Simian virus 40 (SV40) infections of semipermissive human cells and permissive monkey cells were compared. Viral specific events were examined on per cell basis using immunocytochemistry to detect viral antigen-producing cells and in situ hybridization to identify viral DNA-replicating cells. The SV40 infection aborted before T-antigen expression in many cells of each of the human cell lines examined. This was further investigated by in situ hybridizations to probe for early SV40 message. The results showed that the same number of cells synthesize early message as produce T antigen indicating that the block to T-antigen expression is prior to transcription. In all but one of the human cell lines studied, most of the T-antigen-producing cells replicated viral DNA. However, in the A172 line of human glial cells only a small percentage of the T-antigen-expressing cells replicated viral DNA. Different structural and functional classes of T antigen have been characterized using anti-T monoclonal antibodies. Therefore, infected A172 cells were examined with a panel of ten anti-T monoclonal antibodies to determine if viral DNA replication might correlate with the expression of a particular epitope of T antigen. One anti-T monoclonal, PAb 100, did specifically recognize that subset of A172 cells which replicated SV40 DNA. The percentage of PAb 100-reactive A172 cells was dramatically increased by the DNA synthesis inhibitors hydroxyurea and aphidicolin. Removal of the hydroxyurea was followed by an increase in the percentages of cells replicating viral DNA corresponding to the increased percentage reactive with PAb 100. The pattern of SV40 infection in A172 cells was not altered by infection with viable viral mutants containing lesions in the small t protein, the agnoprotein, or the enhancer region. Additional studies on carrier systems of SV40-infected A172 and rhesus cells are also described.

About 400 million individuals worldwide are currently infected with filarial parasites. To find a cure or prevention for the infections, studies of the mechanisms of pathogenesis and the survival stratagies of the parasites are necessary. At this early stage of investigation, the lack of an animal model and an in vitro culture system make it impossible to define the important genes for the parasites by any functional analysis (forward genetics). For the purpose of unraveling the survival strategies of O. volvulus, the best approach is to gain a broad knowledge of the genes that are expressed in the parasite and then select those that may be important for survival (reverse genetics). cDNA libraries of infective third stage larvae of the two major filarial parasites, Onchocerca volvulus and Brugia malyai, were constructed. EST (Expressed Sequence Tag) analysis was perfomed on randomly selected clones. 200 ESTs were analyzed from each cDNA library. Seven identical ESTs out of the first 200 ESTs from the cDNA library of Onchocerca volvulus infective stage larvae were found to be very similar to a newly discovered antioxidant enzyme: Thioredoxin Peroxidase (TPx). Two genes that match with TPx were also identified from the cDNA library of Brugia malayi infective third stage larvae through more EST analysis of the ongoing Brugia malayi genome project. The detoxification ability of parasites has always been interesting to the parasitologist because it may be a parasite survival strategy in the face of host generated oxidative stress. In this thesis, I report my success in characterizing the TPx gene and protein of Onchocerca volvulus and I discuss its possible role in parasite survival. The complete sequence of the TPx cDNA of O. volvulus reveals an ORF (Open Reading Frame) that encodes a polypeptide of 199 amino acid residues with a calculated molecular weight of 21,890 daltons. The TPx mRNA represents roughly 2% of total transcripts in the infective third stage larvae of Onchocerca volvulus. The TPx gene was expressed in the pRSET prokaryotic gene expression system and the expressed product was shown to have antioxidant activity. The antiserum raised against the expressed TPx gene product recognized a native protein from both the infective third stage larvae and adult extract that has a molecular weight of 22 kD, in agreement with the calculated molecular weight. The localization of the O. volvulus TPx protein at different stages of parasite development was detected immunochemically using the O. volvulus TPx specific antiserum. It was found to be surface located in infective third stage larvae, microfilariae and probably young adult worms. Therefore, TPx protein in O. volvulus may protect the parasites from being damaged by host generated oxidative stress. The results of immunohistochemistry combined with the results of in situ hybridization for mRNA detection, support the hypothesis that microfilariae are protected by a layer of TPx protein on their surface which is provided by the adult female worms. Evidence suggests that the expression of the TPx is induced initially inside of the intermediate host (blackfly).