Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Abstract

Bunyamwera virus replication was examined in A. albopictus (mosquito) cell cultures, in which a persistent infection is established, and in cytopathically infected BHK [hamster kidney] cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relativey high titers (.simeq. 107 PFU[plaque-forming units]/ml), and autointerference was not observed. Three virus-specific RNA (L, M and S) and 2 virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA and viral protein synthesis were extremely low, corresponding to < 2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h post-infection and was shut down to barely detectable levels at 24 h post-infection. Virus-specific RNA and nucleocapsid synthesis showed similar patterns of change, but later in infection. The proportions of cells able to release 1 PFU at 3, 6, and 54 days post-infection were 100, 50 and 1.5%, respectively. Titers fell to 103-105 PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus, but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days post-infection and increased gradually until they were 75-95% of the total infectious virus at 66 days post-infection. Temperature-sensitive mutants appeared 23-49 days post-infection. No antiviral activity, similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus, was detected in culture fluids by 3 mo. after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of .simeq. 10. Virus-specific RNA and protein synthesis were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. These results were discussed in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.