ORIGINAL RESEARCH article
Front. Vet. Sci.
Sec. Veterinary Infectious Diseases
Volume 12 - 2025 | doi: 10.3389/fvets.2025.1671591
Development of a reverse genetics system for West Nile Virus (Kunjin type)
Provisionally accepted
Zhen WuTao HuZhou ZhouYu HeTao Wang
Mingshu WangRenyong Jia
Dekang Zhu
Mafeng LiuXinxin Zhao
Qiao YangYing Wu
Shaqiu Zhang
Juan Huang
Xumin OuDi Sun
Bin Tian
Anchun Cheng*
Shun Chen*- Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
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Kunjin virus (KUNV), a naturally attenuated strain of West Nile virus (WNV), shares similar transmission modes and hosts-primarily mosquitoes, birds, and horses. Globally, reverse genetics is the principal methodology for characterising the molecular aetiology of flaviviruses. In this study, cytomegalovirus (CMV) promoter-driven KUNV reporter replicons were engineered to incorporate three distinct reporter genes: Nanoluc, oxGFP, and mCherry. These replicons demonstrated successful translation and replication in mammalian (BHK-21), avian (DEF), and avian hepatic (LMH) cell lines. Additionally, an in vitro pseudovirus packaging system for KUNV was established using helper plasmids expressing either full-length C-prM/E or a truncated C-terminal variant (C 18 -prM/E). Both plasmids efficiently packaged replicon RNA into pseudoviruses, with C 18 -prM/E exhibiting notably higher packaging efficiency than full-length C-prM/E. Furthermore, leveraging a previously developed full-length infectious KUNV clone, a stable reporter virus was generated by inserting the Nanoluc luciferase gene. The reporter virus maintained genomic integrity over five serial passages with no loss of the reporter gene. Collectively, these experiments establish robust in vitro reverse genetics systems for KUNV. These tools constitute valuable molecular resources for investigating the KUNV lifecycle, advancing antiviral drug screening, and facilitating vaccine development.
Keywords: Kunjin virus, Reporter replicon, Packaging system, Reporter virus, Reverse genetics system
Received: 23 Jul 2025; Accepted: 08 Aug 2025.
Copyright: © 2025 Wu, Hu, Zhou, He, Wang, Wang, Jia, Zhu, Liu, Zhao, Yang, Wu, Zhang, Huang, Ou, Sun, Tian, Cheng and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Anchun Cheng, Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
Shun Chen, Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
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