By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.Papillomaviruses (PVs) are a large group of epitheliotropic pathogens that cause proliferations of the stratified squamous epithelium in a wide variety of host species. Ninety-three different types of human PV (HPV), causing a wide spectrum of genotype-specific lesions (22), have been completely genomically characterized (9). Although only a limited number of nonhuman PVs have been fully characterized, they cover a broad range of host species (9,14,16). Considering the numerous partial sequences of putative novel animal PVs published recently (2) and the large number of suspected PV infections in animals, based on clinical, histopathological, electron microscopic, and immunohistochemical findings (14), the PV genotype variety in nonhuman vertebrates might exceed that of the HPVs, and every vertebrate species could have its own set of PVs.The Florida manatee (Trichechus manatus latirostris) belongs to the order of Sirenia and is presently listed as an endangered species. Natural disease in manatees is uncommon (3,7,8), and the majority of manatee deaths are directly or indirectly attributable to human activities, as is the case for collisions with watercrafts and habitat degradation (3). In 1997, seven captive manatees from the Homosassa Springs State Wildlife Park, Homosassa, Florida, developed multiple pedunculated cutaneous papillomas, distributed over the leading contact regions of the anterior body. Four of these animals subsequently developed topically and clinically distinct sessile papillomas which were more diffuse and numerous and frequently showed a linear distribution along lines of scratch marks or trauma. In both lesion types, PVs were suspected as a causative agent, based on histological, ultrastructural, and immunohistochemical data (5). Papillomatous skin lesions have since been observed in free-ranging Florida manatees, and fragmental PV DNA sequences were obtained from these lesions by degenerate primer PCR (