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Entry into and production of the Japanese encephalitis virus from C6/36 cells
Verawan Boonsanay1, Duncan R Smith
1Molecular Pathology Laboratory, Institute of Molecular Biology and Genetics, Mahidol University, Nakorn Pathom, Thailand.
Objectives:
The mosquito-borne Japanese encephalitis virus is a leading cause of encephalitis worldwide. However, few studies have investigated the kinetics of Japanese encephalitis virus internalization and production in mosquito cells, and fewer still have investigated the nature of the molecules involved in the binding of the virus to mosquito cells.
Methods:
Using the Aedes albopictus/Stegomyia albopicta-derived C6/36 cell line, Japanese encephalitis virus internalization and production were assayed by standard plaque assay, and virus binding was investigated through preinfection enzymatic treatment of cells and virus overlay protein-binding assay of membrane fractions in native and denaturing gels.
Results:
The internalization of the virus was nonlinear, and intracellular infective viruses were detected 8 h after infection and exported to the medium 10 h after infection. The internalization of the virus was primarily mediated by protein elements, and several bands were observed after overlay assay to membrane proteins, although mass spectroscopy was unable to identify candidate proteins. Soluble laminin produced a marginal, but dose-dependent inhibition of infection.
Conclusions:
These results suggest that the mechanism of Japanese encephalitis entry, production, attachment and receptor usage are distinct from those employed by the dengue viruses.
Insights
Japanese encephalitis virus (JEV) internalization in mosquito cells is protein-mediated and occurs non-linearly. JEV production and release kinetics differ from dengue viruses, suggesting unique entry mechanisms.
Area of Science:
- Virology
- Cell Biology
- Arthropod-borne Viruses
Background:
- Japanese encephalitis virus (JEV) is a significant cause of encephalitis globally.
- Limited research exists on JEV kinetics in mosquito cells and its binding molecules.
Purpose of the Study:
- Investigate JEV internalization and production kinetics in Aedes albopictus C6/36 cells.
- Characterize the molecular basis of JEV binding to mosquito cells.
Main Methods:
- Standard plaque assays for virus internalization and production.
- Enzymatic treatment and virus overlay protein-binding assays for studying virus attachment.
- Analysis of membrane proteins using native and denaturing gels.
Main Results:
- JEV internalization was nonlinear, with detection at 8 hours post-infection and release at 10 hours.
- Virus internalization is primarily mediated by protein components.
- Mass spectrometry failed to identify specific binding proteins, but soluble laminin showed minor inhibitory effects.
Conclusions:
- JEV entry, production, attachment, and receptor usage mechanisms are distinct from dengue viruses.
- Further research is needed to identify specific JEV binding proteins in mosquito cells.
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