Mosquito cells infected with Japanese encephalitis virus release slowly-sedimenting hemagglutinin particles in association with intracellular formation of smooth membrane structures

Arthropod-borne flaviviruses can grow in both arthropod and mammalian cells. Virion morphogenesis, though well studied in mammalian cells, is still unclear in arthropod cells. Here, we compared a mosquito cell line C6/36 and a mammalian cell line Vero in extracellular virus particles and intracellular ultrastructures triggered by infection with Japanese encephalitis virus (JEV). Sedimentation analyses of virion and slowly-sedimenting hemagglutinin (SHA) particles released by infection with the Nakayama strain revealed that C6/36 cells produced higher envelope (E) antigen levels in the SHA than the virion fraction in contrast to Vero cells that showed the opposite pattern. Specific infectivities per ng of E were similar in both cells, whereas specific hemagglutinating activities in the SHA fraction were lower in C6/36 than Vero cells. The precursor membrane protein was less efficiently cleaved to the membrane protein in SHA particles released from C6/36 than Vero cells. Ultrastructural studies showed more remarkable production of smooth membrane structures (SMSs) in C6/36 than in Vero cells. The differences in sedimentation patterns of extracellular virus particles between Nakayama-infected C6/36 and Vero cells were consistently observed in 5 other strains (Beijing P1, Beijing P3, JaTH-160, KE-093 and JaGAr-O1), except for KE-093-infected C6/36 cells which exhibited the Vero-type sedimentation profile under conditions of open cultivation. By electron microscopy, the production of SMSs from KE-093-infected C6/36 cells under open conditions was markedly less than that under closed conditions where the cells exhibited the C6/36-type sedimentation profile. Thus, intracellular SMS formations were associated with extracellular SHA production in JEV-infected mosquito cells.