The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M
protein of BEFV with
clathrin or
dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic
dye, 3,30-dilinoleyloxacarbocyanine
perchlorate (DiO), further suggesting that BEFV uses a
clathrin-mediated endocytosis pathway. Our results suggest that
clathrin-mediated and
dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a
short hairpin RNA (
shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent
infection in MDBK and Vero cells. Treatment of BEFV-infected cells with
nocodazole significantly decreased the M
protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive
infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV
infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by
clathrin-mediated and
dynamin 2-dependent endocytosis in a pH-dependent manner.