PRELIMINARY MOLECULAR CHARACTERIZATION OF MANATEE PAPILLOMA VIRUS FROM SKIN LESIONS OF THE FLORIDA MANATEE
(Trichechus manatus latirostris)

R. A. Woodruff¹, R. K. Bonde² and C. H. Romero^1
1Department of Pathobiology, University of Florida, Gainesville, Florida
²U.S. Geological Survey, Gainesville, Florida

Presented at the International Association of Aquatic Animal Medicine Conference,
10-14 May 2003, Waikaloa, Hawaii.

Abstract

Skin lesions suggestive of papillomatosis were sampled from captive and free-ranging Florida manatees (Trichechus manatus latirostris) from Crystal River and Homosassa River, Florida. Total DNA was extracted from these lesions and amplified by polymerase chain reaction (PCR) using primers MY09 and MY11 routinely used in humans for the diagnosis of papilloma virus (HPV) infection in cervical carcinomas. These primers targeted sequences located within the L1 capsid protein gene and amplified a DNA fragment of 458-bp that translated into a peptide of 152 amino acid residues. GenBank searches identified this peptide as a highly conserved domain within the late L1 capsid protein of manatee papilloma virus (TmlPV). A second DNA fragment of approximately 1.5 kb was unexpectedly obtained, using the same set of primers, from skin lesions that had been conserved in DMSO for at least a year.  Sequencing of one of these fragments showed that it contained the open reading frame (ORF) that codes for the E6 oncogenic early protein gene known to bind the p53 tumor suppressor protein in addition to the L1 capsid protein fragment. These preliminary results provide molecular evidence of the association of a papilloma virus, unique to the Florida manatee, with papillomatous skin lesions seen in captive and free- ranging manatees.   

Introduction

The Florida manatee (Trichechus manatus latirostris) is one of the most threatened marine mammals in U.S. waters and is listed as endangered at both the state and federal levels.⁴ Its highly developed immune system is believed to play a very important role in the protection against the harsh marine environment and possibly, natural disease.² While the majority of manatee deaths are due to watercraft casualties, a large number of deaths remain undetermined.³

Recently, the first natural viral infection of manatees was documented and reported as caused by a papilloma virus.² Microscopic lesions indicative of papillomatosis and the presence of numerous particles in the nuclei characteristic of papilloma viruses were observed in skin lesions of seven captive manatees in Homosassa Springs State Wildlife Park, Homosassa, Florida. These manatees developed raised, firm, whitish-gray lesions distributed over the leading contact regions of the anterior body, including the pectoral flippers, upper lips, external nares, and periorbital regions.² Papilloma viruses of terrestrial mammals are small (~52-55 nm), double-stranded DNA viruses associated with the development of skin and mucosal tumors in animals and humans.⁵ Immunohistochemical staining with polyclonal antibodies raised against bovine papilloma virus (BPV-1) has shown the presence of papilloma virus structural antigens within the nuclei of koilocytes and other keratinocytes of the manatee's stratum granulosum.²

Thus, papilloma virus infections in captive manatees have been well documented. In the present report, we extend these findings and provide molecular evidence of the presence of papilloma viruses in skin lesions obtained from free-ranging manatees. The polymerase chain reaction (PCR) followed by direct sequencing of amplified DNA fragments and sequence analyses were used to confirm the presence of papilloma viruses in skin lesions clinically diagnosed as papillomatosis.

Materials and Methods

     Skin lesion samples

Skin lesions clinically indicative of papilloma virus infection were sampled from four captive Florida manatees (Trichechus manatus latirostris) housed in Homosassa Springs Wildlife State Park (HSSWP), Homosassa, Florida.  All four samples (V369, V374, V375, and P31) were obtained from adult female Florida manatees.  Samples V369, V374, V375 and P31 had been collected in 1998 and preserved in 10% neutral buffered formalin at room temperature until extracted in January, 2003.  Recently, samples were harvested from papillomatous skin lesions of two free-ranging Florida manatees (V378 and V396). Sample V378 was obtained from a 250 cm in length adult male manatee in Crystal River, FL. Sample V396 was harvested from a male manatee calf in Homosassa River, FL. Fresh samples of skin lesions from these two manatees were chilled in ice, transported to the laboratory and total DNA was extracted and stored frozen at – 80^oC. Samples V378 and V396 were obtained and extracted in January, 2003, and February, 2003, respectively.

