Avian Flavivirus Enters BHK-21 Cells by a Low pH-Dependent Endosomal Pathway

Duck Tembusu virus (DTMUV), a pathogenic member of the Flavivirus family, was first discovered in the coastal provinces of South-Eastern China in 2010. Many previous reports have clearly shown that some Flaviviruses utilize several endocytic pathways to enter the host cells, however, the detailed mechanism of DTMUV entry into BHK-21 cells, which is usually employed to produce commercial veterinary vaccines for DTMUV, as well as of other Flaviviruses by serial passages, is still unknown. In this study, DTMUV entry into BHK-21 cells was found to be inhibited by noncytotoxic concentrations of the agents chloroquine, NH₄Cl, and Bafilomycin A1, which blocked the acidification of the endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of DTMUV virions to pH 5.0 in the absence of host cell membranes decreased entry into cells by 65%. Furthermore, DTMUV infection was significantly decreased by chlorpromazine treatment, or by knockdown of the clathrin heavy chain (CHC) through RNA interference, which suggested that DTMUV entry depends on clathrin. Taken together, these findings highlight that a low endosomal pH is an important route of entry for DTMUV.