Molnupiravir: A Prodrug of the Ribonucleoside β‐D‐N4‐Hydroxycytidine Triphosphate Inhibits the Propagation of Nervous Necrosis Virus in Grouper Brain Cells

ABSTRACT Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis, otherwise known as viral encephalopathy and retinopathy in larval and juvenile marine fish worldwide. The pandemic outbreaks have caused nearly 100% mortality in hatcheries, leading to significant economic losses in the aquaculture industry. Since NNV attacks the insufficient immune competence fish at early developmental stages, there is an urgent need for effective antiviral drugs. Several nucleoside analogues, including Molnupiravir, have been developed to target viral RNA‐dependent RNA polymerase (RdRp). This study examines the antiviral activity of Molnupiravir against NNV isolated from giant grouper (GG), Epinephelus lanceolatus (Bloch), using grouper brain (GB) cells as an infection model. The inhibition concentration for 50% of maximal effect (EC 50 ) of Molnupiravir on GGNNV propagation in GB cells was determined as 1.87 μM. Surprisingly, the titre of GGNNV reduced 50,000 at 100 μM of Molnupiravir treatment at 4‐day post‐infection, whereas treatment with 200 μM of Molnupiravir resulted in a 10 5 reduction in viral titre. The inhibition of viral replication and translation was further examined using RNA‐fluorescence in situ hybridization (FISH) and immunocytochemistry detection, respectively. Moreover, administration of 100 μM Molnupiravir at early time 0 hpi resulted in a dramatic decrease in viral load, reducing cell‐associated virus by approximately 4000‐fold and released virus by 6000‐fold. Finally, the molecular docking simulation model supports the interaction between NHC‐TP and GGNNV RdRp for the successful therapeutics. These results demonstrated that Molnupiravir is a very effective therapeutic drug for controlling viral nervous necrosis caused by NNV infection.