Infectivity and cell death of microglia in response to Japanese encephalitis virus

Abstract

Japanese encephalitis virus (JEV), an arbovirus that belongs to the family Flaviviridae, is endemic to large parts of Asia and Pacific. Even though neurons have been proposed to be the principle JEV target cells in the central nervous system, microglia are activated and secreted many mediators in response to JEV infection. Recently, autophagy, a cellular process involved in the degradation of organelles and aggregated long-lived proteins, has been reported to play a significant role in viral infection. This study aimed to determine the involvement of autophagy in response to JEV infection in human microglial (CHME-5) cells. Upon JEV infection at a multiplicity of infection of 10, a small deficit in total cell number, compared to that of mock-infected CHME-5 cells was observed. However, with the multiplicity of infection of 100, a significant reduction in total cell number was observed since day 2 post infection. Interestingly, the percentage of cell death, determined by tryptan blue dye exclusion assay, was relatively the same (less than 10%) in both levels of multiplicity of infection, indicating a slower proliferation rate in JEV-infected microglial culture with a higher multiplicity of infection. By immunocytochemistry, JEV virions were clearly detected in cytoplasm of microglial cells, with an estimated of 78% infectivity at day 2 post infection. Electron micrographs demonstrated autophagosomes with JEV virions. Besides, an increase in the expression of LC3-II in response to JEV infection was detected by indirect immunofluorescence and western blotting. In addition, inhibition of autophagy, using 3-methyladenine, led to a reduction of the JEV production suggesting autophagy mediated viral replication. Collectively, these results indicate that human microglia are susceptible to JEV and autophagy is induced during JEV infection.