Channel catfish virus ORF25 and ORF63 genes are essential for viral replication in vitro.

ABSTRACT

The channel catfish virus (CCV) is a lethal pathogen to aquatic animals that can provoke severe haemorrhagic disease in juvenile channel catfish. Although the CCV genome has been fully sequenced, the molecular mechanisms of CCV infection and pathogenesis are less well known. Genomic DNA replication is a necessary and key event for the CCV life cycle. In this study, the impacts of the putative helicase and primase encoded by viral ORF25 and ORF63 on CCV genome replication and infection were evaluated in channel catfish ovary (CCO) cells. The results showed that the number of CCV genome copies was decreased significantly in virus-infected CCO cells after knockdown of ORF25 and ORF63 using RNA interference. In contrast, the overexpression of ORF25 and ORF63 led to slight increase in the number of virus genome copies. Consistent with the above results, the present results also showed that the expressions of CCV true-late genes which strictly depend on viral DNA replication, were significantly increased or repressed by overexpression or RNA interference targeting viral ORF25 and ORF63 genes in virus-infected CCO cells. In addition, knockdown of ORF25 and ORF63 remarkably inhibited CCV-induced cytopathic effects and decreased progeny virus titres in CCO cells. Moreover, transmission electron microscopy observation of CCO cells infected with CCV accompanied by siRNA targeting the viral ORF25 and ORF63 genes showed that the number of virus particles was remarkably reduced. Taken together, these results indicated that ORF25 and ORF63 are essential for regulating CCV genome replication and CCV-induced infection. Our findings will provide an understanding of the replication mechanisms of CCV and contribute to the development of antiviral strategies for controlling CCV infection in channel catfish culture.