Download Sequence and classification of FdPV2, a papillomavirus isolated

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Year: 2009
Sequence and classification of FdPV2, a papillomavirus isolated
from feline Bowenoid in situ carcinomas
Lange, C E; Tobler, K; Markau, T; Alhaidari, Z; Bornand, V; Stöckli, R; Trüssel, M;
Ackermann, M; Favrot, C
Lange, C E; Tobler, K; Markau, T; Alhaidari, Z; Bornand, V; Stöckli, R; Trüssel, M; Ackermann, M; Favrot, C
(2009). Sequence and classification of FdPV2, a papillomavirus isolated from feline Bowenoid in situ carcinomas.
Veterinary Microbiology, 137(1-2):60-65.
Postprint available at:
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Posted at the Zurich Open Repository and Archive, University of Zurich.
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Originally published at:
Veterinary Microbiology 2009, 137(1-2):60-65.
Sequence and classification of FdPV2, a papillomavirus isolated
from feline Bowenoid in situ carcinomas
Abstract
Bowenoid in situ squamous cell carcinoma (BISC) is a rare feline skin disorder, which has been
described as often associated with papillomavirus infection. It is clinically characterized by solitary or
multiple hyperkeratotic plaques affecting older cats. Papillomavirus (PV) sequences amplified from
feline viral plaques, and BISC lesions seldom correspond to FdPV1. The goal of the present study was
to investigate three cases of BISC and to carry out initial genomic analysis of the associated viral DNA.
Samples of skin biopsies taken from three BISC cats were histologically characterized. DNA was
extracted and rolling-circle amplification was performed on the skin samples. Restriction enzyme
analysis of the amplified DNA revealed the presence of a putative unknown PV. The whole genome was
subsequently sequenced and cloned. Alignments with previously described feline PV sequences were
carried out and phylogenetic trees were generated. The circular 7899 base pair sequence of Felis
domesticus PV type 2 (FdPV2) contains a typical noncoding region and characteristic open reading
frames (ORF) for six putative viral proteins. Phylogenetic analysis based on the nucleotide alignment of
L1 genes or the amino acid alignment of E1 proteins of FdPV2 and 52 other PV types indicates that
FdPV2 might represent a new genus.
1 Sequence and classification of FdPV-2, a papillomavirus isolated from feline Bowenoid
2 in situ carcinomas
3 4 C. E. Langea,b,*, K. Toblerb, T Markauc, Z. Alhaidarid, V. Bornande, R. Stöcklif, M. Trüsselg,
5 M. Ackermannb and C. Favrota
6 7 8 a
9 Switzerland
Dermatology Unit, Clinic for Small Animal internal Medicine, Vetsuisse Faculty, Zurich,
10 b
Institute of Virology, Vetsuisse Faculty, Zurich, Switzerland
11 c
Small animal practice Markau and Lauer, Im Rudert 14, 35043 Marburg, Germany
12 d
Veterinary hospital, Route Nationale 85, 06330 Roquefort les pins, France
13 e
Institut of veterinary pathology, Vetsuisse Faculty, Berne, Switzerland
14 f
Cyto/Histodiagnostics, Freienstein, Switzerland
15 g
16 * Corresponding author. Tel.: +41 44 6358489; E-mail address: [email protected]
Small animal practice, Friedhofstrasse 21, Bremgarten, Switzerland
1
17 Abstract
18 Bowenoid in situ squamous cell carcinoma (BISC) is a rare feline skin disorder, which has
19 been described as often associated with papillomavirus infection. It is clinically characterized
20 by solitary or multiple hyperkeratotic plaques affecting older cats. PV-sequences amplified
21 from feline viral plaques, and BISC lesions seldom correspond to FdPV1.
22 The goal of the present study was to investigate three cases of BISC and to carry out initial
23 genomic analysis of the associated viral DNA.
24 Samples of skin biopsies taken from three BISC cats were histologically characterized. DNA
25 was extracted and rolling circle amplification was performed on the skin samples. Restriction
26 enzyme analysis of the amplified DNA revealed the presence of a putative unknown PV. The
27 whole genome was subsequently sequenced and cloned. Alignments with previously
28 described feline PV sequences were carried out and phylogenetic trees were generated.
