Abstract
Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic orthoflavivirus that poses a significant global health threat. It causes severe neuroinflammatory disease in humans and reproductive failure in swine. Because of the broad host range and cell tropism of JEV, identifying animal cell lines resistant to infection has been a persistent challenge. In this study, we demonstrate that Madin-Darby bovine kidney (MDBK) cells are resistant to JEV infection yet remain fully permissive to viral replication when transfected with viral genomic RNA. Using immunoblotting, immunofluorescence, and flow cytometry, we show that MDBK cells, unlike the highly susceptible baby hamster kidney (BHK-21) cells used as controls, do not support viral entry but sustain all post-entry stages of the replication cycle. Further investigation confirmed that MDBK cells possess a functional clathrin-mediated endocytic pathway, as evidenced by their susceptibility to bovine viral diarrhea virus, which relies on clathrin-dependent endocytosis for host cell entry. These findings establish MDBK cells as a nonsusceptible cell line for JEV entry despite intact endocytic function, providing a valuable platform for studying virus-host cell interactions and for identifying and validating host cell entry factors, a major challenge in JEV research.