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.

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While both in vivo and in vitro evidence has suggested that liver cells undergo apoptosis in response to dengue virus infection, little is known about the mechanism of induction. Given that the p53 tumour suppressor gene is a key mediator of apoptosis, we sought to define the role of this gene in response to dengue virus infection. After infection, a p53 wild type liver cell line (HepG2) showed changes consistent with apoptosis including alterations of cell morphology, cellular detachment and DNA laddering. However, p53 was neither up-regulated, nor showed any evidence of complexing with dengue virus proteins as determined by immunoprecipitation. Infection of a p53 null liver cell line (Hep3B) also produced changes consistent with the induction of apoptosis. While the profile of the cells undergoing apoptosis in each cell line was similar as determined by flow cytometry, the absolute levels were markedly different with up to 90% of Hep3B cells undergoing apoptosis compared to only 20% of HepG2 cells at day 5 post infection. By day 7, all Hep3B infected cells were dead. In contrast, it proved possible to culture dengue virus infected HepG2 cells for 3 months. Viral progeny released from the p53 null cell line were nine-fold higher per attached cell than from the p53 wild type cell line. These results suggest that, while induction of apoptosis in liver cells is mediated by a non-p53 regulated pathway, p53 may play a role in restricting the level of viral progeny to below a critical level at which apoptosis is triggered.

Despite the availability of effective vaccines, Japanese encephalitis virus (JEV) infections remain a leading cause of encephalitis in many Asian countries. The virus is transmitted to humans by Culex mosquitoes, and, while the majority of human infections are asymptomatic, up to 30% of JE cases admitted to hospital die and 50% of the survivors suffer from neurological sequelae. Microglia are brain-resident macrophages that play key roles in both the innate and adaptive immune responses in the CNS and are thus of importance in determining the pathology of encephalitis as a result of JEV infection.

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