that the number of pig JEV antibody positive rate showed first decreased and then increased,the positive rate were 83.3%,66.7%,41.6%,25%,50%,91.6%;37 mosquito samples,16 samples were JEV positive,the positive rate is as high as 43.2%,which in July a total of 14 samples were detected in 7 samples of mosquito samples,JEV positive rate up to 50%.In the JE epidemic season,when piglets of JEV antibody decreased lowest susceptible to JEV,then bited by mosquito carried with JEV,leading to pigs JEV antibody positive rate increased.Virus isolation from 45 positive mosquitoes samples,and 10 positive brain tissue samples,monoclonal cell lines were inoculated in BHK21(BHK-3),a total of 3 isolated strains of JEV,the new strains named HNML1(mosquito source),HNML2(mosquito source)HNML3 strain(swine source).3 strains of the homology between E gene nucleotide sequence and vaccine strain SA14-14-2 is respectively were 86.9%,86.8%,86.9%,the deduced amino acid homology were 97%,96.8%,97%.There were 15,16 and 15 amino acid differences with SA14-14-2,including 8 amino acids related to critical virulence.Gene homology analysis showed that JEV isolates were I type JEV strains.Notably,the HMML3 strain is genetically related to the LN02-102 strain isolated from the human body in 2002 in Liaoning,china.Using BL21(DE3)expression EDIII protein in prokaryotic system,the immunogenicity of EDIII,ICR mice were injected by EDIII protein twice,preparation of mouse anti EDIII recombinant protein immune serum antibody and Western Blotting results showed that the recombinant protein in serum can have obvious reaction.Indirect immunofluorescence(IFA)showed that the immune serum and intracellular virus specific reaction;Calculation of neutralization titer in accordance with Reed-Muench method,serum neutralizing antibody titer was 1:44.6.By ELISA,neutralization test and Western Blotting,it was proved that ED III protein had immunogenicity.The EDIII protein as coated antigen,JEV(IgG)to determine the optimum reaction conditions of indirect ELISA method,compared it with immunofluorescence results the coincidence rate of 93.4%.In order to better determine the regular of the growth and decline of maternal antibodies and the onset of antibody infection in pigs,(IgM)antibody indirect ELISA method was used to establish JEV antigen.In the JE epidemic season,we collected 6 batches of piglets a total of 840 serum,this trial established JEV(IgG,IgM)ELISA method for testing results show JEV antibody and JE seasonal epidemic regularity is consistent. |
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