Superinfection Exclusion Between Japanese Encephalitis Virus Genotypes I and III Occurs During Viral Entry

Superinfection exclusion occurs when a primary viral infection prevents subsequent infection of the same cell. Japanese encephalitis virus (JEV) comprises several genotypes that differ primarily in their envelope protein sequences, and a shift in predominance from genotype III (GIII) to genotype I (GI) has been observed in nature. However, the mechanisms contributing to this genotype replacement remain unclear. In this study, we examined superinfection exclusion between JEV GI and GIII in mammalian BHK-21 cells and mosquito C6/36 cells. Reciprocal exclusion was observed in both cell types. Further analysis in BHK-21 cells, using GIII as the primary virus and GI as the secondary virus, indicated that inhibition occurred at the viral entry stage. The extent of exclusion increased as the interval between primary GIII infection and secondary GI infection was prolonged, indicating that superinfection exclusion became progressively stronger over time. Quantitative analysis using fluorescently labeled GI virus and RT-qPCR further showed that GI entry was significantly reduced in cells previously infected with GIII compared with mock-infected controls. Transferrin uptake remained readily detectable in JEV-infected cells, suggesting that prior infection did not grossly impair transferrin internalization under the conditions examined. Together, these findings demonstrate superinfection exclusion between JEV GI and GIII and indicate that interference occurs predominantly during viral entry. The potential contribution of this phenomenon to JEV genotype replacement in nature warrants further investigation.