Psoriasis: A Possible Reservoir for Human Papillomavirus Type 5, the Virus Associated with Skin Carcinomas of Epidermodysplasia Verruciformis
1998; Elsevier BV; Volume: 110; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.1998.00164.x
ISSN1523-1747
AutoresMichel Favre, Gérard Orth, Slavomir Majewski, Anna Pura, Stefania Jabłońska, Samia Baloul,
Tópico(s)Viral-associated cancers and disorders
ResumoRecent polymerase chain reaction data have shown that most human papillomavirus (HPV) genotypes associated with epidermodysplasia verruciformis (EV) are widespread; however, HPV5 associated with EV skin carcinomas has only rarely been detected in non-EV patients. To identify the reservoir of this virus, we examined 335 sera from different groups of patients for the presence of HPV5 antibodies by an enzyme-linked immunosorbent assay test based on HPV5 virus-like particles. The prevalence of antibodies reacting with HPV5 virus-like particles was found to be significantly higher in psoriatic patients (24.5%) than in other groups (2–5%), including patients with atopic dermatitis and renal transplant recipients. Analysis of scrapings of lesional and uninvolved skin by a nested polymerase chain reaction method, using degenerate EV HPV primers, disclosed HPV DNA in 91.7% of 48 psoriatic skin samples and 35.5% of 31 atopic dermatitis specimens. Eleven EV HPV genotypes, most frequently HPV5 and HPV36, and a putative novel genotype (PsoX1) were identified in psoriasis. Five EV HPV genotypes and two putative novel genotypes (ADX1 and ADX2) were detected in atopic dermatitis patients. HPV5 was not found in atopic dermatitis patients. Using type specific primers, HPV5, HPV36, and HPV1 were found in 89.4%, 84.2%, and 42.1% of specimens from psoriatic patients, whereas HPV36 was detected in 22.5% of specimens from atopic dermatitis patients. HPV16 was never detected. On the whole, 27 HPV5 and 13 HPV36 DNA variants were disclosed after sequencing amplification products. Our data confirm that EV HPV are widespread and point to psoriasis as a reservoir for HPV5. Whether HPV5 is involved in the pathogenesis of psoriasis remains to be determined. Recent polymerase chain reaction data have shown that most human papillomavirus (HPV) genotypes associated with epidermodysplasia verruciformis (EV) are widespread; however, HPV5 associated with EV skin carcinomas has only rarely been detected in non-EV patients. To identify the reservoir of this virus, we examined 335 sera from different groups of patients for the presence of HPV5 antibodies by an enzyme-linked immunosorbent assay test based on HPV5 virus-like particles. The prevalence of antibodies reacting with HPV5 virus-like particles was found to be significantly higher in psoriatic patients (24.5%) than in other groups (2–5%), including patients with atopic dermatitis and renal transplant recipients. Analysis of scrapings of lesional and uninvolved skin by a nested polymerase chain reaction method, using degenerate EV HPV primers, disclosed HPV DNA in 91.7% of 48 psoriatic skin samples and 35.5% of 31 atopic dermatitis specimens. Eleven EV HPV genotypes, most frequently HPV5 and HPV36, and a putative novel genotype (PsoX1) were identified in psoriasis. Five EV HPV genotypes and two putative novel genotypes (ADX1 and ADX2) were detected in atopic dermatitis patients. HPV5 was not found in atopic dermatitis patients. Using type specific primers, HPV5, HPV36, and HPV1 were found in 89.4%, 84.2%, and 42.1% of specimens from psoriatic patients, whereas HPV36 was detected in 22.5% of specimens from atopic dermatitis patients. HPV16 was never detected. On the whole, 27 HPV5 and 13 HPV36 DNA variants were disclosed after sequencing amplification products. Our data confirm that EV HPV are widespread and point to psoriasis as a reservoir for HPV5. Whether HPV5 is involved in the pathogenesis of psoriasis remains to be determined. atopic dermatitis epidermodysplasia verruciformis renal