29 The circular 7899 base pair sequence of Felis domestius PV type 2 (FdPV-2) contains a
30 typical noncoding region and characteristic open reading frames (ORF) for six putative viral
31 proteins. Phylogenetic analysis based on the nucleotide alignment of L1 genes or the amino
32 acid alignment of E1 proteins of FdPV-2 and 52 other PV types indicates that FdPV-2 might
33 represent a new genus.
34 35 Keywords: Bowenoid in situ squamous cell carcinoma; Papillomavirus; cat; Phylogenetic
36 analysis
37 Introduction:
38 Papillomaviruses (PVs) are small non-enveloped, double-stranded DNA viruses with mucosal
39 and skin tropism, and are associated with various hyperplastic, dysplastic and neoplastic
2
40 conditions in humans and animals. More than 100 human PVs have been isolated, cloned and
41 sequenced completely, and many more partially (de Villiers et al., 2004). New tools in
42 molecular biology such as broad range PCR assays and multiply primed rolling-circle
43 amplification (RCA) as well as the increasing interest in animal PVs have permitted the
44 detection and characterization of several new animal PVs (Munday et al., 2007; Nespeca et
45 al., 2006; Rector et al., 2008; Rector et al., 2005a; Rector et al., 2004; Rector et al., 2005b;
46 Stevens et al., 2008; Tobler et al., 2006; Tobler et al., 2008).
47 Bowenoid in situ squamous cell carcinoma (BISC) is a rare feline skin disorder that is often
48 associated with papillomavirus infection (Munday et al., 2007; Nespeca et al., 2006; Wilhelm
49 et al., 2006). This condition is clinically characterized by solitary or multiple hyperkeratotic
50 plaques affecting older cats (Baer and Helton, 1993; Gross et al., 2005; Miller et al., 1992).
51 BISC has been shown to sometimes occur in individuals with viral plaques, another PV-
52 associated condition (Wilhelm et al., 2006). Although some lines of evidence suggest that
53 PVs could play an active role in the development of both conditions, it is currently still
54 uncertain whether PV infection causes lesion development (Munday et al., 2007; Nespeca et
55 al., 2006; Wilhelm et al., 2006). DNA from several different PVs has been isolated from
56 lesioned skin. Differences in these DNA sequences suggest that more than one virus could be
57 involved in the underlying pathogenesis of those conditions (Munday et al., 2007; Nespeca et
58 al., 2006).
59 Although PV genomes have been isolated from several species of the family Felidae, namely
60 Lynx rufus (LrPV1), Pantera leo (PlPV1), Puma concolor (PcPV) and Uncia uncia (UuPV1),
61 the entire genomic DNA sequence has thus far been determined for only one feline PV, i.e.,
62 FdPV1 (Sundberg et al., 2000; Tachezy et al., 2002; Terai and Burk, 2002). Of note, PV
63 sequences that have been amplified from other feline viral plaques and BISC lesions do not
64 correspond to FdPV1 (Munday et al., 2008; Munday et al., 2007; Nespeca et al., 2006).
3
65 The goal of the present study was to investigate three additional cases of BISC and to carry
66 out genomic analysis. Taking advantage of the circular organization of the PV genome,
67 rolling circle amplification was carried out. Restriction enzyme digestion demonstrated that
68 the sequence corresponds to an unknown PV. The whole genome was subsequently
69 sequenced and cloned. Alignments with previously described feline, as well as with other PV
70 sequences, were carried out. Based on the L1 nucleotide and the E1 protein sequences,
71 phylogenetic trees were generated.
72 Materials and Methods:
73 Animals and samples:
74 Three cats presented with pigmented plaques in three different veterinary practices in Europe,
75 namely Marburg, Germany, Roquefort-les-pins, France and Bremgarten, Switzerland (Table
76 1). In order to make a diagnosis, 6 mm skin biopsies were taken. These skin samples were
77 sent to three different histopathology laboratories and processed routinely (haematoxylin &
78 eosin staining). Additionally, in cases one and two, because of the suspicion of viral infection,
79 additional samples were frozen immediately after excision and sent to the Dermatology Unit
80 of the Vetsuisse Faculty in Zurich, Switzerland. In case three, paraffin-embedded tissue was
81 sent for virological analysis after histological examination.
