乙型脑炎减毒活疫苗病毒SA14-14-2株经三带喙库蚊胸腔接种后的生物学和分子生物学特性

目的研究乙型脑炎病毒减毒活疫苗SA14-14-2株经三带喙库蚊胸腔接种繁殖后的生物学和分子生物学特性,进一步评价该株病毒的稳定性和安全性。方法将乙型脑炎病毒减毒活疫苗SA14-14-2株胸腔接种的三带喙库蚊研磨,离心收集上清液,接种于BHK21细胞中培养,得到SA14-14-2M1C1病毒液。应用空斑形成单位法测定病毒滴度,再进行小鼠脑内毒力测定和乳鼠脑内回传后毒力返祖试验。提取病毒RNA,RT-PCR扩增E基因片段,测序后与SA14-14-2原株的E基因序列进行比较分析。结果SA14-14-2M1C1病毒液滴度达1.4×107PFU/ml,小鼠脑内毒力测定结果无小鼠发病死亡,乳鼠脑内传代对小鼠的脑内致病力很低,毒力为1.9LgLD50/0.03ml,小于《中国药典》规定的3.0LgLD50,且皮下注射未见小鼠发病死亡。扩增出的E基因片段序列与原株相比较,只有E区1340位一个核苷酸的变化A→G,导致E447位1个氨基酸的改变D(Asp)→G(Gly),此差异位点不是SA14-14-2株发生基因变异的8个位点之一。结论SA14-14-2疫苗株病毒经三带喙库蚊胸腔接种繁殖后,其生物学(弱毒)特性和基因特征(E区8个氨基酸突变)未发生改变,进一步证实了该病毒的稳定性。

Biological and Molecular Characteristics of Live Attenuated Japanese Encephalitis Vaccine Virus Strain SA14-14-2 Inoculated Intrathoracically to Culex tritaeniorhynchus

Objective To further evaluate the stability and safety of live attenuated Japanese encephalitis(JE)vaccine virus strain SA14-14-2 by studying the biological and molecular characteristics of the virus after intrathoracical inoculation to Culex tritaeniorhynchus.Methods Grind the Culex tritaeniorhynchus inoculated intrathoracically with strain SA14-14-2 then centrifuge.Collect the supernatant and inoculate to BHK21 cells for virus propagation.The obtained SA14-14-2 M1C1 virus liquid was determined for titer by PFU method,then subjected to neurovirulence test in mice and virulence reversion test in suckling mice.Extract viral RNA for amplification of E gene fragment by RT-PCR.Sequence the amplified gene fragment and compare with that of E gene of original SA14-14-2 strain.Results The titer of SA14-14-2 M1C1 virus liquid reached 1.4×107 PFU/ml.In neurovirulence test,no onset or death of mice was observed.In virulence reversion test,the virus titer after intracerebral passage in suckling mice was 1.9 LgLD50/0.03ml,which was less than 3.0 LgLD50 described in Chinese Pharmacopeia.No onset or death of suckling mice inoculated s.c.with the strain was observed.Compared with that of original SA14-14-2 strain,the nucleotide at site 1 340 of E region of amplified E gene changed from A to G,resulting the change of amino acid at E447 from D(Asp)to G(Gly).However,E447 was not one of the eight sites of gene mutation of SA14-14-2 strain during attenuation.Conclusion After intrathoracical inoculation to Culex tritaeniorhynchus,neither biological nor molecular characteristic of SA14-14-2 strain showed significant change,which further proved the safety of the JE vaccine virus strain.