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To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis (JE) virus strain SA14-14-2 used in the live-attenuated JE vaccine prepared at different years, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years (1987-2001) were unchanged, including the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines prepared are quite stable without any reversion noted.
To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis (JE) virus strain SA14-14-2 used in the live-attenuated JE vaccine prepared at different years, the viral titer was titrated by plaque formation in BHK- 21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT- PCR with the designed primers, and then it was found that the loss of viral titer of vaccines stored at -20 ° C for longer than 10 years was less than 0.5 Lg PFU / ml . No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years (1987-2001 ) were unchanged, includin g the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines prepared are quite stable without any reversion noted.