Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry

Abstract

The mode of entry of West Nile virus (WNV) into the macrophage-like cell line P388D1 was investigated at the electron microscopical level using synchronized infections. The presence of the antiviral monoclonal antibody F6/16A at a concentration that enhanced viral attachment to P388D1 cells ninefold made no difference to the entry pathway of WNV. In both the absence and presence of F6/16A the initial uptake of single viral particles was mediated by coated pits, and started within 30 s of warming the cells to 37 degrees C. Viral particles later appeared in fully or partially coated vesicles and later in uncoated prelysosomal endocytic vacuoles before degradation in lysosomes. However, aggregates of viral particles (five or more virus particles in cross-section), appeared to be phagocytosed whole by cells in a process which involved aggregates being engulfed by extensions of the plasma membrane. This process exhibited a slower time course than the uptake of single viral particles, becoming prominent 15 to 30 min after warming the cells to 37 degrees C. The involvement of a prelysosomal vacuolar compartment in the entry process was shown by a failure to stain for acid phosphatase. This compartment could be specifically loaded with viral particles when viral internalization occurred at 20 degrees C in the presence of 50 mM-ammonium chloride.