transplant recipients virus-like particles Epidermodysplasia verruciformis (EV) is a rare genetic disease characterized by an abnormal susceptibility to a specific group of related human papillomavirus (HPV) types and the frequent occurrence of cutaneous malignancies mainly associated with HPV5 (Orth, 1987Orth G. Epidermodysplasia verruciformis.in: Howley P.M. Salzman N.P. The Papillomaviruses. vol. 2. Plenum Press, New York1987: 199-243Google Scholar; Majewski and Jablonska, 1995Majewski S. Jablonska S. Epidermodysplasia verruciformis as a model of human papillomavirus-induced genetic cancer of the skin.Arch Dermatol. 1995; 131: 1312-1318Crossref PubMed Scopus (195) Google Scholar). EV HPV DNA sequences have only rarely been detected by molecular hybridization experiments in cutaneous premalignant tumors and skin cancers in the general population (Kawashima et al., 1990Kawashima M. Favre M. Obalek S. Jablonska S. Orth G. Premalignant lesions and cancers of the skin in the general population. Evaluation of the role of human papillomaviruses.J Invest Dermatol. 1990; 95: 537-542Abstract Full Text PDF PubMed Google Scholar); however, recent studies using highly sensitive nested polymerase chain reaction (PCR) have disclosed the presence of DNA sequences of EV HPV or related HPV types in nonmelanoma skin tumors of immunosuppressed and immunocompetent individuals (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar; Pfister and ter Schegget, 1997Pfister H. ter Schegget J. Role of HPV in cutaneous premalignant and malignant tumors.Clin Dermatol. 1997; 15: 335-348Abstract Full Text PDF PubMed Scopus (111) Google Scholar). EV HPV DNA sequences have also been found highly prevalent in plucked hairs from renal transplant recipients (RTR) and healthy volunteers (Boxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Abstract Full Text PDF PubMed Scopus (271) Google Scholar). This indicates that EV HPV cause widespread latent infections; however, their role in skin carcinogenesis in non-EV patients remains to be substantiated. Strikingly, using the same approach, HPV5 has only occasionally been detected in skin tumors of non-EV patients (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar; Shamanin et al., 1996Shamanin V. zur Hausen H. Lavergne D. et al.Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients.J Nat Cancer Inst. 1996; 88: 802-811Crossref PubMed Scopus (282) Google Scholar; Pfister and ter Schegget, 1997Pfister H. ter Schegget J. Role of HPV in cutaneous premalignant and malignant tumors.Clin Dermatol. 1997; 15: 335-348Abstract Full Text PDF PubMed Scopus (111) Google Scholar) and in plucked hairs (Boxman et al., 1997Boxman I.L.A. Berkhout R.J.M. Mulder L.H.C. Wolkers M.C. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers.J Invest Dermatol. 1997; 108: 712-715Abstract Full Text PDF PubMed Scopus (271) Google Scholar). This raises the question of the reservoir for HPV5. Therefore, our first aim was to search for antibodies to HPV5 in patients with common cutaneous disorders [psoriasis and atopic dermatitis (AD)], in RTR (a population at risk for the development of skin neoplasia), in patients with anal warts, and in subjects with no known history of HPV infection. We disclosed a significant prevalence of anti-HPV5 antibodies only in patients with psoriasis. Psoriasis is characterized by an epidermal proliferation (Christophers and Sterry, 1993Christophers E. Sterry W. Psoriasis.in: Fitzpatrick T.B. Eisen A.Z. Wolff K. Dermatology in General Medicine. McGraw-Hill, New York1993: 489-514Google Scholar), which appears to be a secondary event related to activation of T lymphocytes and production of proinflammatory cytokines and growth factors (Griffiths and Voorhees, 1996Griffiths C.E. Voorhees J.J. Psoriasis, T cells and autoimmunity.J R Soc Med. 1996; 89: 315-319PubMed Google Scholar). To find out whether this inflammatory hyperproliferative epidermal process could permit HPV5 DNA