82 Virological analyses:
83 Amplification and cloning of the genome.
84 Total DNA from 25 mg tissue samples (cases one and two) and paraffin-embedded tissue
85 (case three) of three cats was isolated using a DNeasy extraction kit (Qiagen) according to the
86 manufacturer's recommendations. DNA (1 µl) was used for RCA (Rector et al., 2004), using a
87 TempliPhi Amplification kit (General Electrics Biosciences). The protocol supplied by the
88 manufacturer was used, with slight modifications: 1 µl of 10 mM dNTPs was added and the
4
89 reaction time was prolonged to 16 h at 30°C. Amplified DNA was cloned into the XbaI or the
90 ClaI site of pBluescript II-KS+ (Stratagene) using standard procedures.
91 Sequence analysis.
92 The nucleotide sequence of cloned DNA and the precipitated RCA product was determined
93 commercially (Microsynth) by cycle sequencing using an ABI 377 sequencer (Applied
94 Biosystems). The primary sequences were assembled using Contigexpress (Vector NTI
95 Informax). Multiple sequence alignments using CLUSTAL_X version 1.83 (Jeanmougin et
96 al., 1998) were calculated with the default parameters (nucleotide: UIB DNA matrix; amino
97 acid: Gonnet 250 matrix; nucleotide and amino acid: pair-wise: gap-open 10.0, gap-extd. 0.1,
98 multiple: gap-open 10.0, gap-extd. 0.2). Bootstrapped (1000 datasets) phylogenetic trees were
99 constructed with PHYLIP version 3.63 (Felsenstein, 2008), sequentially using the programs
100 SEQBOOT, either DNADIST (F84 matrix, transition/transversion 2.0) or PROTDIST (JTT
101 matrix), NEIGHBOR, CONSENSE and DRAWGRAMM. Pairwise sequence alignments
102 were made with Needle (EMBOSS; nucleotide: UIB matrix, amino acid: PAM250 matrix;
103 nucleotide and amino acid: gap-open 10.0, gap-extd. 0.1). The nucleotide sequence data of
104 FdPV2 were deposited in GenBank under accession no. EU796884.
105 PCR
106 To specifically detect FdPV2 DNA, a primer pair amplifying an 858 nt fragment of the L1
107 ORF was used (A16: 5'-T T C A G C A A A C A C G G A C A A A G-3' and A37: 5'-C G G
108 G C A A C C T C A A A G T A A G A-3'). Amplification was carried out in a PTC-200
109 thermo cycler (MJ Research, Watertown, MA, USA) under conditions of 94°C for 3 min
110 followed by 45 cycles of 95°C 30 for sec, 55°C for 30 sec and 72°C for 30 sec.
111 Electrophoresis in a 1% agarose gel containing ethidium bromide was used to detect the
112 amplified fragment.
5
113 114 Results:
115 Clinical and histological data:
116 Table 1 provides an overview of the BISC cases. All three cats developed round,
117 hyperpigmented lesions with surface crusting on the face (figure 1), the neck and in one case
118 the paws.
119 Histological examination of samples from the three cats revealed similar changes. The
120 epidermis was irregularly hyperplastic (with rete ridges formation) and presented ortho- and
121 parakeratotic hyperkeratosis. Both epidermis and hair follicles were affected by a loss of
122 nuclear polarity; hyperchromatic nuclei were visible in the basal and suprabasal layers. In all
123 three samples, some koilocytes (cells with shrunken, irregular nuclei surrounded by clear
124 spaces) were present (figure 2). A diagnosis of Bowenoid in situ carcinoma was made. All
125 laboratories were in agreement with the classification of BISC.
