Molecular evolution of fibropapilloma-associated herpesviruses infecting juvenile green and loggerhead sea turtles.

Chelonid Alphaherpesvirus 5 (ChHV5) has long been associated with fibropapillomatosis (FP) tumor disease in marine turtles. Presenting primarily in juvenile animals, FP results in fibromas of the skin, connective tissue, and internal organs, which may indirectly affect fitness by obstructing normal turtle processes. ChHV5 is near-universally present in tumorous tissues taken from affected animals, often at very high concentrations. However, there is also considerable asymptomatic carriage amongst healthy marine turtles, suggesting that asymptomatic hosts play an important role in disease ecology. Currently, there is a paucity of studies investigating variation in viral genetics between diseased and asymptomatic hosts, which could potentially explain why only some ChHV5 infections lead to tumor formation. Here, we generated a database containing DNA from over 400 tissue samples taken from green and loggerhead marine turtles, including multiple tissue types, a twenty year time span, and both diseased and asymptomatic animals. We used two molecular detection techniques, quantitative (q)PCR and nested PCR, to characterize the presence and genetic lineage of ChHV5 in each sample. We found that nested PCR across multiple loci out-performed qPCR and is a more powerful technique for determining infection status. Phylogenetic reconstruction of three viral loci from all ChHV5-positive samples indicated widespread panmixia of viral lineages, with samples taken across decades, species, disease states, and tissues all falling within the same evolutionary lineages. Haplotype networks produced similar results in that viral haplotypes were shared across species, tissue types and disease states with no evidence that viral lineages associated significantly with disease dynamics. Additionally, tests of selection on viral gene trees indicated signals of selection dividing major clades, though this selection did not divide sample categories. Based on these data, neither the presence of ChHV5 infection nor neutral genetic divergence between viral lineages infecting a juvenile marine turtle is sufficient to explain the development of FP within an individual.