乙脑/登革4型嵌合病毒的构建及其小鼠神经毒力测定

Construction and neurovirulence test for dengue serotype 4 chimeric virus using Japanese encephalitis vaccine strain SA 14-14-2 as backbone

目的构建以乙型脑炎病毒(Japanese encephalitis virus,JEV)疫苗株SA14-14-2为基因骨架的乙脑/登革4型嵌合病毒,并分析该嵌合病毒对小鼠的神经毒力。方法通过重叠PCR方法扩增含有登革病毒4型(DENV-4)H241株pr ME基因序列和乙型脑炎病毒疫苗株SA14-14-2的NS1蛋白前177个核苷酸的融合片段,用Nar I和Bgl II双酶切后替换乙型脑炎病毒疫苗株SA14-14-2全长克隆中的相应区域,构建成乙脑/登革4型嵌合全长克隆,通过体外转录和转染BHK21细胞获得嵌合病毒(JEV/DENV-4 chimeric virus,JD4)。通过测定嵌合病毒JD4和2个母本株JEV SA14-14-2株及DENV-4 H241株蚀斑大小、小鼠脑内神经毒力和皮下感染入脑能力、乳鼠脑内神经毒力,比较JD4和母本株之间的差异。通过将JD4在原代地鼠肾(primary hamster kidney,PHK)细胞传代30次,分析传代后嵌合病毒的神经毒力是否减弱及减弱的程度。结果测序结果表明,构建的嵌合病毒JD4基因组序列和预期一致,没有产生新的位点突变。JD4蚀斑较SA14-14-2明显偏小,但和DENV-4 H241株没有明显区别。JD4对3周龄小鼠具有较强的脑内神经毒力,和母本株DENV-4 H241没有差异,对小鼠没有神经侵袭力。乳鼠实验结果表明,嵌合病毒JD4脑内神经毒力虽然略低于母本株DENV-4 H241,但两者之间没有明显差异,都明显强于乙脑疫苗株SA14-14-2。在PHK细胞传代30次后,小鼠神经毒力虽然有所减低,但并不明显。结论成功构建了嵌合病毒JD4,通过测定并比较JD4与母本株的蚀斑特征、小鼠及乳鼠神经毒力等试验,为分析登革疫苗候选株安全性研究奠定了基础。

Objective To constuct a chimeric virus of Japanese encephalitis /dengue (JD4)as a vaccine candidate using the Japanese encephalitis virus (JEV) vaccine strain as the genetic skeleton, and to investigate resulted neurovirulence for mouse. Methods The gene fragments containing the prME gene of dengue virus serotype 4(DENV-4)H241 strain and the partial NS1 gene of JEV SA14-14-2 strain were amplified by overlap-PCR. By replacing the corresponding area of genome of JEV strain SA14-14-2, the JD4 chimeric clone was constructed, then by in vitro transcription and transfection of primary rat kidney (PHK) cells, the chimeric virus JD4 was rescued. The chimeric virus was identified by a complete genome sequencing. Plaque morphology, neurovirulence and neuroinvasiveness were tested in 3-week-old mice, neurovirulence in 3-5-day-old suckling mice, respectively. Neurovirulence was detected in mice again after 30 passages in PHK cells. Results Sequencing showed that the sequence of JD4 was consistent with that of two parent strians and no a new site mutation was generated. The plaque size of the JD4 virus was much smaller than that of JEV strain SA14-14-2 and similar to DENV-4 H241. Neurovirulence showed that there was no a significant difference between DENV-4 H241 and the JD4 virus. However, neuroinvasiveness was not detected in 3-week-old mice. Neurovirulence of JD4 virus in 3-5-day-old suckling mice was a little weaker than that of DENV-4 H241, but was significantly higher than that of JEV strain SA14-14-2. Neurovirulence in mice was not obviously decreased after 30 passages in PHK cells. Conclusion JD4 was successfully generated based on JEV strain SA14-14-2, and the related neurovirulence and neuroinvasiveness were detected and compared with the parent strains. It could provide a suitable foundation in further study on the safety of dengue vaccine candidate.