In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas).
Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.
Work
Thierry M
TM
US Geological Survey, National Wildlife Health Center, Honolulu Field Station, PO Box 50167, Honolulu, HI 96850, USA.
Dagenais
Julie
J
US Geological Survey, National Wildlife Health Center, Honolulu Field Station, PO Box 50167, Honolulu, HI 96850, USA.
Balazs
George H
GH
NOAA-National Marine Fisheries Service, Pacific Islands Fisheries Science Center, 2570 Dole Street, Honolulu, HI 96822, USA.
Schumacher
Joanne
J
US Geological Survey, National Wildlife Health Center, Honolulu Field Station, PO Box 50167, Honolulu, HI 96850, USA.
Lewis
Teresa D
TD
Hawaii Institute of Marine Biology, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, PO Box 1346, Kaneohe, HI 96744, USA.
Leong
Jo-Ann C
JC
Hawaii Institute of Marine Biology, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, PO Box 1346, Kaneohe, HI 96744, USA.
Casey
Rufina N
RN
College of Veterinary Medicine, Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853-6401, USA.
Casey
James W
JW
College of Veterinary Medicine, Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853-6401, USA.
D018138:Alphaherpesvirinae / Q000254:growth & development / Q000302:isolation & purification*; D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D005347:Fibroblasts / Q000821:virology; D006254:Hawaii; D006566:Herpesviridae Infections / Q000662:veterinary* / Q000821:virology; D015603:Keratinocytes / Q000821:virology; D010212:Papilloma / Q000662:veterinary* / Q000821:virology; D014426:Turtles / Q000821:virology*; D014775:Virus Activation