Channel catfish virus entry into host cells via clathrin-mediated endocytosis

•The cell invasion mechanism of CCV was studied using biochemical inhibitor assays.•CCV entry not by the caveolae-dependent endocytosis pathway and macropinocytosis.•CCV enters via clathrin-mediated endocytosis in a low-pH-dependent manner. Channel catfish virus (CCV), an important member of the family Alloherpesviridae, causes a lethal infection in channel catfish. As with most animal viruses, the initial step of infection by CCV is entry into host cells, which is also a promising antiviral target for CCV disease. This study investigated the mechanism of host cell invasion by CCV using a series of biochemical inhibitor assays in channel catfish cells. CCV infection in host cells was does-dependently inhibited when cells were treated with endosomal acidification inhibitors (5 μM chloroquine, 50 nM bafilomycin A1, and 1 mM ammonium chloride) and hypertonic medium (50 mM sucrose) , which suggests that CCV invades host cells in a manner dependent on low-pH and the endocytic pathway. Moreover, when the cells were pretreated with inhibitors of clathrin-mediated endocytosis, including chlorpromazine (2 μM) and dynasore (50 μM), the CCV infection in the host cells was strongly inhibited. In contrast, the destruction of cellular cholesterol by methyl-β-cyclodextrin and nystatin and inhibition of macropinocytosis had no effect on viral entry. Altogether, these findings indicate that CCV infects host cells via clathrin-mediated endocytosis in a low-pH-dependent manner, suggesting that this CCV entry pathway offers an antiviral target against CCV disease.