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 prepears, the viral titer was titrated by plaqu...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 prepears, 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, in- cluding 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 sion noted. prepared are quite stable without any reversion noted.展开更多
目的建立纯化乙型脑炎减毒活疫苗(SA14-14-2株)的方法。方法应用超滤浓缩和分子筛凝胶柱层析纯化乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14-2病毒液,检测其病毒滴度、原代地鼠肾细胞残留蛋白含量、牛血清白蛋白残留量、庆...目的建立纯化乙型脑炎减毒活疫苗(SA14-14-2株)的方法。方法应用超滤浓缩和分子筛凝胶柱层析纯化乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14-2病毒液,检测其病毒滴度、原代地鼠肾细胞残留蛋白含量、牛血清白蛋白残留量、庆大霉素残留量,并进行小鼠脑内致病力试验、乳鼠传代返祖试验、异常毒性试验,制备纯化冻干疫苗,进行热稳定性试验。结果纯化后的JEV病毒滴度高于7.0 lg PFU/ml,平均回收率为23%;牛血清白蛋白残留量、庆大霉素残留量及安全性试验均符合《中国药典》三部(2010版)相关规定,去除了约95%的原代地鼠肾细胞残留蛋白;制备的冻干疫苗及热稳定性试验中的病毒滴度均高于5.7 lg PFU/ml,且热稳定性试验中病毒滴度下降未超过1.0 lg,符合《中国药典》三部(2010版)规定。结论建立了乙型脑炎减毒活疫苗(SA14-14-2株)纯化工艺,但病毒回收率较低,不适宜规模化生产,对乙型脑炎病毒减毒活疫苗质量控制具有指导意义。展开更多
The incidence of Japanese encephalitis(JE)has significantly decreased in China due to JE vaccines.In this study,we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against...The incidence of Japanese encephalitis(JE)has significantly decreased in China due to JE vaccines.In this study,we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus(JEV)to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children.A total of 300 serum samples were collected from vaccinated children aged 3-10 years in Zhaotong,Yunnan,China.The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test.The highest seropositivity of 82%was observed in vaccinated children during the first 0.5-1.5 years after booster vaccination.Then,the seropositivity began to decline and remained lower than the original level observed in the 0.5-1.5-year group.An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination.Similarly,the neutralizing antibody(nAb)titres gradually decreased over time,and the levels showed a positive correlation with the protective efficacy in mice.This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine.Our results provide data that clarify the persistence and waning of antibodies to JEV,which may help elucidate the pathogenesis of JE.展开更多
文摘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 prepears, 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, in- cluding 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 sion noted. prepared are quite stable without any reversion noted.
文摘目的建立纯化乙型脑炎减毒活疫苗(SA14-14-2株)的方法。方法应用超滤浓缩和分子筛凝胶柱层析纯化乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14-2病毒液,检测其病毒滴度、原代地鼠肾细胞残留蛋白含量、牛血清白蛋白残留量、庆大霉素残留量,并进行小鼠脑内致病力试验、乳鼠传代返祖试验、异常毒性试验,制备纯化冻干疫苗,进行热稳定性试验。结果纯化后的JEV病毒滴度高于7.0 lg PFU/ml,平均回收率为23%;牛血清白蛋白残留量、庆大霉素残留量及安全性试验均符合《中国药典》三部(2010版)相关规定,去除了约95%的原代地鼠肾细胞残留蛋白;制备的冻干疫苗及热稳定性试验中的病毒滴度均高于5.7 lg PFU/ml,且热稳定性试验中病毒滴度下降未超过1.0 lg,符合《中国药典》三部(2010版)规定。结论建立了乙型脑炎减毒活疫苗(SA14-14-2株)纯化工艺,但病毒回收率较低,不适宜规模化生产,对乙型脑炎病毒减毒活疫苗质量控制具有指导意义。
基金supported by grants from the National Natural Science Foundation of China (81471957, 81772172, 81671971, U1602223 and 81871641)a grant from the Open Research Projects of Key Laboratory of Beijing Ditan Hospital, Capital Medical University (DTKF201705)
文摘The incidence of Japanese encephalitis(JE)has significantly decreased in China due to JE vaccines.In this study,we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus(JEV)to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children.A total of 300 serum samples were collected from vaccinated children aged 3-10 years in Zhaotong,Yunnan,China.The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test.The highest seropositivity of 82%was observed in vaccinated children during the first 0.5-1.5 years after booster vaccination.Then,the seropositivity began to decline and remained lower than the original level observed in the 0.5-1.5-year group.An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination.Similarly,the neutralizing antibody(nAb)titres gradually decreased over time,and the levels showed a positive correlation with the protective efficacy in mice.This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine.Our results provide data that clarify the persistence and waning of antibodies to JEV,which may help elucidate the pathogenesis of JE.
