Development of a Mass Antiviral Screening System Using Viral Hemorrhagic Septicemia Virus as an RNA Surrogate and Activity Confirmation with a Fish Rhabdovirus

With emerging viruses and drug resistance on the rise, the discovery and development of innovative antiviral substances and agents are necessary for the effective treatment and control of viral outbreaks. Surrogate viruses are safer alternatives used in research to mimic dangerous or hard-to-culture viruses. They enable efficient, ethical, and cost-effective screening of antiviral compounds. In this study, we used a recombinant viral hemorrhagic septicemia virus (rVHSV) expressing enhanced green fluorescent protein as a surrogate for RNA viruses for the high-throughput screening of antiviral agents. An optimized mixture of viruses and EPC host cells was distributed in 96-well plates containing chemical compounds or plant extracts for screening. Using this system, 44,642 chemical compounds and 8104 plant and marine organism extracts were tested; 140 candidates were selected from primary screening, and 8 compounds and 5 plant extracts were further selected based on the selectivity index (SI), representing the ratio of the cytotoxic concentration (CC₅₀) to the inhibition concentration (IC₅₀). Among these, compound <b>3</b>, which had the highest SI value of 1046, was further tested, considering in vitro activity against VHSV and another fish rhabdovirus, snakehead rhabdovirus (SHRV).