Using electron microscopy to observe ultrastructural changes occurring in BHK-21 cells during cellular infection by Japanese encephalitis virus

Abstract

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus and a leading cause of viral encephalitis in Asia, remodels host cellular membranes extensively during its life cycle. Although classical literature describes JEV budding into the endoplasmic reticulum (ER), accumulating within ER-derived vesicles, and maturing through the secretory pathway, detailed electron microscopy (EM) analyses have been severely limited. BHK-21 cells are widely used for propagating JEV and have served as a model for several virological studies. However, comprehensive ultrastructural characterization of steps in JEV life cycle including egress pathways is still unknown in this cell line. In this study, we have employed resin-embedded ultramicrotomy and room-temperature transmission electron microscopy to visualize JEV-infected BHK-21 cells across multiple stages of infection. Our analyses reveal ER-derived membrane remodeling, including vesicle packets and multilamellar compartments consistent with flavivirus replication organelles, along with accumulation of progeny virions within dilated ER cisternae. We also identify single membrane enclosed vesicles which transport assembled virions in addition to cellular cargo, suggesting that JEV utilizes more than one vesicle-mediated egress route for virion trafficking. These findings expand the limited ultrastructural dataset available for JEV and offer comparative perspectives on flavivirus pathogenesis across different host cell environments.

Keywords: Flavivirus egress; Japanese encephalitis virus; Room temperature thin-section ultramicrotomy; Transmission electron microscopy.