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Download or read book Identification and Characterization of Novel Caenorhabditis Nematode Viruses written by Carl Joseph Franz and published by . This book was released on 2014 with total page 165 pages. Available in PDF, EPUB and Kindle. Book excerpt: The model organism, Caenorhabditis elegans, has been used in basic research for the last 50 years, providing a wealth of knowledge in a range of fields spanning genetics, development, neurobiology, and computational biology just naming a few. However, only in the last ~15 years has this model organism been broadly considered for investigating host-pathogen interactions. During this time a number of bacterial, fungal, and microsporidial pathogens were shown to infect C. elegans; however, until recently, no natural viruses were known, resulting in a limited understanding of virus-host interactions in C. elegans. In lieu of a known C. elegans virus, several studies were conducted by establishing an unnatural virus infection or creating transgenic viral replicon systems. In 2011, we along with our collaborators reported the discovery of the first known viruses that naturally infect Caenorhabditis nematodes. This dissertation encompasses the identification and characterization of the first viruses known to naturally infect C. elegans and its closest known relative, C. briggsae. Using Next generation 454 pyrosequencing, a novel virus (Orsay virus) was found in a wild isolate of C. elegans (JU1580) and 2 novel viruses (Santeuil and Le Blanc viruses) were found in wild isolates of C. briggsae (JU1264 and JU1498). Phylogenetic analysis revealed the closest known relatives of Orsay, Santeuil, and Le Blanc were viruses in the family Nodaviridae: viruses that naturally infect insects (alphanodaviruses) and fishes (betanodaviruses). Comparative genome analysis revealed substantial differences in genome size and genome organization between the Caenorhabditis viruses and nodaviruses. For instance, the Caenorhabditis viruses have bigger genomes partly due to the presence of a larger 5' untranslated region (UTR) in the RNA1 segment and an additional open reading frame (ORF delta) in the RNA2 segment. While ORF delta has no known function, evidence demonstrates it is translated fused to the capsid protein by a slippery sequence-mediated frameshift. An immunofluorescence assay (IFA) was established targeting viral proteins in infected Caenorhabditis nematodes. Viral protein could be detected as early as 6 hours post-infection (hpi) and the number of infected animals steadily rose between 6 to 12 hpi. In both Orsay virus-infected C. elegans and Santeuil and Le Blanc virus-infected C. briggsae, the site of infection localized primarily to the intestinal tissue. Strikingly, only 1 to 6 cells of the total 20 nonregenerating intestinal cells were positive by IFA for viral protein, suggesting that infection is confined to a subset of the intestine. A transcriptional profile analysis was carried out to investigate which host genes in Caenorhabditis nematodes were responding to viral infection, yielding ~200 genes that were specifically induced by virus in C. elegans. These candidate genes were then individually subjected to RNA interference revealing several characterized and uncharacterized genes that conferred an increased infection phenotype upon knockdown and may play a previously undescribed role in antiviral defense. The discovery and characterization of these novel Caenorhabditis nematode viruses lay the groundwork for the introduction of C. elegans as a powerful model organism to study virus-host interactions.