Cultivation And Immune Characteristics Research Of Attenuated Strain Of Japanese Encephalitis Virus Genetype ?

Japanese encephalitis virus(JEV)is a phago-neurogenic virus,causing acute viral encephalitis in human and reproductive failure in sows and orchitis in boars,which mainly spread through mosquito bites.In recent years,the dominant genotype of Japanese encephalitis virus is gradually changing from genotype III to genotype I.In view of its serious public hazard,this study was based on genetype I Japanese encephalitis virus SCYA201201 strain to breed an attenuated strain,through serially intracellular subculture,plaque purification and virulence screening.Then the molecular basis of the attenuated virulence was analyzed by molecular biology technique.Meanwhile,The immunological characteristics of the attenuated strain were studied by cross immunization challenge test,indirect ELISA test and lymphocyte proliferation test.1.Cultivation of attenuated strain of Japanese encephalitis virus genetype IThrough the optimization of inoculative dose and harvest time of Japanese encephalitis virus SCYA201201 strain,the results showed that the highest viral titers was obtained after 36h post infection with MOI-0.05?0.1.The virus of JEV SCYA201201 strain was serially passaged on BHK-21 cell lines from F86 to F120,and purified on F106,F117 utilizing plaque assay respectively.Based on 4 times screening of neurovirulence,1 time screening of nerveinvasiveness and 1 time screening of immunogenicity,an attenuated strain with the weakest virulence was successfully screened out at F118.To detected the neurovirulence and nerveinvasiveness of attenuated strain YA1-0901 passaged to F120,3-weeks-old mice were inoculated by intracerebral inoculation and intraperitoneal inoculation,respectively.Compared with F86,the lethality of neurovirulence was reduced form 30%to 0,and the lethality of neuroinvasiveness was reduced form 10%to 0 detected.The results showed that the safety of attenuated strain YA1-0901 was relatively good.Attenuated strain YA1-0901 was continually passaged 5 times on 3?5-days-old suckling mice,one time on BHK-21.Then the neurovirulence and nerveinvasiveness of YA1-0901-SN5 and YA1-0901-SN5-1 were detected in the same methods as described above.The results showed that the lethality of neurovirulence of YA1-0901-SN5 strain and YA1-0901-SN5 strain was both recovered to 100%,but the lethality of neuroinvasiveness was still retained 0.The results indicated that the neurovirulence of attenuated strain YA1-0901 could recover after serially passages on suckling mice,and the neuroinvasiveness was not changed significantly.Using designed 8 pairs primers before,the genomic sequence of attenuated strain YA1-0901 and re-virulent strain YA1-0901-SN5 were determined.By comparing the coding region sequences of YA-0901,YA1-0901-SN5 and F86,we found that 14 nucleotides were mutated.Among them,5 mutations resulted in changes in the amino acid.Combined with coding region sequences of F1 and F46,we found that these amino acids belong to reverse mutation which located in C-111,E-72,E-176,NS2b-95,respectively,and E-273 can be stably inherited during passages in suckling mice brain.After comparing the difference of nurovirulence and neuroinvasiveness of attenuated strain YA1-0901 and re-virulent strain YA1-0901-SN5,we preliminary determine that E-72,E-176 are associated with virulence of JEV.2.Immune characteristics research of attenuated strain YA1-09013-weeks-old mice were immunized with attenuated strain YA1-0901 by intraperitoneal inoculation and intracranial inoculation,respectively.14 days after immunization,these mice were challenged intraperitoneally with JEV virulent strain of YA1-SN10(7.881gPFU/ml)and SC2013-SN5(7.791gPFU/ml),respectively.In the test of intraperitoneal inoculation,the protective rate of YA1-0901 was both 30%in the virus titer of 8.56 IgPFU/ml,and 10%and 30%in 7.29 1gPFU/ml respectively after challenging by JEV virulent strain of genetype ? and genetype ?,separately.Nevertheless,the protective rate was 90%and 100%in 8.56 1gPFU/ml,70%and 100%in 7.29 1gPFU/ml with intracranial inoculation.This results indicated that the protective effect of intracerebral immunization of attenuated YA 1-0901 was significantly higher than that of intraperitoneal inoculation.Then the IgG antibody of mice serum and transformation rate of mice spleen lymphocytes after adding the irritant of inactivated JEV or ConA were detected 14 days later,when mice were immunized the virus of attenuated strain YA1-0901 by intracerebral immunization and intraperitoneal inoculation,respectively.The results showed that IgG antibody level of intracerebral immunization was both lower than that of intraperitoneal inoculation at the virus titer of 8.56 1gPFU/ml and 7.291gPFU/ml,but the transformation rate of spleen lymphocytes of intracerebral immunization was higher than that of intraperitoneal immunization after adding irritant.