126 127 Amplification and sequencing reveal papillomavirus-like DNA
128 DNA extracted from the samples was amplified by RCA as described in the Methods. The
129 resulting concatemeric DNA was digested with a set of restriction enzymes, including EcoRI
130 and XbaI, to find a potential single-recognition site that would allow cloning of the
131 papillomavirus genome in its entirety. However, such a site was not initially found (data not
132 shown). Therefore, a subgenomic XbaI fragment was cloned. Digestion of RCA-amplified
133 DNA with XbaI resulted in two (5314 and 2585 bp) fragments; the smaller one was cloned
134 into pBluescript II-KS+. Both strands of the cloned fragment were sequenced. The non-cloned
135 sequence of the putative papillomavirus DNA was obtained by primer-walking on the RCA
136 product. ClaI was subsequently identified as a single cutter and used to clone the whole
6
137 genome into pBluescript II-KS+. This was confirmed by partial sequencing of the clone using
138 the standard primers T7 and M13r as well as by restriction-enzyme analysis of the cloned
139 DNA using ClaI, EcoRI, HindIII, and XbaI.
140 Whole sequence analysis confirmed that papillomavirus DNA had actually been detected; this
141 DNA consisted of 7899 bp and had a GC content of 53%. Open reading frame (ORF) analysis
142 revealed the presence of four long and two short ORFs. These ORFs potentially encode the
143 E1, E2, L2, L1, E6 and E7 proteins, respectively (Fig 3). Deduced amino acid sequences of
144 the putative proteins revealed an ATP-dependent helicase motif (GPPNTGKS) in E1, two
145 putative metal-binding motifs (CX2CX29CX2C) in E6, one such motif in E7, and one pRb
146 binding domain (LXCXE) (Münger et al., 2004; Wilson et al., 2002). The deduced amino acid
147 sequences of both structural proteins L1 and L2 were predicted to harbour a basic tail at their
148 C termini. Furthermore, the long control region (LCR) between the stop-codon of the L1 gene
149 and the start-codon of the E6 gene was 615 bp in length. The XbaI sites used for cloning were
150 located at the beginning of the ORF encoding the E1 protein and in the middle of the ORF
151 encoding the E2 protein; the ClaI site used for cloning of the whole sequence was located in
152 the LCR.
153 In addition, papillomavirus-specific DNA motifs were identified in the novel genome
154 sequence. Eight consensus sequences for E2 binding (ACCN6GGT) (Androphy et al., 1987)
155 were detected; five of these were located in the LCR (positions 7628–7639, 7654–7665,
156 7749–7760, 7801–7812 and 7875–7886) and three were located within the putative E1 ORF
157 (positions 804–815, 1123–1134 and 1208–1219). Within the LCR, a putative origin of DNA
158 replication was identified, consisting of two E2-binding regions (positions 7801–7812 and
159 7875–7886) flanking an A/T-rich region (positions 7814–7835) and dyad-symmetry repeats
160 (TTGTTGTTAACAACAA; positions 7836–7851). Within the 5' end of the L2 ORF, a
161 polyadenylation consensus sequence (AATAAA) for processing the early protein pre-mRNA
7
162 was identified at positions 4315–4320. Thus, all important elements present in the
163 papillomavirus genome were detected. Therefore, the PV with the presently determined
164 sequence was termed FdPV type 2 (FdPV2: Felis domesticus PV2).
165 166 Analysis of the other two samples
167 The DNA isolated from the three cases was compared using restriction enzyme digest
168 analysis of RCA products and FdPV2-specific PCR. The restriction patterns of sample 1 and
169 sample 2 were identical upon digestion with ClaI, EcoRV, NdeI and XbaI (Fig. 4), but RCA
170 failed in case 3. To confirm the presence of the same virus in all three samples, PCR was
171 performed on DNA extracted from the skin samples of all three cases. This amplification with
172 the primer pair A16/A37 was successful for all three samples (table 1). Subsequent
173 sequencing of the RCA product in case 2 and PCR product in case 3 with the primers used for
174 amplification also revealed a DNA sequence identical to that detected in the entirely
175 sequenced case 1 sample. Overall, the results indicate the presence of FdPV2 DNA in all
176 three cases.
