1 Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
2 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Ontario N1G 2W1, Canada
3 Toronto Zoo, Ontario M1B 5K7, Canada
4 The Jackson Laboratory, Bar Harbor, ME 04609-1500, USA
Correspondence
Marc Van Ranst
marc.vanranst{at}uz.kuleuven.ac.be
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession number of the sequence reported in this paper is AY763115.
Supplementary material is available in JGV Online.
Present address: Copenhagen Zoo, Copenhagen, Denmark.
Present address: Kristiansand Zoo, Kristiansand, Norway.
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
Papillomatosis has been documented in a number of carnivores, mainly Canidae and Felidae (Sundberg, 1987; Sundberg et al., 2000
). In 1994, the genomic sequence of the canine oral PV (COPV), associated with oropharyngeal papillomatosis in dogs, coyotes and wolves, was characterized (Delius et al., 1994
). COPV is the largest of all known PV genomes, and contains a unique second non-coding region (NCR2) between the early and late protein region. Recently, a second carnivore PV genome, containing a similar NCR2, was isolated from a cutaneous lesion of a Persian cat (Felis domesticus PV type 1, FdPV-1) (Tachezy et al., 2002a
). COPV and FdPV-1 share a high degree of sequence similarity, and belong to the genus Lambda PV.
Papillomavirus infection has been reported in raccoons (Procyon lotor), small carnivores that are widely distributed throughout North America. In one study, 2 of 53 wild trapped raccoons showed proliferative skin lesions that stained positive for PV group-specific antigens (Hamir et al., 1995). Since dogs and raccoons both belong to the suborder Caniformia of the Carnivora, investigation of the relationship between the canine COPV and the raccoon PV would be interesting to test the hypothesis that PVs have co-evolved with their host species.
We report here the complete genomic sequence and phylogenetic position of the first raccoon PV (Procyon lotor papillomavirus type 1, PlPV-1), isolated from papillomatous skin lesions of an adult free-ranging raccoon that was trapped and euthanized at the Toronto Zoo (Ontario, Canada). This animal showed numerous small dermal proliferations on the palmar surface of both front feet. Histological examination of these skin lesions showed numerous small exophytic epidermal nodules, characterized by epithelial dysplasia, hyperplasia and orthokeratotic hyperkeratosis. Koilocytes, with ballooning degeneration and amphophilic intranuclear inclusion bodies, were present in the stratum granulosum. Total genomic DNA was extracted from a lesion biopsy as described previously (Tachezy et al., 2002a), and amplicons suggestive of PV-specific amplification were generated by PCR using degenerate PV-specific primers (Supplementary material in JGV Online). The PCR products were sequenced with the same primers as used for PCR. Similarity searches, performed with the National Center for Biotechnology Information (NCBI) BLAST (Basic local alignment search tool) server on GenBank DNA database release 132.0 (Altschul et al., 1990
), showed that partial L1 and E1 sequences of a novel PV were amplified. Primers for long template PCR were chosen in these partial E1 and L1 sequences in order to amplify the complete genome of the raccoon PlPV-1 in two overlapping long PCR fragments: RAC1 of approximately 3·7 kb, amplified with forward and reverse primers RAC-longF1 (5'-TATGGGCTGTTCGTGGTTTAGAGATTGGTCGTGG-3') and RAC-longR1 (5'-CTGCGAGTGGCTGCAACCAGAACTGACTCTTGC-3'), and fragment RAC2 of approximately 6·2 kb, amplified with primers RAC-longF2 (5'-GGCTAGAGCTGAGGCAGGTAAGACACTGTTGC-3') and RAC-longR2 (5'-CTACATGTCTCATGTAGCACCTATAGTCTGCAGGG-3'). Long template PCR was performed with the Expand Long Template PCR system (Roche Diagnostics). The RAC1 and RAC2 PCR products were purified on a 0·8 % agarose gel with crystal violet staining, and isolated from the gel by using SNAP purification columns (TOPO XL PCR Cloning kit; Invitrogen). The fragments were ligated into a pCR-XL-TOPO vector, followed by transformation into One Shot TOP10 competent cells (TOPO XL PCR Cloning Kit; Invitrogen). The bacteria were selectively grown on LuriaBroth agar plates containing 50 µg kanamycin ml1, and one clone containing the 3·7 kb RAC1 PCR fragment and one containing the 6·2 kb PCR fragment were selected. The complete nucleotide sequences of the cloned RAC1 and RAC2 PCR products were determined by primer-walking sequencing, starting from the universal primers in the pCR-XL-TOPO vector, and using 32 primers to cover the two fragments on both strands. Sequencing was performed on an ABI Prism 3100 Genetic Analyser (Perkin-Elmer Applied Biosystems), chromatogram sequencing files were inspected with Chromas 2.2 (Technelysium), and contigs were prepared using SeqMan II (DNASTAR). The complete nucleotide sequence of the PlPV-1 genome consists of 8170 bp, and was deposited in GenBank under the accession no. AY763115.
