Abstract
Damselfish neurofibromatosis is a disease affecting bicolor damselfish (Stegastes partitus) of Caribbean reefs. The causative agent of this disease has been identified and is termed the damselfish virus-like agent (DVLA), a small previously uncharacterized DNA-containing agent whose extrachromosomal DNA genome adopts a complicated secondary structure. This body of work focused on characterizing the RNAs of this agent and determining the role these might play in tumor formation. Five distinctly sized RNAs (0.3, 0.55, 0.8, 1.0 and 1.4 kb) were identified within established cell lines and tissues infected with DVLA. These transcripts were arranged in an overlapping fashion and this arrangement prevented identification of the strand of transcription for all but the largest transcript. Two possible positions were proposed for this 1.4 kb transcript based on the results from the strand analysis. 5' RACE data identified a putative 5' end for one of these 1.4 kb transcripts, asyell as the 5' ends for two of the smaller transcripts (0.3 and 0.8 kb). A 3' end was identified for the other 1.4 kb transcript. This data suggested that both 1.4 kb-sized transcripts were in fact present within infected cell culture lines. Sequence analysis detected the presence of multiple potential open reading frames; however most were of a small size and poorly correlated with the positions of the RNA transcripts. No conserved domains or sequence homology to published protein sequence was found and no DVLA-specific proteins were detected in any analysis suggesting a possible alternative role for these RNAs. The intense secondary structure suggests that these RNAs may act by RNA interference pathways. All transcripts were present within tumor tissue of infected fish but subsets of this complete pattern were found in other non-tumored tissue. Non-tumored tissues were found to contain both DVLA RNA and DNA although this material was lost following growth of the tissue in culture suggesting that infection of these tissues is halted. Analysis of the DVLA RNA in vivo indicated a correlation between the 1.4 kb transcript(s) and the presence of tumors in infected fish possibly suggesting a role of these transcripts in the process of tumorigenesis.