Micropterus salmoides rhabdovirus enters cells via clathrin-mediated endocytosis pathway in a pH-, dynamin-, microtubule-, rab5-, and rab7-dependent manner.

Micropterus salmoides rhabdovirus (MSRV) is an important fish pathogen that infects largemouth bass. To date, the entry process for MSRV remains obscure. Here, the dynamic process of MSRV entry and internalization was analyzed using biochemical inhibitors, RNA interference, and single-virus tracking technology. Accordingly, DiD was used as a fluorescent label for sensitive, long-term tracking of MSRV entry in living cells. The motion analysis suggested that MSRV initially experiences slow movement in the cell periphery, while it undergoes relatively faster and directed motion toward the cell interior, dependent on the microtubule. Besides, our data demonstrated that the MSRV enters epithelioma papulosum cyprinid (EPC) cells via clathrin-mediated endocytosis in a low pH-, dynamin-, and clathrin-dependent manner. Furthermore, after endocytosing into EPC cells, MSRV moves along the classical endosome/lysosome trajectory. This study reveals the entry pathway and intracellular dynamics of MSRV in EPC cells, providing new insights into the infection mechanism of rhabdoviruses. IMPORTANCE Although Micropterus salmoides rhabdovirus (MSRV) causes serious fish epidemics worldwide, the detailed mechanism of MSRV entry into host cells remains unknown. Here, we comprehensively investigated the mechanism of MSRV entry into epithelioma papulosum cyprinid (EPC) cells. This study demonstrated that MSRV enters EPC cells via a low pH, dynamin-dependent, microtubule-dependent, and clathrin-mediated endocytosis. Subsequently, MSRV transports from early endosomes to late endosomes and further into lysosomes in a microtubule-dependent manner. The characterization of MSRV entry will further advance the understanding of rhabdovirus cellular entry pathways and provide novel targets for antiviral drug against MSRV infection.