177 178 Comparison with PV sequences obtained from cats
179 Several published yet partial PV sequences isolated from cats were compared with the
180 complete genome of FdPV2 to assess the recent finding. In one study, we recently reported
181 the amplification and sequencing of 8 PV specific sequences (E1 gene) from samples of skin
182 biopsies of cats showing signs of invasive SCC or BISC (Nespeca et al., 2006). Pairwise
183 alignments of 7 of these sequences (GenBank DQ085782, DQ085783, DQ085784,
184 DQ085785, DQ085786, DQ085787, DQ085788) with the corresponding part of the FdPV2
185 genome reveals identities below 60%. Interestingly, one sequence (GenBank DQ085789)
8
186 showed a nucleotide sequence identity of about 92%. In another study, samples taken from
187 several BISC-affected cats were examined by PCR (Munday et al., 2007). Of the sequences
188 obtained from five of these cases, a 235 bp composite sequence (GenBank EF447284) was
189 created. Alignment of that sequence to the corresponding L1-FdPV2 genome portion revealed
190 an identity of 99.1%. Only recently, the same group discovered in 24 invasive squamous cell
191 carcinoma (ISCC) and BISC samples PV sequences sharing 98-99% similarity with the
192 composite sequence (Munday et al., 2008).
193 Comparison of FdPV2 with known PV genomes
194 In order to address the possible classification of FdPV2, alignments were made at the
195 nucleotide sequence level for single L1 ORFs, and the translated individual E1 ORFs were
196 compared at the amino acid sequence level. First, a phylogenetic tree was constructed based
197 on the multiple sequence alignment of L1 nucleotide sequences (Fig. 5A). FdPV2 distantly
198 groups together with the recently discovered polar bear papillomavirus (UmPV1) and the
199 Alpha -papillomaviruses. CPV3 (unclassified), HPV32 (Alpha -PV), HPV18 (Alpha -PV),
200 UmPV1 and CPV4 (both unclassified) shared the greatest identity in the L1 nucleotide
201 sequence (between 58.1 and 57.1% as determined by pairwise nucleotide sequence
202 alignments). Comparison with the only other published cat PV FdPV1 revealed only 51.1%
203 identity. Furthermore, an alternative phylogenetic tree was constructed based on the multiple
204 sequence alignment of E1 (Fig 5B). As the E1 ORF is less conserved than the L1 ORF, amino
205 acid sequences were used. In this E1-based tree, FdPV2 grouped with Lambda, Kappa and
206 Mu papillomaviruses. The highest identities at the E1 ORF amino acid level were found
207 among the papillomaviruses of felide species, namely PcPV1, FdPV1, LrPV1 and UuPV1,
208 which shared an identity between 51.5 and 50.2% as determined by pairwise amino acid
209 sequence alignments.
210 9
211 Discussion
212 The clinical conditions of BISC are described as solitary or multiple hyperkeratotic plaques
213 (Baer and Helton, 1993; Gross et al., 2005; Miller et al., 1992). PV-specific sequences have
214 been repeatedly detected in BISC lesions (Munday et al., 2008; Munday et al., 2007; Nespeca
215 et al., 2006). However, none of these sequences matched that of the only entirely sequenced
216 feline papillomavirus (FdPV-1).
217 Here we describe three cases of BISC where we carried out genomic analysis of PVs. The
218 whole genome of an as yet unknown feline PV (FdPV-2) was amplified and sequenced. A few
219 years ago, FdPV1 DNA was isolated and sequenced from feline viral plaques (Tachezy et al.,
220 2002), which have been proposed to be the precursor lesion for BISC (Wilhelm et al., 2006).
221 However, FdPV-1 and 2 only share 51.1% identity in the L1 ORF and thus belong to two
222 different genera. PV DNA has also been found in various BISCs, ISCCs and also on the
223 healthy skin of cats (Munday at al., 2007; Munday at al., 2008; Nespeca et al., 2006). Several
224 of these sequences are very similar (>90%) to FdPV2, while others remain unclassified.
225 Among all published cases where PV DNA was amplified from BISCs or ISCCs and
226 sequenced, FdPV2 or very similar sequences seem to be the most prevalent by far. This
227 suggests that the virus might either be involved in the development of mentioned lesions,
228 which is supported by signs of viral replication in the three cases described here, or might be
229 very prevalent in general.
230 According to the rules of the International Committee on Taxonomy of Viruses (ICTV), the
231 degree of the calculated L1 coding sequence identity is used to taxonomically classify PVs.
232 PVs belonging to different genera are defined as showing less than 60% homology and those
233 belonging to different species as showing less than 70% homology. In fact, pairwise
234 alignment of the FdPV2 L1 ORF sequence to the closest related PV L1 ORF sequences
10
235 revealed 57.1 to 58.1% identity. FdPV2 might consequently be regarded as the prototype of a
236 new genus.
