Development of the reverse genetics system for viral hemorrhagic septicemia virus genotype IVa and its application in antiviral compound screening

  • Virol Sin. 2025 Dec 27:S1995-820X(25)00177-4. doi: 10.1016/j.virs.2025.12.010.
Hao Huang  1 ,  Xiaobing Lu  2 ,  Tianlai Hong  1 ,  Yihong Chen  3 ,  Meisheng Yi  4 ,  Kuntong Jia  5
Affiliations
  • 1. School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China.
  • 2. School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China; Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519082, China; Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Zhuhai 519082, China.
  • 3. Institute of Modern Aquaculture Science and Engineering (IMASE)/Guangzhou Key Laboratory of Subtropical Biodiversity and Biomonitoring, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China.
  • 4. School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China; Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519082, China; Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Zhuhai 519082, China. Electronic address: [email protected].
  • 5. School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China; Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519082, China; Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Zhuhai 519082, China. Electronic address: [email protected].
PMID: 41461367 DOI: 10.1016/j.virs.2025.12.010
Abstract

Viral hemorrhagic septicemia virus (VHSV) is a major pathogen affecting freshwater and marine fish species, posing a significant threat to global aquaculture. Reverse genetics systems are essential for studying viral replication, and host interactions, as well as developing vaccines and therapeutics. In this study, we developed a reverse genetics platform for VHSVLB2018 strain, a genetically distinct VHSV genotype IVa strain which exhibits low genomic identity with Other Asian isolates, using a dual RNA polymerase I/II transcription vector. We successfully rescued recombinant VHSV in mammalian (B7GG) and fish (FHM and EPC) cell lines, and engineered recombinant VHSV strains expressing EGFP (rVHSV-EGFP) and cherry (rVHSV-Cherry) fluorescent proteins. Phenotypic analysis revealed that unmodified recombinant VHSV (rVHSV) exhibited growth kinetics and virulence similar to the wild-type VHSV, while fluorescent protein-expressing variants showed attenuated replication and virulence, with the rVHSV-EGFP strain displaying the greatest attenuation. Utilizing the rVHSV-EGFP strain, we conducted Antiviral compound screening and identified three promising inhibitors-xanthohumol, octyl gallate, and rottlerin that effectively inhibit VHSV replication. Time-of-addition assays further revealed that xanthohumol and rottlerin targeted the viral replication stage, while octyl gallate interfered with viral internalization. This reverse genetics system provides a versatile platform for studying VHSV pathogenesis, developing live-attenuated vaccines, and screening Antiviral compounds, enhancing our understanding of this pathogen and offering new tools for aquaculture disease management.

Keywords
Viral hemorrhagic septicemia virus (VHSV); antiviral compound screening; reverse genetics system.
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