Ammonium Produced from Shewanella Strain 0409 Contributes to Antiviral Activity Against Betanodavirus

Nervous necrosis virus (NNV) (genus: Betanodavirus) attacks aquacultured fish. Biocontrol may prevent and control the disease caused by NNV infection. Shewanella strain 0409 was isolated from the micmbiome of grouper intestines, and its culture supernatant (CS0409) could inhibit NNV replication in vitro. The present study elucidated the antiviral mechanism of Shewanella strain 0409 against NNV in vitro. In NNV-infected GF-1 cells, CS0409 reduced both NNV RNA synthesis and the percentage of cells expressing NNV RNA-dependent RNA polymerase (RdRp) within 24 hpi. However, NNV RdRp activity was not inhibited by CS0490 treatment. The most favorable time for CS0409 treatment in NNV-infected GF-1 cells was the period of viral adsorption, which revealed a high inhibition level of NNV RNA synthesis. Shewanella strain 0409 produced ammonium in CS0409. When ammonium in CS0409 was removed by heating with evaporation, the anti-NNV activity of CS0409 decreased. Moreover, ammonium in CS0409 inhibited the acidification of acidic organelles. Furthermore, the morphology of NNV virions changed to empty particles after exposure to pH 5 and 6, indicating that NNV uncoating required the acidic environment of endosomes. Therefore, ammonium in CS0409 is responsible for the anti-NNV activity in vitro by inhibiting the acidification of endosomes and then suppressing NNV uncoating.