237 Phylogenetic analysis based on the L1 coding sequences or the E1 amino acid sequences
238 classified FdPV2 differently. This may be due to a lack of close relatives among the included
239 PVs, which can also be expected to be the reason for the relatively low bootstrap support. It
240 should be noted that individual PV genes seem to evolve at different rates and remain
241 somewhat independent of each other (Garcia-Vallve et al., 2005). The two trees nevertheless
242 suggested distant relatedness of FdPV-2 to Alpha- or Lambda-papillomaviruses. However, we
243 were not able to identify a potential E5 ORF on the FdPV2 genomic sequence; the presence of
244 this ORF is a typical characteristic of Alpha-papillomaviruses. Furthermore, the FdPV-2
245 genome lacks the Lambda PV specific second long control region, which is found in all
246 felidae PVs described thus far (Rector et al., 2007). These findings further support the
247 grouping of FdPV2 into a new genus.
248 249 FdPV2 has been found in three different European countries. Short DNA sequences amplified
250 from 5 BISC cats from the USA share more than 99% homology with the present genome;
251 additionally, 24 sequences from BISC- and ISCC-cats from the USA and New Zealand also
252 share an equal homology. This suggests that FdPV2 could have a worldwide distribution and
253 may be involved in the development of certain neoplastic diseases.
254 References
255 256 257 258 259 260 Baer, K.E., Helton, K., 1993, Multicentric squamous cell carcinoma in situ resembling Bowen's disease in cats. Vet Pathol 30, 535‐543. de Villiers, E.‐M., Fauquet, C., Broker, T.R., Bernard, H.‐U., zur Hausen, H., 2004, Classification of papillomaviruses. Virology 324, 17‐27. Garcia‐Vallve, S., Alonso, A., Bravo, I.G., 2005, Papillomaviruses: different genes have different histories. Trends Microbiol 13, 514‐521. 11
261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 Gross, T.L., Ihrke, P.J., Walder, E.J., Affolter, V.K., 2005, Chapter 7. Hyperplastic diseases of the epidermis, In: Gross, T.L., Ihrke, P.J., Walder, E.J., Affolter, V.K. (Eds.) Skin diseases of the dog and cat. Blackwell Publishing, Oxford, pp. 136‐160. Miller, W.H., Jr., Affolter, V.K., Scott, D.W., Suter, M.M., 1992, Multicentric squamous cell carcinomas in situ resembling Bowen's disease in five cats. Vet. Dermatol. 3, 177‐182. Munday, J.S., Kiupel, M., French, A.F., Howe, L., 2008, Amplification of papillomaviral DNA sequences from high proportion of feline cutaneous in situ and invasive squamous cell carcinomas using a nested polymerase chain reaction. Veterinary Dermatology 19, 259‐263. Munday, J.S., Kiupel, M., French, A.F., Howe, L., Squires, R.A., 2007, Detection of papillomaviral sequences in feline Bowenoid in situ carcinoma using consensus primers. Veterinary Dermatology 18, 241‐245. Münger, K., Baldwin A, Hayakawa, H., Nguyen, C.L., Owens, M., Grace, M., Huh, K., 2004, Mechanisms of Human Papillomavirus‐Induced Oncogenesis. J. Virol. 78, 11451‐11460. Nespeca, G., Tobler, K., Grest, P., Ackermann, M., Favrot, C., 2006, Detection of novel
papillomaviruses in paraffine-embedded samples of feline invasive and in situ squamous cell
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317 Table 1: Overview of the BISC cases included in the study Case 1
Marburg-D
Case 2
Roquefort-F
Case 3
Bremgarten-CH
Maine Coon
Siamese cross
European shorthair
male
male castrated
male castrated
11
11
13
Localization
Face, lips, forefoot
Face (temporal)
Ventral neck, face
Lesion
multiple pigmented
plaques
solitary pigmented
plaque
multiple pigmented
plaques
Histological
findings
irregular epidermal
hyperplasia, loss of
polarity, some
koilocytes
irregular epidermal
hyperplasia, loss of
polarity, some
koilocytes
irregular epidermal
hyperplasia, loss of
polarity, some
koilocytes
PCR* and
sequencing
FdPV2 positive
FdPV2 positive
FdPV2 positive
Breed
Gender
Age at onset (years)
318 319 *A16/A37 primers amplifying an 858 nt fragment of the L1 ORF were used.