replication and favor an immune humoral response, we screened for the presence of HPV5 and other EV HPV in scrapings of lesional and/or uninvolved skin in psoriatic patients and, as control, in patients with AD, using a nested PCR technique (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar). Our data disclosed the presence of HPV5 DNA in most patients with psoriasis but not in AD patients. Sera were obtained from 155 Polish psoriatic patients (62 females and 93 males, mean age 49.2 ± 17.9 y). The series studied consisted of 129 patients with plaque psoriasis, 18 with guttate psoriasis, five with psoriatic erythroderma, and three with pustular psoriasis. Onset of the disease, its duration, extent, and activity of lesions, persistence of the last relapse, and previous treatments were recorded. Sera were also obtained from 44 Polish patients with AD (mean age 29.7 ± 12.1 y) and 14 Polish patients with EV (mean age 39.7 ± 17.1 y). Southern blot hybridization experiments had shown that 10 of the EV patients were infected with HPV5, one was infected with HPV12, and three were infected with HPV3, including the sister of an HPV5 infected patient. All but one of the HPV5 infected patients were found to harbor at least another EV HPV genotype (Orth G, and Jablonska S, unpublished results). Sera from 38 Polish RTR (mean age 40.8 ± 11.9 y), 38 French patients with anal condylomata acuminata induced by HPV6 or HPV11 (mean age 30.1 ± 8.4 y), and 60 French individuals with no known history of HPV infection (mean age 36.1 ± 23.5 y) were retrieved from three serum collections. Scrapings were taken from a single lesion at various anatomical sites (hand, forearm, back, thorax, groin, leg) of Polish patients with plaque psoriasis (28 cases), guttate psoriasis (nine cases), and AD (16 cases). Scrapings were collected from two lesions in four patients with plaque psoriasis and one patient with guttate psoriasis and from noninvolved skin of six patients with plaque psoriasis. Scrapings were also taken from uninvolved and lesional skin of 12 Polish patients with AD and from uninvolved or lesional skin of three and four additional Polish patients, respectively. Recombinant baculoviruses expressing the L1 capsid protein of HPV5 variants E10 (subtype a), E11 (subtype c), and E5 (a subtype b+ variant) (Kawase et al., 1996Kawase M. Orth G. Jablonska S. Blanchet-bardon C. Rueda L.-A. Favre M. Variability and phylogeny of the L1 capsid protein gene of human papillomavirus type 5: contribution of clusters of nonsynonymous mutations and of a 30-nucleotide duplication.Virol. 1996; 221: 189-198Crossref PubMed Scopus (20) Google Scholar) were constructed and expressed in Sf9 cells. Culture media were collected 72 h after infection and clarified by low speed centrifugation. After ultracentrifugation of the supernatants at 100,000 ×g, pellets were resuspended in phosphate buffered saline and L1 proteins were purified by equilibrium centrifugation in CsCl gradients. When observed under an electron microscope, purified L1 preparations contained mostly well assembled virus-like particles (VLP) (Kawase M, Orth G, and Favre M, in preparation). HPV1 virions were purified from pooled plantar warts as described previously (Favre et al., 1975Favre M. Breitburd F. Croissant O. Orth G. Structural polypeptides of rabbit, bovine, and human papillomaviruses.J Virol. 