Identification of G-quadruplex forming sequences in three manatee papillomaviruses.

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

Molecular characterization of novel mucosotropic papillomaviruses from a Florida manatee (Trichechus manatus latirostris).

We isolated two new manatee papillomavirus (PV) types, TmPV3 and TmPV4, from a Florida manatee (Trichechus manatus latirostris). Two PV types were previously isolated from this species. TmPV1 is widely dispersed amongst manatees and a close-to-root PV; not much is known about TmPV2. The genomes of TmPV3 and TmPV4 were 7622 and 7771 bp in size, respectively. Both PVs had a genomic organization characteristic of all PVs, with one non-coding region and seven ORFs, including the E7 ORF that is absent in other cetacean PVs. Although these PVs were isolated from separate genital lesions of the same manatee, an enlarged E2/E4 ORF was found only in the TmPV4 genome. The full genome and L1 sequence similarities between TmPV3 and TmPV4 were 63.2 and 70.3 %, respectively. These genomes shared only 49.1 and 50.2 % similarity with TmPV1. The pairwise alignment of L1 nucleotide sequences indicated that the two new PVs nested in a monophyletic group of the genus Rhopapillomavirus, together with the cutaneotropic TmPV1 and TmPV2.

Seroepidemiology of TmPV1 infection in captive and wild Florida manatees (Trichechus manatus latirostris).

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.

Six novel gammaherpesviruses of Afrotheria provide insight into the early divergence of the Gammaherpesvirinae.

The Afrotheria represent an early branching of placental mammals. Only two herpesviruses from Afrotheria have been previously identified, and the genus Proboscivirus in the subfamily Betaherpesvirinae has been proposed for them. Six novel gammaherpesviruses were identified in four species in the superorder Afrotheria by detection and analysis of their DNA polymerase genes. Elephantid herpesvirus 3 (ElHV3) and Elephantid herpesvirus 4 (ElHV4) were identified from conjunctival swabs from Asian elephants (Elephas maximus). ElHV3 was also found in a vaginal swab from one elephant with vaginitis. Elephantid herpesvirus 5 (ElHV5) was identified from vaginal swabs of two Asian elephants with vaginal plaques. Elephantid herpesvirus 6 was discovered in a conjunctival swab from an African elephant (Loxodonta africana). Procavid herpesvirus 1 (PrHV1) was found in spleen and conjunctival swabs of rock hyrax (Procavia capensis). Trichechid herpesvirus 1 (TrHV1) was identified from skin and buffy coats of Florida manatees (Trichechus manatus latirostris). ElHV3 and ElHV4 form a distinct cluster, and ElHV5, ElHV6, TrHV1, and PrHV1 form a second cluster. These viruses may have codiverged with their host species. Phylogenetic analysis of these novel herpesviruses suggests that two separate groups of gammaherpesviruses may have codiverged with the Afrotheria.

Molecular identification of a papilloma virus from cutaneous lesions of captive and free-ranging Florida manatees.

Cutaneous papillomatous lesions were biopsied from three captive Florida manatees (Trichechus manatus latirostris) at Homosassa Springs State Wildlife Park (HSSWP), Homosassa, Florida, USA, and from six free-ranging Florida manatees from Crystal and Homosassa rivers, Florida. Total DNA extracted from these lesions was assayed for the presence of papilloma virus genomes using the polymerase chain reaction (PCR) with primers that target the L1 capsid protein gene. The amplification generated DNA fragments 458 base pairs in length that encompassed a highly conserved domain within the L1 capsid protein and translated into identical polypeptides of 152 amino acids, suggesting the involvement of a single papilloma virus genotype. Multiple amino acid sequence and phylogenetic analyses of the L1 fragment indicated that the Florida manatee papilloma virus is a unique and quite distinct papillomavirus from other known papilloma viruses. The emergence of this new pathogen raises concerns about its potential impact on the already endangered Florida manatee.

Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1.

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.

Viral papillomatosis in Florida manatees (Trichechus manatus latirostris).

The Florida manatee (Trichechus manatus latirostris) is one of the most endangered marine mammals in American coastal waters. Naturally resistant to infectious disease, the manatee immune system appears highly developed to protect it against the harsh marine environment and the effects of human-related injury. In 1997, seven captive Florida manatees developed multiple, cutaneous, pedunculated papillomas over a period of 6 months. Approximately 3 years later, four of the seven manatees developed multiple, cutaneous, sessile papillomas topically and clinically distinct from the initial lesions, some of which are still present. Histologic, ultrastructural, and immunohistochemical features indicated that the two distinct phenotypic lesions were caused by papillomaviruses (PVs). Preliminary immunologic data correlated with daily clinical observations suggested that the manatees were immunologically suppressed and that the papillomas were caused by activation of latent PV infections and reinoculation from active infections. The emergence of PV-induced papillomas in captive manatees, the possibility of activation of latent infection or transmission of active infection to free-ranging manatees, and the underlying cause of immune suppression predisposing manatees to develop viral papillomatosis are serious concerns for the future management of this highly endangered species.