Abstract
The Chelonid fibropapilloma-associated herpesvirus (CFPHV; ChHV5) is believed to be the causative agent of fibropapillomatosis (FP), a neoplastic disease of marine turtles. While clinical signs and pathology of FP are well known, research on ChHV5 has been impeded because no cell culture system for its propagation exists. We have cloned a BAC containing ChHV5 in pTARBAC2.1 and determined its nucleotide sequence. Accordingly, ChHV5 has a type D genome and its predominant gene order is typical for the varicellovirus genus within the alphaherpesvirinae. However, at least four genes that are atypical for an alphaherpesvirus genome were also detected, i.e. two members of the C-type lectin-like domain superfamily (F-lec1, F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase (F-sial). Four lines of evidence suggest that these atypical genes are truly part of the ChHV5 genome: (1) the pTARBAC insertion interrupted the UL52 ORF, leaving parts of the gene to either side of the insertion and suggesting that an intact molecule had been cloned. (2) Using FP-associated UL52 (F-UL52) as an anchor and the BAC-derived sequences as a means to generate primers, overlapping PCR was performed with tumor-derived DNA as template, which confirmed the presence of the same stretch of “atypical” DNA in independent FP cases. (3) Pyrosequencing of DNA from independent tumors did not reveal previously undetected viral sequences, suggesting that no apparent loss of viral sequence had happened due to the cloning strategy. (4) The simultaneous presence of previously known ChHV5 sequences and F-sial as well as F-M04 sequences was also confirmed in geographically distinct Australian cases of FP. Finally, transcripts of F-sial and F-M04 but not transcripts of lytic viral genes were detected in tumors from Hawaiian FP-cases. Therefore, we suggest that F-sial and F-M04 may play a role in FP pathogenesis.
Highlights
The Chelonid fibropapilloma-associated herpesvirus (CFPHV; Chelonid herpesvirus 5, ChHV5) is strongly associated with fibropapillomatosis (FP), a neoplastic disease of marine turtles [1]
While it is almost standard procedure to clone Bacterial Artificial Chromosome (BAC) from replication competent herpesviruses, this is, to our knowledge, the first time that a genomic herpesvirus BAC has been created directly from infected tissue and in the absence of any means to propagate the agent in culture [7,18,19,23,24,25]
The nucleotide sequence determination of the BAC revealed that the overall structure of the cloned molecule corresponded in its size (132.233 bp) and configuration (TRS-unique short sequence (US)-inverted repeat sequences (IRS)-Unique Long (UL)) to a Type D genome, which is typical for members of the varicellovirus genus of the alphaherpesvirinae
Summary
The Chelonid fibropapilloma-associated herpesvirus (CFPHV; Chelonid herpesvirus 5, ChHV5) is strongly associated with fibropapillomatosis (FP), a neoplastic disease of marine turtles [1]. Skin tumors are characterized as fibropapillomas [13], whereas internal tumors are characterized as fibromas, myxofibromas or fibrosarcomas of low grade malignancy [10]. All these tumor-types are ChHV5 DNA-positive and have extensive collagen deposition in the extracellular matrix with myxofibromas having deposition of sulfated proteoglycans between collagen fibers [10]. At present, it remains unclear, whether these alterations to the extracellular matrix are being caused by altered metabolism of the fibroblasts by transformation or directly by the function of unidentified viral enzymes that may be secreted from infected cells. Perivascular mononuclear cell infiltration has been observed frequently in the dermal layers of tumors [13]
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