African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis

• We investigated ASFV entry mechanism in fully characterized porcine macrophages, the natural host cell, and compared to vaccinia virus.
• ASFV uses multiple dynamin-dependent endocytic pathways for entry: clathrin-mediated and cholesterol-dependent endocytosis.
• This mode of entry is strongly pH-dependent and involves PI3K.
• In contrast, vaccinia virus entry is clathrin- and pH-independent.

The main cellular target for African swine fever virus (ASFV) is the porcine macrophage. However, existing data about the early phases of infection were previously characterized in non-leukocyte cells such as Vero cells. Here, we report that ASFV enters the natural host cell using dynamin-dependent and clathrin-mediated endocytosis. This pathway is strongly pH-dependent during the first steps of infection in porcine macrophages. We investigated the effect of drugs inhibiting several endocytic pathways in macrophages and compared ASFV with vaccinia virus (VV), which apparently involves different entry pathways. The presence of cholesterol in cellular membranes was found to be essential for a productive ASFV infection while actin-dependent endocytosis and the participation of phosphoinositide-3-kinase (PI3K) activity were other cellular factors required in the process of viral entry. These findings improved our understanding of the ASFV interactions with macrophages that allow for successful viral replication.