Replication of Bunyamwera Virus in Aedes albopictus C6/36 Cells: Establishment and Maintenance of a Persistent Infection

Bunyamwera virus is a member of the Bunyaviridae, a large family of mainly arthropod-borne viruses which possess a tri-segmented RNA genome comprising three segments of single-stranded RNA, designated L (large), M (medium) and S (small). There are five recognised genera within the Bunyaviridae-Bunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. Bunyamwera virus is the prototype virus of the Bunyavirus genus and each of its genome segments are of negative polarity. In common with other arboviruses Bunyamwera virus replicates in its vector species (the mosquito) without causing overt detrimental effects and establishes a persistent infection which lasts for the life time of the mosquito. Upon transmission to a susceptible vertebrate host, Bunyamwera virus causes an acute infection. These disparate host-dependent outcomes of infection can be reproduced in tissue culture. Bunyamwera virus undergoes a lytic replication cycle in mammalian cells which results in cell death, but establishes a long-term, non-cytocidal, persistent infection in cultures of mosquito cells. In this thesis, the molecular mechanisms leading to the establishment of persistent Bunyamwera virus infection of a cloned mosquito cell line, Aedes albopictus C6/36 (C6/36) cells, and the factors responsible for the maintenance of the persistent infection, were investigated. Viral protein and RNA synthesis were compared in BHK and C6/36 cells infected with Bunyamwera virus. In BHK cells, host protein synthesis was inhibited and viral protein synthesis was detected until the cells died. In C6/36 cells, host protein synthesis continued throughout the infection, but viral protein synthesis declined from maximum levels at 24 h pi to barely detectable levels at 36 h pi, despite the presence of translatable S mRNA. The levels of encapsidated L and M RNAs in the C6/36 cells declined after 24 h pi, but encapsidated S RNA species continued to accumulate. In the BHK cell infection, encapsidated L, M and S RNA species continued to accumulate up to 48 h pi. Overall, relatively more S segment RNA than L or M segment RNA accumulated in infected C6/36 cells compared to BHK cells. The decline in viral protein syrithesis and genome replication observed in the C6/36 cells after 24 h pi, was paralleled by an accumulation of encapsidated S mRNA. Encapsidation of S mRNA may have a role to play in preventing the cytopathic effects of Bunyamwera virus infection in C6/36 cells by limiting translation of this viral mRNA. A persistent infection of C6/36 cells was established with a biologically cloned stock of Bunyamwera virus (wtL9BUN virus) and the persistently infected cell line (C6/36/BUN) was passaged at weekly intervals for over a year without cytopathic effects. The titre of virus released from the cells, the susceptibility of the cells to superinfection with homologous virus, and the level of viral RNA in the cells at different passages fluctuated markedly, but there was no simple relationship between virus titre, superinfectibility and levels of viral RNA. There was an alteration in the plaque-phenotype of virus released from the persistently infected culture with increasing passage level. By passage 15 the virus released from the C6/36/BUN cell line produced small, cloudy plaques on BHK cell monolayers, in contrast to the clear lytic plaques produced by the wtL9BUN virus. Direct RNA sequence analysis was carried out on the S RNA segments of viruses plaque-purified from the supernatant culture fluid of the C6/36-PI LO cell line established by Elliott and Wilkie. Only one nucleotide substitution, in a non-conserved region of the viral genome, was detected in viral isolate BUN18.10 (which was isolated from the persistently infected culture after it had been passaged for four months) suggesting that genetic drift during persistent Bunyamwera virus infection of C6/36 cells was minimal. However, Northern blot analysis of viral RNA extracted from different passage levels of the C6/36/BUN cell line revealed that polymerase errors which generated subgenomic L RNAs, and S RNA species larger than the standard S RNA segment, did occur in the persistently infected cells. The defective viral RNA species were not efficiently encapsidated by N protein. There was a strong selection for encapsidation of full-length S RNAs in the persistently infected cells and the majority of intracellular nucleocapsids contained standard-sized S RNA species. Stable cell lines were established with cells cloned from the persistently infected C6/36/BUN cell line. The cloned cells were heterogeneous in their ability to produce virus, and their susceptibility to superinfection with wtL9BUN virus, and in the levels of intracellular viral RNA they contained. Individual clones contained a single prominent defective L RNA species and resistance of the cells to superinfection appeared to correlate more with the levels of full-length viral genomic RNA resident in the cells than with the amount or occurence of defective viral RNAs