14
320 Figure captions:
321 Figure 1: Cat two with temporal crusted and hyperpigmented lesion
322 Figure 2: Histological aspect of bowenoid in situ squamous cell carcinoma (BISC) in cat one.
323 Haired skin: note viral cytopathic effects such as giant keratohyaline granules (white arrow
324 heads) and koilocytes (black arrows), which are large keratinocytes with cleared cytoplasm,
325 shrunken, irregular nuclei and nucleoli. Haematoxylin and Eosin. Bar = 10 µm.
326 Figure 3: Schematic presentation of the FdPV2 genome and open reading frames (ORFs).
327 The genome is divided into three sections: early genes (Early), late genes (Late) and long
328 control region (LCR). Numbers indicate nucleotide positions of the start- and stop-codons of
329 the ORF.
330 Figure 4: Agarose gel electrophoresis of digested multiply primed rolling-circle amplification
331 (RCA) product from samples 1 and 2. The restriction endonucleases ClaI, EcoRV, NdeI and
332 XbaI were used as indicated above the lanes. A 1kb ladder (Invitrogen) was loaded as size
333 marker.
334 Figure 5: Neighbour-joining phylogenetic trees of the L1 nucleotide sequences (A) and the
335 E1 amino acid sequences (B) of FdPV2 and 52 other papillomaviruses (PVs). To date
336 unclassified PVs are marked with an asterisk. The PV-types (with their GenBank accession
337 numbers) included bovine BPV1 (NC_001522), BPV2 (M20219), BPV5 (NC_004195),
338 bandicoot papillomatosis carcinomatosis BPCV1 (NC_010107), BPCV2 (NC_010817),
339 canine oral COPV (NC_001619), canine CPV2 (NC_006564), CPV3 (NC_008297), CPV4
340 (NC_010226), deer DPV1 (NC_001523), Equus caballus EcPV1 (NC_003748), European elk
341 EEPV1 (NC_001524), Fringilla coelebs FcPV1 (NC_004068), Felis domesticus FdPV1
342 (NC_004765), human HPV3 (NC_001588), HPV4 (NC_001457), HPV5 (NC_001531),
343 HPV6 (AF092932), HPV7 (NC_001595), HPV9 (NC_001596), HPV13 (NC_001349),
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344 HPV15 (NC_001579), HPV16 (NC_001526), HPV18 (NC_001357), HPV22 (NC_001681),
345 HPV29 (NC_001685), HPV32 (X74475), HPV41 (NC_001354), HPV49 (NC_001591),
346 HPV50 (NC_001691), HPV52 (NC_001592), HPV54 (U37488), HPV56 (NC_001594),
347 HPV60 (NC_001693), HPV63 (NC_001458), HPV75 (Y15173), HPV95 (AJ620210), Lynx
348 rufus LrPV1 (AY904722), Mastomys natalensis MnPV1 (NC_001605), Ovine OvPV1
349 (NC_001789), OvPV2 (U83595), Puma concolor PcPV1 (AY904723), Psittacus erithacus
350 PePV1 (NC_003973), Procyon lotor PlPV1 (AY763115), Phocoena spinipinnis PsPV1
351 (NC_003348), rabbit oral ROPV (NC_002232), Rousettus aeyptiacus RaPV1 (NC_008298),
352 rhesus monkey RhPV1 (NC_001678), Trichechus manatus latirostris TmPV1 (NC_006563),
353 Tursiops truncatus TrPV2 (NC_008184), Ursus maritimus UmPV1 (NC_010739), Uncia
354 uncia UuPV1 (DQ180494). BPCV1 and BPCV2 are not included into the E1 phylogenetic
355 tree, due to lack of such ORFs. Numbers at internal nodes represent the bootstrap support
356 values in percent. Only bootstrap probabilities above 50% are shown.
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357 358 Figure 1
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359 360 Figure 2
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361 362 Figure 3
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363 364 Figure 4
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365 366 Figure 5
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