Sequence duplication in 3′ UTR modulates virus replication and virulence of Japanese encephalitis virus

ABSTRACT

Japanese encephalitis virus (JEV), an important neurotropic pathogen, belongs to the genus Flavivirus of the family Flaviviridae 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.

KEYWORDS:

• Japanese encephalitis virus
• 3′ UTR
• virus replication
• DB duplication
• viral virulence

Acknowledgements

The authors thank Center for Animal Experiment staff (Xue-fang An, Fan Zhang, He Zhao, and Li Li) at Wuhan Institute of Virology and Wuhan Key Laboratory of Special Pathogens and Biosafety for their helpful supports during the course of the work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This work was supported by National Natural Science Foundation of China: [Grant Number 32100110, 31600141, 32002297]; Guangzhou Municipal Science and Technology Project: [Grant Number 202102020241].