1975; 15: 1239-1247Crossref PubMed Google Scholar). Wells of microplates (Nunc-immuno module maxisorp F16, Inter Med, Roskilde, Denmark) were coated with VLP or HPV1 virions (0.1 μg) and sera were tested at a 1/100 dilution (Kirnbauer et al., 1994Kirnbauer R. Hubbert N.L. Wheeler C.M. Becker T.M. Lowy D.R. Schiller J.T. A virus-like particle enzyme-linked immunosorbent assay detects serum antibodies in a majority of women infected with human papillomavirus type 16.J Natl Cancer Inst. 1994; 86: 494-499Crossref PubMed Scopus (333) Google Scholar). To determine the nonspecific reactivity of sera, each serum was tested in duplicate in wells without antigen. After incubation for 2 h at 37°C, goat anti-human IgG conjugated to horseradish peroxidase (Biosys, Compiègne, France) were used as described by the supplier. The plates were developed with ortho-phenylene-diamine (0.4 mg per ml) in 0.1 M citrate buffer (pH 4.0) in the presence of 0.01% hydrogen peroxyde. The A490 nm values were recorded on a microplaque reader (Metertech S 960). For each serum, the mean value obtained with buffer coated wells was subtracted from the mean value of wells coated with each antigen. To score antibody positive and antibody negative sera, the mean and the SD were calculated for the 60 sera obtained from subjects with no known history of HPV infection and a cut-off value (mean + 3 SD) was deduced after exclusion of outliers (Müller et al., 1995Müller M. Viscidi R.P. Ulken V. et al.Antibodies to the E4, E6, and E7 proteins of human papillomavirus (HPV) type 16 in patients with HPV-associated diseases and in the normal population.J Invest Dermatol. 1995; 104: 138-141Crossref PubMed Scopus (26) Google Scholar). In each experiment, 20 of the negative control sera were included as standards. The proportions of positive HPV5 or HPV1 sera between the different groups tested were compared using the Yates’ corrected chi square test. Tests were considered significant at the p = 0.05 level. Total cellular DNA was extracted from scrapings as previously described (Kawase et al., 1996Kawase M. Orth G. Jablonska S. Blanchet-bardon C. Rueda L.-A. Favre M. Variability and phylogeny of the L1 capsid protein gene of human papillomavirus type 5: contribution of clusters of nonsynonymous mutations and of a 30-nucleotide duplication.Virol. 1996; 221: 189-198Crossref PubMed Scopus (20) Google Scholar). The adequacy of DNA preparations was tested by PCR using β globin primers (Greer et al., 1991Greer C.E. Peterson S.L. Kiviat N.B. Manos M.M. PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.Am J Clin Pathol. 1991; 95: 117-124PubMed Google Scholar). EV associated HPV were detected by a nested PCR method (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar) with two sets of degenerate primers (CP65/70 and CP66/69) located in the L1 open reading frame. To detect specific HPV types, a nested PCR was also performed using primers located in the L1 open reading frame of HPV1 (1 L1), HPV5 (5 L1), HPV16 (16 L1), and HPV36 (36 L1). The nucleotide (nt) positions of the two pairs of primers (1 and 2; 3 and 4) are for 1 L1, nt 5542–5561 and 6137–6156; nt 5593–5612 and 6066–6085, for 5 L1, nt 5913–5932 and 7015–7034; nt 6124–6143 and 6563–6582, for 16 L1, nt 5909–5928 and 6477–6492; nt 5981–6000 and 6350–6369, and for 36 L1, nt 6005–6024 and 6602–6621; nt 6098–6117 and 6537–6556 (Myers et al., 1995Myers G. Delius H. Icenogel J. Bernard H.-U. Baker C. Halpern A. Wheeler C. Human Papillomaviruses 1995. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos1995Google Scholar). PCR reaction mixtures contained 0.5–1 μg total cellular DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 100 mM each deoxynucleotide triphosphate, 2.5 mM MgCl2, 25 pM of each primer of the first pair (primers 1 and 2), and 2 units of Thermus aquaticus DNA polymerase (Perkin-Elmer, Emeryville, CA). Mixtures were incubated for 5 min at 95°C for DNA denaturation and 35 cycles of amplification were performed with a PCR processor (Perkin-Elmer Cetus 9600) (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar). Every eight PCR reactions, a tube containing water instead of DNA was included as negative control. Cloned HPV1, 5, 16, and 36 DNA (1 ng) were used as positive controls. Subsequently, 5 μl of the first PCR were amplified with the second set of primers (primers 3 and 4). PCR products were submitted to agarose gel electrophoresis, stained with ethidium bromide to visualize amplimers, alkali denatured and transferred onto Hybond N+ membranes (Amersham, Les