In this study, genetic analyses were performed to study the interaction of RNA silencing and viral infection in Drosophila melanogaster. Genetic mutants containing loss-of-function mutations in the key RNA silencing pathway genes, dicer-2 (dcr-2) or r2d2, exhibited enhanced disease susceptibility (EDS) to two evolutionarily diverse viruses: FHV and cricket paralysis virus (CrPV). Both viruses accumulated to higher levels in dcr-2 and r2d2 mutant flies than wild type (wt) flies. Thus, my findings established a key role for the dcr-2-dependent siRNA pathway in the antiviral immunity in adult D. melanogaster. In the subsequent study, FHV viral transcripts or virions were microinjected into embryos to investigate the mechanism of antiviral silencing pathways. Small RNA blots showed that FHV viral-derived siRNAs (viRNAs) from infected embryos were mostly Dcr-2-dependant and that Dcr-1 and Ago-2 played no detectable role in the biogenesis of viRNAs. Cloning and sequencing data showed that approximately equal ratios of (+) and (-) strand viRNAs were identified from S2 cells infected with a B2-deficient mutant of FHV (FHVΔB2). The majority of viRNAs were specifically located in the 5'-terminal approximate 400 nucleotide region of FHV RNAs only when no functional B2 was expressed. It was suggested that the 5' nascent dsRNA replicative intermediates formed during initiation of viral progeny RNA synthesis may trigger Dcr-2-dependent antiviral immunity in D. melanogaster and that this triggering process of the viral immunity is suppressed by B2.

Finally, the pathogenesis and infectivity of FHVΔB2 in adult flies were analyzed. FHVΔB2 virions could not accumulate in wt flies, but it could be partially rescued in adult flies containing loss-of-function mutations in the antiviral siRNA pathway genes. This viral mutant then provided me with a useful tool to analyze the possible antiviral roles of other RNA silencing genes. I found that replication of FHVΔB2 resulted in detectable viral accumulation in Loquacious (loqs) mutant flies and in homozygous dcr-1 and ago-1 mutant embryos. Further, the accumulation of FHVΔB2 was also rescued in S2 cells by over-expression of Loqs, known to physically interact with Dcr-1 and Dcr-2. These results suggested that, besides dcr-2, r2d2 and ago-2, other genes in the RNA silencing pathway including dcr-1, ago-1 and loqs, could also be involved in antiviral immunity against FHV. In contrast, FHVΔB2 could not be rescued in flies deficient in any one of the piRNA pathway genes: aubergine (aub), spendle-E (spn-E) or armitage ( armi). The interaction of FHV infection and transposon silencing mediated by piRNA was also examined in this study.

In summary, my studies described in this dissertation establish a D. melanogaster model for studying the innate immunity against viruses in animals. Dcr-2/r2d2/Ago-2-dependent small RNA pathway play key roles in antiviral immunity; other genes in other small RNA pathways may be also involved in the control or regulation of viral infection in D. melanogaster .