Cultivation And Molecular Mutation Analysis Of The Attenuated Strain Of Swine Japanese Encephalitis Virus

Japanese encephalitis(JE)is a mosquito-borne zoonosis,which was caused by the Japanese encephalitis virus(JEV).Swines infected with JEV mainly caused reproductive failure in sows and orchitis in boars.The strain SA14-14-2 is the JE attenuated live vaccine,which belongs to genetype ?.While genetype I also is the main popular genetype now.Therefore,development of gene type I Japanese encephalitis attenuated live vaccine has value.In the study,the genetype I swine JEV strain SCYA201201 has serially passed on BHK-21 cell to attenuate and detect culture characteristics,virulence,gene variation of different generations viruses and analysis of the molecular mutation of attenuated viruses,which could lay the solid foundation for development of the SCYA201201 strain of the attenuated vaccine strain as well as in-depth study of the mechanism of attenuated virus.1.Cultivation of Japanese encephalitis attenuated strain of swineThe Japanese encephalitis virus strain SCYA201201 serially passed on BHK-21 cells in low temperature and room temperature culture for 86 times and three times plaque purification were undertaken on F37,F77 and F79 viruses,respectively.We have chosen F1,F16,F26,F36,F46,F56,F66,F76,F86 viruses to determine their properties including viral titer,growth curve and median lethal dose.The results showed that the viral titer has inceased from 104.71PFU/ml(F16)to 1010.83PFU/ml(F86)on BHK-21 cells,the median lethal dose in suckling mice has increased from 10-5.68 LD50/0.03ml(F1)to 10-2.88LD50/0.03ml(F86)and the peak of viral titer was about 48?72 h post-infection.This study has proved that the virus titer of SCYA201201 strain in BHK-21 cells was significantly increased,and the virulence was significantly decreased at the same time.To evaluated the neurovirulence and neuroinvasiveness of F1 and F86,groups of 2,3,4.5-week-old Kunming mice were inoculated by the intracerebral inoculation(i.c.)and the intraperitoneal(i.p.)respectively.The results showed that virulence of the F1 was stronger than the F86,which could indicate serially passing on BHK-21 cells reduced the neurovirulence or neuroinvasiveness virulence of the strain SCYA201201.Immunogenicity of the F86 virus has been determined.Firstly,the F86 virus diluted 5,10-fold has tested in mice by intraperitoneal inoculation and serum antibody levels in mice has deteminated by ELISA.The results showed that mice immunized with F86 could produce JE antibody positive after two weeks and with prolonged immunization,antibody levels in mice gradually increased in 8 weeks.Then,three groups of immunized mice challenge protection test.Two weeks after immunization,mice were challenged intracerebrally with 50 50%lethal doses(50 LD50)of JEV YA1 strain.The result showed the protection rate of F86 diluted 5-fold was 62.5%,the protection rate of F86 diluted 10-fold and SA14-14-2 strain vaccine was 50%.which incadite the F86 virus has good immunogenicity.This study successfully bred swine japanese encephalitis virus attenuated strain,which provides basis for the development of Japanese encephalitis attenuated vaccine.2.Molecular mutation analysis of the attenuated strain of Japanese encephalitis virus(1)Analysis of E gene sequenceDuring the cultivation of SCYA201201 strain,the E protein gene of the viruses of strain F1,F16,F26,F36,F46,F56,F66,F76,F86 were sequenced and compared with each other.We found four nucleotide acids leaded to four amino acid changes in the E protein,of which E-123?E-451 were the reverse mutations and E-72?E-251 were adaptive and stable mutations.Two stable mutations located in domain ? of E protein.(2)Cloning and analysis of the coding sequenceDuring the cultivation of SCYA201201 strain,the coding sequence(gene C,M,E.NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5)of the viruses F1,F46,F86 were sequenced and alignment analysised the deduced amino acid sequence.The result showed 30 nucleotide leaded to 14 amino acids changes from F1 to F86 and these amino acids were distributed in structural proteins C,M,E and non-structural proteins NS1,NS2B,NS4A,NS5.At the same time,it was found that the change of the nucleotide 3594 position from C to A,which affected the stem loop structure of NS1 protein.In this study,a total of 14 amino acid changes were found from the molecular mutation of the virus,which may be closely related to the characteristics of culture and the decrease of virulence of SCYA201201 strain.