     Extraction of total DNA from skin lesions 

Total DNA was extracted from the entire skin lesion (~25 mg) using the DNeasy tissue kit (Qiagen) following the manufacturer's protocol. Tissues were grinded in 1.5 ml micro centrifuge tubes and suspended in 180 µl of digestion buffer ATL, 20 µl proteinase K and incubated at 55°C overnight. Then, 200µl of buffer AL and 200 µl of 100% ethanol were added to the samples and the mixture run through a DNeasy spin column to bind the DNA. The membrane was then washed with 500µl of buffer AW1 and 500µl of buffer AW2. Finally, 200 µl of buffer AE was added to the membrane and centrifuged to elute the DNA. The final DNA product was quantitated by spectrophotometry and stored frozen at -80°C.

     Papillomavirus PCR

Total DNA extracted from skin lesions suggestive of papillomatosis was assayed by PCR targeting the L1 capsid protein gene with primers MY09 and MY11¹. These primers amplify a DNA fragment of ~ 458-bp when used with DNA from most of the more than 85 types of human papilloma viruses described. The PCR reaction mixture in a final volume of 100µl contained 200 nM of each primer, 2 mM MgCl₂, 100 M dNTP's, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and 2 units of Taq DNA polymerase. Approximately 500ng to 1g of total DNA was incorporated in the PCR reaction. After an initial denaturation at 94°C for 1 min, reactions were subjected to 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min.  A final cycle incorporated an extension at 72°C for 10 min. Small aliquots of the amplified DNA fragments were resolved by horizontal electrophoresis in 1.2 % agarose containing ethidium bromide (0.5 g/ml) and visualized by transillumination under UV light using a gel documentation system.

   DNA sequencing

Amplified PCR fragments were resolved by electrophoresis in low melting point (LMP) agarose (1.0 %), and purified with the Wizard SV Gel kit (Promega).  Approximately 200 fmol of total DNA was sequenced using the forward and reverse primers and the Beckman Coulter 2000 XL sequencing instrument. Sequences were confirmed using the Chromas software (Technelysium), and the sequences analyzed using the Gap, Seqed, Translate, and Lineup functions of the GCG Wisconsin Package (University of Wisconsin) and the EditSeq and MegAlign functions of the Lasergene software (DNASTAR, Inc.).

click figures to enlarge

click figures to enlarge

Results

PCR diagnosis of papilloma virus infections

Targeting the L1 capsid protein gene

• PCR amplification of total DNA extracted from skin lesions (V369, V375, and P31) of captive manatees and free-ranging manatees (V378 and V396) utilizing primers MY09 and MY11 yielded DNA fragments 458-bp in size. 
   
• Sequencing of the 458-bp fragments from these five samples yielded nucleotide sequences that were 100% identical. These sequences translated correctly from the second nucleotide of the fragments on the bottom DNA strand (Figure 1).
   
• Comparison of the deduced amino acid sequence of the L1 capsid protein fragment of TmlPV to several homologous fragments from mammalian papilloma viruses showed 58.4-65.3 % homology to several types of HPV and 58.0-61.6 % homology to non-human papilloma viruses. Comparison of several types of HPV to TmlPV revealed the following percentages of amino acid homology: HPV-68 (65.3 %), HPV-18 (63.6 %), HPV-1 (62.6 %), HPV-5 (61.8 %), HPV-13 (61.6 %), HPV-11 (61.1 %), HPV-59 (60.2 %), and HPV-16 (58.4 %).  Comparison of TmlPV to non-human papilloma viruses revealed the following percentages of amino acid homology: Pigmy chimpanzee PV (61.6 %), Bovine PV-1 (61.3 %), Rhesus monkey PV (60.9 %), Equine PV (59.6 %), European elk PV (58.3 %), and Ovine PV-1 (58.0 %). A phylogenetic tree shows the genetic relatedness among these papilloma viruses based on the L1 capsid amino acid sequences (Figure 5).

Targeting the E6 oncogenic protein gene

• PCR amplification of total DNA extracted from skin lesions of captive manatee samples V374 and V375 using primers MY09 and MY11 yielded 1.5 Kb DNA fragments in addition to the 458-bp fragment of the L1 protein gene (Figure 6).
   
• Partial sequencing of the 1.5 Kb fragments from sample V374 revealed that the fragment contained the gene that codes for the open reading frame (ORF) of the E6 oncogenic gene.
   
