2. Academic Validation
  3. Construction and application of infectious cDNA clone, subgenomic replicon and packaging system for Zika virus and Dengue virus

Construction and application of infectious cDNA clone, subgenomic replicon and packaging system for Zika virus and Dengue virus

  • J Virol Methods. 2025 Jun 18:338:115207. doi: 10.1016/j.jviromet.2025.115207.
Yu He 1,  Yibin Tang 2,  Xiaoli Wang 2,  Zhen Wu 1,  Tao Wang 3,  Mingshu Wang 3,  Renyong Jia 3,  Dekang Zhu 3,  Mafeng Liu 3,  Xinxin Zhao 3,  Qiao Yang 3,  Ying Wu 3,  Shaqiu Zhang 3,  Juan Huang 3,  Bing Tian 3,  Xumin Ou 1,  Di Sun 3,  Anchun Cheng 4,  Shun Chen 5
Affiliations

Affiliations

  • 1 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Agricultural Bioinformatics, Ministry of Education, Chengdu, Sichuan 611130, China.
  • 2 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
  • 3 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
  • 4 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China. Electronic address: chenganchun@vip.163.com.
  • 5 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Agricultural Bioinformatics, Ministry of Education, Chengdu, Sichuan 611130, China. Electronic address: shunchen@sicau.edu.cn.
PMID: 40541574 DOI: 10.1016/j.jviromet.2025.115207
Abstract

Flaviviruses pose a significant global health threat due to their rapid spread and potential to cause severe clinical manifestations. Comprehending the mechanisms of replication of these pathogens and developing effective Antiviral strategies are essential for combating these pathogens. In the present study, full-length infectious cDNA clones were generated for Zika virus (ZIKV) and Dengue Virus (DENV), respectively. Recombinant viruses were successfully produced by transfecting cDNA clone plasmids. Using the infectious clone, ZIKV and DENV subgenomic replicons were also generated, which lack the prM-E gene and instead expressing a luciferase or fluorescent marker (OXGFP or mCherry). The replicons exhibited efficient replication in BHK-21 cells. Through the utilization of ZIKV and DENV-2 replicons that express luciferase, three potential Antiviral agents were identified. These agents demonstrated activity against DENV-2 and ZIKV, while not inducing significant cytotoxic effects. This demonstrates the significance of these replicons in the screening of Antiviral agents. Moreover, DENV-2 and ZIKV single-round infectious particles (SRIPs) were produced by co-transfecting packaging plasmids and replicons into BHK-21 cells. The packaging assay demonstrated that flaviviruses can utilize the prM-E protein from Other species to generate SRIPs. The specific binding of the nucleocapsid (NC) to the prM-E protein varies among different flaviviruses. The reverse genetics tools established in this study will facilitate research on DENV-2 and ZIKV virus replication and the development of Antiviral medications targeting these two arboviruses.

Keywords

Antiviral assay; Flavivirus; Packaging system; Reverse genetics system; Subgenomic replicon.

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