Safety And Immunogenicity Of Genotype ? Attenuated Strain SD-F120 Of Japanese Encephalitis Virus In Swine

Japanese encephalitis(JE),a common viral encephalitis in Asia,is a mosquito-borne zoonotic infection caused by the Japanese encephalitis virus(JEV),and pigs are the major source of infection and amplifying host.China is a large pig-raising country.JE is quite prevalent in pig herds.Although it does not cause a high mortality rate after infection,it can lead to a series of reproductive diseases such as boar orchitis and abortion of pregnant sows,caused huge economic losses to the pig industry.In this study,RT-PCR,Indirect Immunoinfluscent Assay and virus isolation laboratory techniques were used to detect a pig sample suspected of being infected with JE at a farm in Fengxian District,Shanghai.A JEV was isolated from the brain tissue of aborted fetus and named SD12 Strains,and sequence analysis of the entire gene to construct a genetic evolution tree,identified as G? JEV.On this basis,a mouse infection model was established to study its dynamic distribution in mice,determine the virus titer and biological characteristics,and analyze its molecular evolution.In view of the current trend of the prevalence of G?/? JEV in China,in order to detect the JEV load and genotype in clinical and experimental samples rapidly and efficiently,this study compared the sequences of the strains of WHV,DENV,ZIKV and G?/? JEV,a Taq Man RT-q PCR assay was developed to differentiate G?/? JEV by using a specific probe.It is proved that this method good sensitivity,repeatability,and specificity,which will lay a foundation for the follow-up experimental research.hasSo far,there is no specific antiviral therapy for JE,and vaccination is still the only important and effective prevention and control measure.The JE vaccine used to immunize pigs in China is a mouse brain inactivated HW1 vaccine based on SA14-14-2 strain and a cell-cultured live attenuated SA14-14-2 vaccine,both of which belong to G? JE vaccine.SA14-14-2 strain is an attenuated strain of the SA14 strain isolated in the 1950 s.The differences of nucleotide and amino acid in genome are obvious between SA14-14-2 strain and the new epidemic strain,and due to the large differences in virulence of different strains,there is an urgent need to assess their ability to neutralize new epidemic strains.In this study,mice immunized with SA14-14-2 attenuated vaccine strain were infected with strain of G? JEV NJ28 and strain of G? JEV SD12 that are lethal doses.The immune protective effect was evaluated by observing clinical symptoms,morbidity and mortality of mice and titrating neutralizing antibody levels.The results showed that the survival rate of mice infected by NJ28 was 100% after two weeks of immunization,but that of mice infectedby SD12 strain was 90%,and the level of neutralizing antibody was generally low.Based on the passage of G? JEV SD12 strain through BHK-21 cells,the virus plaque was purified,and an attenuated strain named SD-F120 strain was screened by animal experiment.In this study,the mouse immune model was used to evaluate its safety in mice according to clinical symptoms,morbidity and mortality of mice and viral load in tissues.To evaluate the immunogenicity of NJ28 and SD12 strains,the Immunoprotective effect of NJ28 and SD12 strains were tested.The results showed that SD-F120 was safe in mice.The mice infected with NJ28 and SD12 could be protected completely after two weeks of immunization.The survival rates of the mice in the blank control group and the immune group were 100%.The mice in the infected control group had typical clinical symptoms and death,and the survival rate was 0%.It showed that the SD-F120 have good immunogenicity to G?/? JEV virulent strain,and the preliminary data were established for carrying out immune protection test in pigs.Through carrying out in vivo immunity experiments of small boars,the safety is evaluated based on clinical symptoms,viremia,tissue lesions and detoxification.Through the challenge protection test,the antibody and neutralizing antibody levels of pig serum samples were detected to evaluate the immune protection effect of the attenuated strain SD-F120 on infection with the SD12 strain.The infected control group showed symptoms such as redness and swelling of the testicles and nerve excitement,while the pigs vaccinated with SD-F120 showed no clinical symptoms related to JE and had low viremia and no detoxification.7 days after single dose of SD-F120 of 5.0 Lg PFU,the neutralizing antibody could exceed the protective level(1:10)to 1:20,and 14 days to 1:113.The neutralizing antibody level in control group was less than 1:10.The result showed that attenuated strain SD-F120 have good safety and immunogenicity to boars.