The purpose of this study was to understand the stability and possibility of back mutation of Japanese encephalitis （JE） attenuated vaccine virus strain SA14-14-2 HKs on molecular level. The E genes of the SA14-14-2...The purpose of this study was to understand the stability and possibility of back mutation of Japanese encephalitis （JE） attenuated vaccine virus strain SA14-14-2 HKs on molecular level. The E genes of the SA14-14-2 HKs vaccine virus and its PHK cells passaged virus （SA14-14-2 HK17 ）, its mouse brain passaged virus （SA14-14-2 SM1 ） were sequenced and compared with the E gene of parental SAI4 virus. The total RNA was extracted from infected Vero cells and amplified by RT-PCR. The RT-PCR products were purified and cloned into T-vector. Positive clones were screened, identified and sequenced. There were twelve nucleotides and eight amino acids substitutions between SA14 parent virus and SA14-34-2 PHKs vaccine virus. The SA14-14-2 PHK17 virus showed two additional mutations （E-331 and E-398） which were not back mutations. Although five additional mutations were found in SA14-34-2 SMt virus, only one （E-307） was back mutation. Genetic characteristics of the attenuated vaccine virus SA14- 34-2 were stable when it was passaged 37 times on PHK cells or one time in mouse brains.展开更多
The aim of this study was to explore the molecular basis for the attenuation of the Japanese encephalitis vires （JEV） vaccine strain SA14-14-2. The virulence of SA14 wild Japanese encephalitis virus （JEV） and its ...The aim of this study was to explore the molecular basis for the attenuation of the Japanese encephalitis vires （JEV） vaccine strain SA14-14-2. The virulence of SA14 wild Japanese encephalitis virus （JEV） and its several attenuated viruses was tested by intracerebral （i. c. ） or intraperitonial （i. p. ） inoculation of 10-12 g mice. The stability of neumattenuation was tested by one passage in suckling mouse brain. The E protein genes of those viruses were amplified by PCR, sequenced and compared. Three attenuated virus strains, SA14-14-2 vaccine virus, SA14-9-7 and SA14-5-3, did not exhibit lethal infections by i.c. or i.p. inoculation of 10-12 g mice and revert to the virulence. The other virus strain, SA14-12- 1-7, showed no neuminvasiveness by i.p. inoculation but residual neurovimlence by i.c. inoculation and reverted to high virulence after one brain passage. Comparison of the E protein gene sequences of the five virus strains indicated that there were differences of twelve nucleotides and eight amino acids between the parent strain SA14 and vaccine strain SA14-14-2, of which six amino acids （E-107, E-176, E-439, E-138, E-279, E-315） exhibited changes common to those of SA14-9-7 and SA14-5-3, three substitutions common to SA14-12-1-7. Two amino acid substitutions at the sites E177 （T→A） and E264 （Q→H） are unique to the SA14-14-2 vaccine virus. The results suggest that the mutations of E-107 （Leu→Phe）, E- 176 （Ile→Val）, and E-439 （Lys→Arg） may contribute for the attenuation of neuminvasiveness and partially for the attenuation of neumvirulence, the mutations of E-138, E-279, E-315 may not only critical to the neumattenuation but also to its stability.展开更多
文摘The purpose of this study was to understand the stability and possibility of back mutation of Japanese encephalitis （JE） attenuated vaccine virus strain SA14-14-2 HKs on molecular level. The E genes of the SA14-14-2 HKs vaccine virus and its PHK cells passaged virus （SA14-14-2 HK17 ）, its mouse brain passaged virus （SA14-14-2 SM1 ） were sequenced and compared with the E gene of parental SAI4 virus. The total RNA was extracted from infected Vero cells and amplified by RT-PCR. The RT-PCR products were purified and cloned into T-vector. Positive clones were screened, identified and sequenced. There were twelve nucleotides and eight amino acids substitutions between SA14 parent virus and SA14-34-2 PHKs vaccine virus. The SA14-14-2 PHK17 virus showed two additional mutations （E-331 and E-398） which were not back mutations. Although five additional mutations were found in SA14-34-2 SMt virus, only one （E-307） was back mutation. Genetic characteristics of the attenuated vaccine virus SA14- 34-2 were stable when it was passaged 37 times on PHK cells or one time in mouse brains.
文摘The aim of this study was to explore the molecular basis for the attenuation of the Japanese encephalitis vires （JEV） vaccine strain SA14-14-2. The virulence of SA14 wild Japanese encephalitis virus （JEV） and its several attenuated viruses was tested by intracerebral （i. c. ） or intraperitonial （i. p. ） inoculation of 10-12 g mice. The stability of neumattenuation was tested by one passage in suckling mouse brain. The E protein genes of those viruses were amplified by PCR, sequenced and compared. Three attenuated virus strains, SA14-14-2 vaccine virus, SA14-9-7 and SA14-5-3, did not exhibit lethal infections by i.c. or i.p. inoculation of 10-12 g mice and revert to the virulence. The other virus strain, SA14-12- 1-7, showed no neuminvasiveness by i.p. inoculation but residual neurovimlence by i.c. inoculation and reverted to high virulence after one brain passage. Comparison of the E protein gene sequences of the five virus strains indicated that there were differences of twelve nucleotides and eight amino acids between the parent strain SA14 and vaccine strain SA14-14-2, of which six amino acids （E-107, E-176, E-439, E-138, E-279, E-315） exhibited changes common to those of SA14-9-7 and SA14-5-3, three substitutions common to SA14-12-1-7. Two amino acid substitutions at the sites E177 （T→A） and E264 （Q→H） are unique to the SA14-14-2 vaccine virus. The results suggest that the mutations of E-107 （Leu→Phe）, E- 176 （Ile→Val）, and E-439 （Lys→Arg） may contribute for the attenuation of neuminvasiveness and partially for the attenuation of neumvirulence, the mutations of E-138, E-279, E-315 may not only critical to the neumattenuation but also to its stability.