Ulis, France), and hybridized with random primed 32P-labeled probes. Probes corresponded to PCR products generated on cloned HPV1, 5, 16, and 36 DNA using the second set of primers. Hybridizations were performed in nonstringent conditions with an HPV5 DNA probe for the detection of EV HPV DNA sequences or in stringent conditions with the homologous probe for the detection of specific HPV types, as previously described (Kawashima et al., 1990Kawashima M. Favre M. Obalek S. Jablonska S. Orth G. Premalignant lesions and cancers of the skin in the general population. Evaluation of the role of human papillomaviruses.J Invest Dermatol. 1990; 95: 537-542Abstract Full Text PDF PubMed Google Scholar). Direct sequencing was performed using the ABI prism dye terminator cycle sequencing core kit with the ampliTaq FS DNA polymerase (Perkin-Elmer, Roissy, France) as recommended by the supplier. Sequence products were analyzed with an ABI Prism 377 DNA sequencer (Perkin-Elmer). All sequences were compared with the available sequences in the EMBL/GenBank Databank using the FASTA program. Multiple alignments of partial HPV5 and HPV36 L1 sequences were performed with the CLUSTAL W program, and phylogenetic trees were constructed by using the maximum parsimony or the maximum likelihood methods with the PHYLIP 3.4 programs (Phylogeny Inference Package) as previously described (Kawase et al., 1996Kawase M. Orth G. Jablonska S. Blanchet-bardon C. Rueda L.-A. Favre M. Variability and phylogeny of the L1 capsid protein gene of human papillomavirus type 5: contribution of clusters of nonsynonymous mutations and of a 30-nucleotide duplication.Virol. 1996; 221: 189-198Crossref PubMed Scopus (20) Google Scholar). Sequences have been deposited at EMBL database under accession numbers Z93786 for EV HPV variant PsoX1, Z93787 to Z93796 for HPV36 variants P36–1 to P36–10, Z93797 to Z93815 for HPV5 variants P5–4 to P5–22, AJ001483 and AJ001484 for EV HPV variants ADX1 and ADX2, and AJ001628 and AJ001629 for HPV36 variants AD 36–2 and AD 36–3. Anti-HPV5 antibodies in patients with psoriasisAnti-HPV5 antibodies were searched for in different groups of patients by an ELISA test using purified HPV5 L1 VLP preparations. VLP corresponded to the most divergent HPV5a and c variants (E10 and E11) and to a subtype b+ variant (E5), with a nine amino acid insert in the L1 protein (Kawase et al., 1996Kawase M. Orth G. Jablonska S. Blanchet-bardon C. Rueda L.-A. Favre M. Variability and phylogeny of the L1 capsid protein gene of human papillomavirus type 5: contribution of clusters of nonsynonymous mutations and of a 30-nucleotide duplication.Virol. 1996; 221: 189-198Crossref PubMed Scopus (20) Google Scholar). Our previous studies had shown that hyperimmune rabbit anti-sera raised against each of the VLP species yielded 4- to 16-fold lower ELISA titres when tested on heterologous VLP and did not cross-react with HPV1 virions. Furthermore, some of the monoclonal antibodies prepared against HPV5b+ VLP were found to cross-react with either HPV5a or HPV5c intact VLP. These distinct antigenic properties disclosed the existence of three HPV5 serotypes (Kawase M, Orth G, and Favre M, in preparation). As control, we screened for anti-HPV1 antibodies using HPV1 virions. Antibodies reacting with two or three HPV5 VLP species, i.e., bi- or trispecific, were detected in 71.4% of 14 EV patients (Table 1 and Table 2, Figure 1). Only the 10 HPV5 infected patients were found seropositive. Antibodies reacting with at least one preparation of HPV5 VLP were detected in 38 (24.5%) of 155 psoriatic patients (Table 1, Figure 1) and the majority of these antibodies were monospecific (Table 1). This prevalence was found to be significantly higher than that (2–5%) observed in the other groups tested, including patients with AD and RTR (p = 0.004). In contrast, no significant difference was observed in the prevalence of anti-HPV1 antibodies (21.7% to 34.2%) in the different