The Joint Center of Translational Precision Medicine, Department of Infectious Diseases, Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, Guangzhou, People's Republic of China.
The Joint Center of Translational Precision Medicine, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China.
Emerg Microbes Infect. 2022 Dec;11(1):123-135. doi: 10.1080/22221751.2021.2016354.
Japanese encephalitis virus (JEV), an important neurotropic pathogen, belongs to the genus of the family and has caused huge threat to public health. It is still obscure regarding the functions of stem-loop (SL) and dumbbell (DB) domains of JEV 3' UTR in viral replication and virulence. In the current study, using the infectious clone of JEV SA14 strain as a backbone, we constructed a series of deletion mutants of 3' UTR to investigate their effects on virus replication. The results showed that partial deletions within SL or DB domain had no apparent effects on virus replication in both mammalian (BHK-21) and mosquito (C6/36) cells, suggesting that they were not involved in viral host-specific replication. However, the entire SL domain deletion (ΔVR) significantly reduced virus replication in both cell lines, indicating the important role of the complete SL domain in virus replication. The revertant of ΔVR mutant virus was obtained by serial passage in BHK-21 cells that acquired a duplication of DB domain (DB-dup) in the 3' UTR, which greatly restored virus replication as well as the capability to produce the subgenomic flavivirus RNAs (sfRNAs). Interestingly, the DB-dup mutant virus was highly attenuated in C57BL/6 mice despite replicating similar to WT JEV. These findings demonstrate the significant roles of the duplicated structures in 3' UTR in JEV replication and provide a novel strategy for the design of live-attenuated vaccines.
日本脑炎病毒（JEV）是一种重要的神经嗜性病原体，属于黄病毒科黄病毒属，对公共卫生造成了巨大威胁。目前，关于 JEV 3'UTR 茎环（SL）和哑铃（DB）结构域在病毒复制和毒力中的功能还不清楚。本研究以 JEV SA14 株的感染性克隆为骨架，构建了一系列 3'UTR 缺失突变体，以研究它们对病毒复制的影响。结果表明，SL 或 DB 结构域内的部分缺失对哺乳动物（BHK-21）和蚊子（C6/36）细胞中的病毒复制没有明显影响，表明它们不参与病毒的宿主特异性复制。然而，完整的 SL 结构域缺失（ΔVR）显著降低了两种细胞系中的病毒复制，表明完整的 SL 结构域在病毒复制中起着重要作用。通过在 BHK-21 细胞中连续传代获得了 ΔVR 突变病毒的回复突变体，该突变体在 3'UTR 中获得了 DB 结构域的重复（DB-dup），大大恢复了病毒复制以及产生亚基因组黄病毒 RNA（sfRNAs）的能力。有趣的是，尽管 DB-dup 突变病毒在 C57BL/6 小鼠中的复制与 WT JEV 相似，但该突变病毒的毒力显著降低。这些发现表明，3'UTR 中重复结构在 JEV 复制中具有重要作用，并为设计活疫苗提供了一种新策略。