PlPV-1 has the fourth largest PV genome, after COPV (8607 bp; GenBank accession no. NC_001619), deer DPV (8374 bp; NC_001523) and FdPV-1 (8300 bp; AF480454), all of which contain two non-coding regions (Delius et al., 1994; Groff & Lancaster, 1985
; Tachezy et al., 2002a
). PlPV-1 contains the seven classical PV major ORFs, encoding five early (E) proteins E1, E2, E4, E6 and E7, and two late (L) capsid proteins L1 and L2. The exact location of the PlPV-1 ORFs and the molecular mass of the predicted proteins are indicated in Fig. 1
. The position of the first nucleotide of the PlPV-1 genome was fixed corresponding to the start of the first major ORF in the early region.
|
|
The classic non-coding region (NCR1) between the stop codon of L1 and the start codon of E6 consists of 494 bp in PlPV-1, from nt 7797 to 120. PVs usually contain an E1 recognition site (E1BS) flanked by two E2-binding sites, for binding of an E1/E2 complex in order to activate the origin of replication. In the PlPV-1 NCR1, an E1BS (TTATTGTTGTTAACAAT) is present at nt 1026. Three typical E2-binding sites (E2BS) with the consensus sequence ACC-N6-GGT are present at nt 79727983, 80938104 and 5566. Three additional modified E2-binding sites (E2BS*), one with the sequence AAC-N6-GGT at nt 7384, and two with the sequence ACC-N6-GCT, at nt 81478158 and 80118022, could be identified through comparison of the NCR1 regions of PlPV-1, COPV and FdPV-1. Since the putative E2BS* at position 81478158 and the E2BS at 5566 are equidistant to the E1BS, this E2BS* might be functionally important, although it is possible that the modifications could result in lower affinity binding. In its 5' end, the NCR1 also contains a polyadenylation site (AATAAA, nt 78897894), 20 bp upstream of a CA dinucleotide, and the G/T cluster, necessary for the processing of the L1 and L2 capsid mRNA transcript. In the 3' end, NCR1 contains a TATA box of the E6 promoter present at nt 43.
The PlPV-1 genome contains a second non-coding region (NCR2) of 1065 bp (nt 36664730) between the early and the late protein region. A similar NCR2 has previously only been characterized in COPV (Delius et al., 1994) and FdPV-1 (Tachezy et al., 2002a
), both also belonging to the genus Lambda, and DPV, which belongs to the genus Delta (Groff & Lancaster, 1985
). It is tempting to speculate that the presence of an NCR2 is a common feature of all Lambda PVs, and that the NCR2 of all three Lambda PVs could originate from a DNA sequence (of unknown function and origin) that was incorporated in their common ancestor. Since this is a non-protein-coding region, numerous point mutations, insertions and deletions have accumulated in this sequence during evolution, and a possible common origin is no longer recognizable above this background. A BLAST search with the PlPV-1 NCR2 failed to detect similarity with the NCR2 of FdPV-1 and COPV, or any other known sequences in GenBank. In contrast to the NCR1, the NCR2 contains no recognizable E1BS and E2BS. There is no classical polyadenylation site for processing of the early viral mRNA transcripts present in this NCR2, but several degenerate polyadenylation sites, followed at an appropriate distance of about 20 bp by a CA dinucleotide, were detected.
