Abstract
Japanese encephalitis virus (JEV) a mosquito‐borne flavivirus is a major cause of viral encephalitis in Asia. While the principle target cells for JEV in the central nervous system are believed to be neurons, microglia are activated in response to JEV and have been proposed to act as a long lasting virus reservoir. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This study sought to identify molecules associated with JEV entry to microglial cells. Virus overlay protein‐binding assay (VOPBA) and liquid chromatography–mass spectrometry (LC/MS/MS) identified the 37/67 kDa high‐affinity laminin receptor protein and nucleolin as a potential JEV‐binding proteins. These proteins were subsequently investigated for a contribution to JEV entry to mouse microglial BV‐2 cells together with other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14, and CD4. In antibody mediated inhibition of infection experiments, both anti‐laminin receptor and anti‐CD4 antibodies significantly reduced virus entry while anti‐Hsp70 and 90 antibodies produced a slight reduction. Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down‐regulation of CD4 and moderate down‐regulation of CD14. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with CD4 playing a major role. J. Med. Virol. 84:615–623, 2012. © 2011 Wiley Periodicals, Inc.