groups, including the EV patients (Table 1, Figure 1). Furthermore, no difference in the prevalence of anti-HPV1 antibodies was observed among HPV5 seropositive and HPV5 seronegative patients (31.5% and 25.6%, respectively), indicating that the ELISA test is reliable enough to discriminate between distantly related HPV types.Table 1Seropositivity to HPV5 L1 VLP and HPV1 virions among different groups of patients and among controlsaAs determined by an ELISA test as described in Materials and Methods.Test groupNumber (%) of positive sera toHPV5HPV1HPV1 + 5EV (n = 14)10 (71.4)4 (28.6)3 (21.4)Psoriasis (n = 155)38 (24.5)42 (27.1)12 (7.7)AD (n = 44)2 (4.5)14 (31.8)1 (2.3)RTR (n = 38)1 (2.6)13 (34.2)1 (2.6)Genital warts (n = 38)1 (2.6)10 (26.3)0Controls (n = 60)3 (5.0)13 (21.7)1 (1.7)a As determined by an ELISA test as described in Materials and Methods. Open table in a new tab Table 2Subtype specificity of patients antibodies to HPV5 L1 VLPaAs determined by an ELISA test as described in Materials and Methods.Test groupReactivity against HPV5a, b+ , and c VLPReactivity against HPV5a, b+ , and c VLPHPV5 positiveReactive to HPV5 VLPab+cPsoriasismonospecific228104(n = 155)bispecific7653trispecific9999total38232416EVmonospecific0000(n = 14)bispecific5334trispecific5555total10889a As determined by an ELISA test as described in Materials and Methods. Open table in a new tab No correlation was observed between the detection rate of anti-HPV5 antibodies and the clinical type of psoriasis (plaque and guttate psoriasis), the age at onset, the duration of the disease, the nature of treatments (previous PUVA therapy, retinoids, methotrexate), and the patients’ age; however, the detection rate was significantly higher (43.2%) for 37 patients with lesions covering more than 50% of body surface than for 65 and 53 patients with lesions covering 20–50% (15.4%, p = 0.004) or less than 20% (22.6%, p = 0.05) of body surface, respectively. Scrapings of lesional or uninvolved skin of 37 patients with psoriasis and, as controls, of 19 patients with AD were analyzed for the presence of HPV5 and EV HPV DNA by a nested PCR method using degenerate consensus primers designed to detect EV HPV types (Table 3) (Berkhout et al., 1995Berkhout R.J.M. Tieben L.M. Smits H.L. Bouwes Bavinck J.N. Vermeer B.J. ter Schegget J. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients.J Clin Microbiol. 1995; 33: 690-695PubMed Google Scholar). Among these patients, 10 psoriatic and two AD patients had detectable anti-HPV5 antibodies. All DNA preparations were negative after the first round of PCR (primers CP65 and 70), even when PCR products were analyzed by blot hybridization (data not shown). In contrast, after the second round of PCR (primers CP66 and 69), a high proportion of DNA preparations obtained from plaque (93.8%) and guttate (80%) psoriasis and all six preparations extracted from uninvolved skin of some of these patients yielded a fragment with the expected size of about 460 bp (Table 3). This fragment was detectable on ethidium bromide stained agarose gels and after hybridization with an HPV5 L1 probe under nonstringent conditions. A lower proportion of preparations obtained from lesional (31.2%) and uninvolved (40%) skin from AD patients were HPV positive. After direct sequencing of the PCR products, 10 known EV HPV genotypes were identified in psoriasis and five in AD patients (Table 3). The most prevalent genotypes were HPV5, HPV36, HPV20, and HPV38 in psoriasis and HPV24 and HPV38 in AD. In addition, four novel putative EV related genotypes were detected, two in psoriatic patients, the recently identified HPV12 related RTRX7 isolate (Höpfl et al., 1997Höpfl R. Bens G. Wieland U. Petter A. Zelger B. Fritsch P. Pfister H. Human papillomavirus DNA in non-melanoma skin cancers of a renal transplant recipient: detection of a new sequence related to epidermodysplasia verruciformis associated types.J Invest Dermatol. 