Isolation, Identification And Attenuation Of Japanese Encephalitis Virus And Establishment Of An Indirect Elisa For Detecting Antibodies

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) is a zoonotic severe acute infectious disease. JEV belongs to the genus Flavivirus which also include West Nile virus and Dengue virus. Culex mosquitoes acted as the primary media of JE。 In humans, JEV mainly causes acute encephalitis with fatality rates ranging from20%to as high as50%and most of the survivors display persistent neurological or psychological sequelae. Swine is an important reservoir and amplifier of Japanese encephalitis virus in the natural environment. Japanese encephalitis is one of the important diseases of Swine, which can cause abortion and stillbirth of sows, orchiditis of boar. The pig industry suffered serious lossed from the viral infection. Prevention of JE in swine can benefit for swine production and health of human being.Hence, we separating Japanese encephalitis virus in pigs and analyzing the molecular characteristics of the attenuated strains. The recombinant E protein was obtained by means of prokaryotic expression system and used to construct an indirect ELISA method to research the pig’s seroepidemiology of ShangHai. So we can provide basis for the establishment of effective detection methods and development swine’s JE vaccine. The contents of the paper contain five parts as following:1. JEV separation was performed from swine’s blood and semen which infected JEV. Six new JEV isolates were obtained by infected mouse brain and BHK-21cell. The new isolates were identified by Indirect Immunofluorescence Assay (IFA) and RT-PCR. They showed special Immunofluorescence. When the new isolates compared with five JEV strains of P3strain, SA14-14-2strain, Beijing-1strain and WHe strain, the homology of M gene were above96%.2. Japanese encephalitis viru NJ08strain was attenuated through Hela cell. We analyzed the change parts of structural protein nucleotide and the deduced amino acid sequences to reveal the rule of NJ08strain from virulent to attenuate during the process of attenuation. Nucleotide sequence of JN08strain,50generation and100generation virus had a few changes on C protein and PrM protein gene. The homology of amino acid sequence reference NJ08strain was up to99%. But the conservation of E protein nucleotide and amino acid sequences was poor.50generation and100generation of strain E protein nucleotide variant region focus on1200bp-1280bp.The amino acid sequence changes in the region located in370aa-440aa. Analyzing of Japanese encephalitis virus E protein3D structure showed that the variant region located on the C-terminal of the E protein. It’s a membrane-proximal region before the transmembrane region. The regional variation may reductbe the virulence of virus.3. Virus genome was extracted from NJ08strain. The full-length E gene was amplified by RT-PCR and cloned into Pcold I vector to construct the recombinant expression vector. The plasmids of E-Pcold I and EDIII-PET-28a were used to express E protein and ED III protein. Both of them were expressed. Western blot showed all the expressed proteins have anti-JEV activity, which laid foundation for the subsequent tests.4. Two indirect ELISA methods were established for the rapid detection of specific Japanese encephalitis virus (JEV) antibodies in swine using a recombinant E protein and domain Ⅲ of E protein. The various factors affected the experiments were optimized. The assay was optimized using0.625μg/ml E DⅢ protein or1.875μg/ml E protein as coat antigen,1:40dilution of testing serum. The critical value of positive and negative of ELISA was D450nm=0.333for ED III protein or D450nm=0.454for E protein. These methodes were appraised to be very specific and sensitive. An indirect ELISA method for specifically detection JEV in serum was successfully established.5. Serum samples collected from Shanghai were detected, using the indirect ELISA method. The results showed that the total JEV positive rate was68.69%.The positive rate of adult pigs was90.14%and the positive rate of piglets was only37.50%. The antibody of the nursery pigs with different weeks was detected. It showed that the level of antibody depressed at first, then elevated. The neutralization activity was determined by plaque reduction test, and most of the positive serums detected by ELISA showed obvious neutralization activity. The indirect ELISA method can reflect antiviral ability of animals.