A neighbour-joining phylogenetic tree was constructed, based on a concatenated E1/E2/L2/L1 nucleotide sequence alignment of PlPV-1 and 45 type species of the different PV genera and species. Multiple nucleotide sequence alignments were performed at the amino acid level by using the CLUSTALW program (Thompson et al., 1994) in the DAMBE software package version 4.2.7 (Xia & Xie, 2001
), after which the nucleotide sequences were aligned according to the aligned amino acid sequences. This was done separately for the different ORFs, and the regions of the genome that contained unambiguously alignable homologous positions (four regions in E1, three in E2, four in L2 and seven in L1) were pasted together in one compiled alignment of 2658 nt. The resulting neighbour-joining phylogenetic tree (Fig. 2
), which was constructed in MEGA version 2.1 (Kumar et al., 2001
), clusters the PVs in the different genera described in the new PV classification (de Villiers et al., 2004
), and the additional Rho and Sigma genera (Rector et al., 2004
; Rector et al., 2005
). In this tree, PlPV-1 clusters with COPV and FdPV-1 in the genus Lambda.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Bininda-Emonds, O. R., Gittleman, J. L. & Purvis, A. (1999). Building large trees by combining phylogenetic information: a complete phylogeny of the extant Carnivora (Mammalia). Biol Rev Camb Philos Soc 74, 143175.[CrossRef][Medline]
Delius, H., Van Ranst, M. A., Jenson, A. B., zur Hausen, H. & Sundberg, J. P. (1994). Canine oral papillomavirus genomic sequence: a unique 1·5-kb intervening sequence between the E2 and L2 open reading frames. Virology 204, 447452.[CrossRef][Medline]
de Villiers, E. M., Fauquet, C., Broker, T. R., Bernard, H. U. & zur Hausen, H. (2004). Classification of papillomaviruses. Virology 324, 1727.[CrossRef][Medline]
Flynn, J. J. & Nedbal, M. A. (1998). Phylogeny of the Carnivora (Mammalia): congruence vs incompatibility among multiple data sets. Mol Phylogenet Evol 9, 414426.[CrossRef][Medline]
Groff, D. E. & Lancaster, W. D. (1985). Molecular cloning and nucleotide sequence of deer papillomavirus. J Virol 56, 8591.[Medline]
Hamir, A. N., Moser, G., Jenson, A. B., Sundberg, J. P., Hanlon, C. & Rupprecht, C. E. (1995). Papillomavirus infection in raccoons (Procyon lotor). J Vet Diagn Invest 7, 549551.[Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 12441245.
Paterson, A. M. & Banks, J. (2001). Analytical approaches to measuring cospeciation of host and parasites: through a glass, darkly. Int J Parasitol 31, 10121022.[CrossRef][Medline]
Rector, A., Bossart, G. D., Ghim, S. J., Sundberg, J. P., Jenson, A. B. & Van Ranst, M. (2004). 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. J Virol 78, 1269812702.
Rector, A., Tachezy, R., Van Doorslaer, K., MacNamara, T., Burk, R. D., Sundberg, J. P. & Van Ranst, M. (2005). Isolation and cloning of a papillomavirus from a North American porcupine by using multiply primed rolling-circle amplification: the Erethizon dorsatum papillomavirus type 1. Virology 331, 449456.[CrossRef][Medline]
Sundberg, J. P. (1987). Papillomavirus infections in animals. In Papillomaviruses and Human Disease. Edited by K. Syrjänen, L. Gissmann & L. G. Koss. Berlin: Springer.
Sundberg, J. P., Van Ranst, M., Burk, R. D. & Jenson, A. B. (1997). The nonhuman (animal) papillomaviruses: host range, epitope conservation, and molecular diversity. In Human Papillomavirus Infections in Dermatovenereology. Edited by G. Gross & G. von Krogh. Boca Raton: CRC Press.
Sundberg, J. P., Van Ranst, M., Montali, R. & 8 other authors (2000). Feline papillomas and papillomaviruses. Vet Pathol 37, 110.
Sundberg, J. P., Van Ranst, M. & Jenson, A. B. (2001). Papillomavirus infections. In Infectious Diseases of Wild Mammals. Edited by E. S. Williams & I. K. Barker. Ames: Iowa State University Press.
Tachezy, R., Duson, G., Rector, A., Jenson, A. B., Sundberg, J. P. & Van Ranst, M. (2002a). Cloning and genomic characterization of Felis domesticus papillomavirus type 1. Virology 301, 313321.[CrossRef][Medline]
Tachezy, R., Rector, A., Havelkova, M., Wollants, E., Fiten, P., Opdenakker, G., Jenson, B., Sundberg, J. & Van Ranst, M. (2002b). Avian papillomaviruses: the parrot Psittacus erithacus papillomavirus (PePV) genome has a unique organization of the early protein region and is phylogenetically related to the chaffinch papillomavirus. BMC Microbiol 2, 19.[CrossRef][Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Van Ranst, M., Kaplan, J. B. & Burk, R. D. (1992). Phylogenetic classification of human papillomaviruses: correlation with clinical manifestations. J Gen Virol 73, 26532660.[Abstract]
Van Ranst, M., Kaplan, J. B., Sundberg, J. P. & Burk, R. D. (1995). Molecular evolution of papillomaviruses. In Molecular Basis of Virus Evolution. Edited by A. Gibbs, C. H. Calisher & F. Garcia-Arenal. Cambridge: Cambridge University Press.
Xia, X. & Xie, Z. (2001). DAMBE: software package for data analysis in molecular biology and evolution. J Hered 92, 371373.
Received 3 January 2005;
accepted 18 March 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |