Xing Jinchao, Zhang Youyue, Lin Ziying, Liu Lele, Xu Qiang, Liang Jiaqi, Yuan Zhaoxia, Huang Cuiqin, Liao Ming, Qi Wenbao
Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.
National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China.
Front Vet Sci. 2021 Aug 2;8:703147. doi: 10.3389/fvets.2021.703147. eCollection 2021.
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV is maintained in an enzootic cycle and causes reproductive failure in pigs. Notably, the shift in JEV genotypes is not fully protected by existing vaccines, so the development of a candidate vaccine is urgently needed. In this study, we compared pathogenicity between Japanese encephalitis virus SA14 and BJB (isolated from humans in the 1970s) strains. We found that the BJB strain was attenuated in mice and that there was no case fatality rate. The growth rate of BJB was higher than SA14 virus in BHK-21 cells. Based on the sequence alignment of the viral genome between the SA14 and BJB virus strains, some mutations at sites 248, 254, 258, and 307 were observed in the 3' untranslated region (3'UTR). The 3'UTR of JEV plays a very important role in the viral life cycle. Furthermore, using a reverse genetic system, we conducted and rescued the parental JEV strain SA14 (T248, A254, and A258) and the mutant virus rSA14-3'UTRmut (T248C, A254G, A258G, and 307G). Through an analysis of the RNA secondary structure model of the 3'UTR, we discovered that the mutations of T248C, A254G, and A258G reduced the apiculus ring and increased the lateral ring significantly in the stem-loop structures IV (SL-IV) structure region of 3'UTR. Moreover, the insertion of 307G added a ring to the dumbbell structure 1 (DB1) structure region. Strikingly, these RNA secondary structure changes in 3'UTR of rSA14-3'UTRmut increased viral negative chain RNA production and enhanced the replication ability of the virus in BHK-21 cells. However, mouse experiments illustrated that the rSA14-3'UTRmut virus significantly decreased the neurovirulence of JEV. These results affirmed that the JEV SL-IV and DB1 regions play an important role in viral proliferation and pathogenicity. Taken together, we complement the study of RNA element function in the 3'UTR region of JEV by providing a new target for the rational design of live attenuated candidate vaccines and the increase of virus production.
日本脑炎病毒（JEV）是一种由蚊子传播的黄病毒，可导致人类致命的神经系统疾病，是具有公共卫生意义的最重要的新兴病原体之一。JEV维持在动物疫源循环中，并导致猪的繁殖失败。值得注意的是，现有疫苗不能完全保护JEV基因型的转变，因此迫切需要开发候选疫苗。在本研究中，我们比较了日本脑炎病毒SA14株和BJB株（20世纪70年代从人类分离）之间的致病性。我们发现BJB株在小鼠中减毒，且无病死率。BJB在BHK-21细胞中的生长速度高于SA14病毒。基于SA14和BJB病毒株之间病毒基因组的序列比对，在3'非翻译区（3'UTR）的248、254、258和307位点观察到一些突变。JEV的3'UTR在病毒生命周期中起着非常重要的作用。此外，我们使用反向遗传系统构建并拯救了亲本JEV株SA14（T248、A254和A258）和突变病毒rSA14-3'UTRmut（T248C、A254G、A258G和307G）。通过对3'UTR的RNA二级结构模型分析，我们发现T248C、A254G和A258G突变在3'UTR的茎环结构IV（SL-IV）结构区域显著减少了尖环并增加了侧环。此外，307G的插入在哑铃结构1（DB1）结构区域增加了一个环。引人注目的是，rSA14-3'UTRmut的3'UTR中的这些RNA二级结构变化增加了病毒负链RNA的产生，并增强了病毒在BHK-21细胞中的复制能力。然而，小鼠实验表明rSA14-3'UTRmut病毒显著降低了JEV的神经毒力。这些结果证实JEV的SL-IV和DB1区域在病毒增殖和致病性中起重要作用。综上所述，我们通过为减毒活候选疫苗的合理设计和病毒产量的提高提供新靶点，补充了JEV 3'UTR区域RNA元件功能的研究。
