Members of the alphavirus genus pose a serious or potential threat to public health in many areas of the world. Nearly all alphaviruses are maintained in nature by transmission cycles that involve alternating replication in a susceptible vertebrate and invertebrate host. The maintenance of this transmission cycle depends on the establishment of a life-long persistent infection in the invertebrate vector host. Although alphavirus replication has been extensively studied in vertebrate models, the strand-specific replication kinetics of alphaviruses during persistent infections of the invertebrate host have not been reported. We investigated the strand-specific replication of different alphavirus genotypes in invertebrate cells. By comparing different detection strategies and chemistries, we identified an optimal ssqPCR assay design for strand-specific quantification of viral RNAs in infected cells and tissues. We found that primer sets incorporating the use of a non-target tag sequence were able to avoid real-time PCR detection of amplicons that were falsely-primed during reverse-transcription. We also determined that DNA hydrolysis probes increased the sensitivity of ssqPCR assays when compared to a double-stranded DNA-specific dye, SYBR Green. Using this information, we determined the replication kinetics of two different genotypes of o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) in infected mosquito cells. We found that (-) strand viral RNAs persisted in invertebrate cells for up to 21 days after infection. We also found that significantly less (-) strand RNA was present in cells infected with opal variants of both ONNV and CHIKV than sense variants at several time points post infection, suggesting that the opal codon has a functional role in (-) strand RNA regulation. We also report the development of an ONNV replicon expression system. In total, the tools we developed for this report will facilitate future replication studies in the mosquito that may shed light on questions regarding the regulatory role of the opal codon and the persistence of (-) strand RNAs during long-term infections. The strand-specific replication kinetics of ONNV and CHIKV genotypes reported here will serve as a foundation for such investigations. / Master of Science in Life Sciences

The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.