• Comparison of the amino acid sequence of the E6 gene fragment of TmlPV to homologous fragments from several mammalian papilloma viruses showed 36.2-50.7 % homology to several types of HPV and 29.1-42.0 % homology to non-human papilloma viruses. Comparison of the E6 fragment of TmlPV to several types of HPV revealed the following percentages of homology: HPV-5 (50.7 %), HPV-16 (46.4 %), HPV-13 (44.9 %), HPV-1a (43.5 %), HPV-18 (43.5 %), HPV-68 (43.5 %), HPV-59 (40.6 %), and HPV-11 (36.2 %). Comparison of TmlPV E6 fragment to several non-human papilloma viruses revealed the following percentages of homology: Phocoena spinipinnis PV (42.0 %), Rhesus monkey PV (40.6 %), Pigmy chimpanzee PV (37.7 %), Ovine PV (36.4 %), Equine PV (33.9 %), and European elk PV (29.1 %). A phylogenetic tree shows the genetic relatedness among these papilloma viruses based on the E6 oncoprotein amino acid sequences (Figure 3).

Figure 6.  Gel electrophoresis of TmlPV PCR fragments

Discussion

Primers utilized for PCR amplification of HPV DNA in the screening for cervical carcinomas in humans¹ have been successfully applied for the detection of TmlPV DNA in skin lesions of captive and free-ranging Florida manatees. Primers MY09 and MY11 target the L1 capsid protein gene of human papilloma viruses¹ and also the homologous L1 protein gene of TmlPV on which they direct the amplification of DNA fragments 458-bp in size that translate into a peptide of 152 amino acid residues.

Our results have shown that TmlPV is not only a viral infection of captive manatees, but also an infection of free-ranging manatees in Florida waters. The implications of these findings on the general health and as an added factor on the long-term survival of the already endangered Florida manatee remain to be uncovered. The discovery of this infection in captive Florida manatees is a recent event² and more time is needed to evaluate the effects of the observed severe skin lesions on the manatee's welfare and on its potential to cause carcinomas, reminiscent of those observed in humans, especially in the case of HPV-16 and HPV-18⁵ . Sequence analyses of both the L1 and E6 gene fragments have indicated that TmlPV is a unique papilloma virus, different from papilloma viruses reported in humans and non-human mammals and, possibly, specific and indigenous to the Florida manatee. The deduced amino acid sequences of the L1 gene fragments in tissues from three captive and two free-ranging manatees were 100.0 % identical (Figure 1), suggesting the existence of only one type of TmlPV in Crystal River and Homosassa Springs, Florida. Amino acid sequence analyses of the L1 gene fragment revealed that the TmlPV is distinct from the L1 protein of other human and non-human papilloma viruses (Figure 4). Furthermore, the analyses of the L1 fragment of TmlPV showed greatest homology to the mucosal HPV-68 (65.3 %) and HPV-18 (63.6 %), and to the cutaneous HPV-1 (62.6 %), papilloma viruses frequently associated with cervical and oropharyngeal carcinomas (HPV-18, HPV-68) and with cutaneous warts (HPV-18) in humans⁵. A second DNA fragment ~ 1.5 Kb in size was also unexpectedly amplified with primers MY09 and MY11 from skin lesions that had been preserved in DMSO. Partial nucleotide sequence analyses have revealed that the 1.5 Kb fragment contains the open reading frame (ORF) that codes for the E6 oncogenic early protein known to bind to the p53 tumor suppressor protein in humans.⁵ Comparison of the deduced amino acid sequence derived from the E6 fragment of TmlPV to homologous fragments of several human and non-human papilloma viruses showed that the TmlPV is a distinct papilloma virus of the Florida manatee (Figure 2), confirming results obtained with the L1 capsid protein fragment. Comparison of the deduced amino acid sequence of the E6 protein fragment showed greatest homology with HPV-5 (50.7 %), HPV-16 (46.4 %), and HPV-13 (44.9 %), results that are distinct from those obtained with the L1 protein fragment. These discrepancies could possibly be explained by different evolutionary rates of these two different genes that code for the external capsid protein L1, a late protein, and for the internal E6 oncoprotein, an early protein. In the present work, the finding of nucleotide sequences corresponding to the L1 and E6 genes in skin lesions of captive and free-ranging manatees supports the association of  papilloma virus infection with the development of papillomas in the skin of the Florida manatee.

References

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3. Florida Marine Research Institute (FMRI). 2002. "Yearly manatee mortality summary report." St. Petersburg, Florida.
4. Florida Fish and Wildlife Conservation Commission (FWC). 2002. "Biological status review of the Florida manatee." St. Petersburg, Fl.
5. zur Hausen, H. 2000. J. Natl Cancer Inst.92: 690-698.