1997; 108: 53-56Abstract Full Text PDF PubMed Scopus (38) Google Scholar) and an as yet unrecognized strain (PsoX1) related to HPV17 (73.4% identical nucleotides among the 354 sequenced), and two isolates in AD patients, sharing 81.4% and 73.4% sequence homology with HPV17 (ADX1) and HPV9 (ADX2), respectively. A double infection was found for three of 26 psoriatic patients with only one lesional skin specimen analyzed (HPV5 + HPV36 or 38, HPV9 + HPV36) and, for patients with two specimens tested, six of 11 psoriatic patients (HPV5 + HPV36, twice, HPV20 + HPV38 or RTRX7, HPV36 + HPV38, and HPV37 + HPV38) and one of 12 AD patients (HPV15 + HPV21). Strikingly, whereas nine (24.3%) of the 37 psoriatic patients were found infected with HPV5, none of the 19 patients with AD (31 specimens) were positive for HPV5.Table 3Detection and identification of HPV DNA sequences in patients with psoriasis and ADaNested PCR using primers specific for EV HPV (Berkhout et al, 1995), HPV1, 5, 16, and 36 and sequencing of PCR products were performed as described in Materials and Methods.Origin of scrapingsNo of patientsHPV detection and typing in scrapings using primers specific forNo of seropositive patientsEV HPVHPV5HPV36HPV1HPV16PCR+HPV typecGenotypes were identified by sequencing PCR products. The number of positive specimens is given in parentheses. RTRX7 corresponds to a recently identified isolate in an RTR (HOpfl et al, 1997). Three putative novel genotypes were characterized in psoriasis (PsoXl) and AD (ADX1 and ADX2). A double infection (HPV5 + 36, HPV5 + 38, and HPV9 + 36) was detected in three cases of plaque psoriasis.PCR+VariantdVariants were identified by comparison of their nucleotide sequence with those of variants identified in EV (Kawase et al, 1996). Variants characterized in psoriasis (P5–1 to – 27, P36–1 to –10) and AD (AD36–1 to –3) were numbered according to the chronologic order of their identification. P5– 1, P5–2, and P5–3 HPV5 variants correspond to variants Jl, E3, and E10 identified in EV (Kawase et al, 1996). P36G HPV36 variant corresponds to variant AD 36–1.PCR+VariantdVariants were identified by comparison of their nucleotide sequence with those of variants identified in EV (Kawase et al, 1996). Variants characterized in psoriasis (P5–1 to – 27, P36–1 to –10) and AD (AD36–1 to –3) were numbered according to the chronologic order of their identification. P5– 1, P5–2, and P5–3 HPV5 variants correspond to variants Jl, E3, and E10 identified in EV (Kawase et al, 1996). P36G HPV36 variant corresponds to variant AD 36–1.PCR +PCR+HPV1HPV5PsoriasisPlaque2830/32bScrapings from two lesions were studied for four patients with plaque psoriasis and one patient with guttate psoriasis.5(×8),9,15,17,20(×6),24,36(×8),37,38(×4),PsoXl,inceinc, inconclusive.30/32bScrapings from two lesions were studied for four patients with plaque psoriasis and one patient with guttate psoriasis.P5–1(×2),-2(×3),-3,-4,-5,-6,-7,-8(×3),-9,-10,-ll,-12,-13,-14,-15,-16,-17,-18,-19,-20,-21,-22, inc(×3)11/14P36–l(×3),-2,-3,-4(×2),-5,-6,-7,-86/140/1488Guttate98/10bScrapings from two lesions were studied for four patients with plaque psoriasis and one patient with guttate psoriasis.5,15(×3),21, 36(×3)8/10bScrapings from two lesions were studied for four patients with plaque psoriasis and one patient with guttate psoriasis.P5–2,-8(×2),-13,-16,-23,-24,-255/5P36–6,-9,-10, inc(×2)2/50/542Uninvolved66/65(×2),20(×2),38, RTRX75/6P5–16,-17,-26,-27, incndfnd, not done.ndnd11ADLesion165/1621,24,38(×2),inc0/164/16AD36–l,-2,-3(×2)0/160/1652Uninvolved156/1515,24(×2),36, ADX1 ,ADX20/153/15AD36–3(×3)0/150/1510a Nested PCR using primers specific for EV HPV (Berkhout et al, 1995), HPV1, 5, 16, and 36 and sequencing of PCR products were performed as described in Materials and Methods.b Scrapings from two lesions were studied for four patients with plaque psoriasis and one patient with guttate psoriasis.c Genotypes were identified by sequencing PCR products. The number of positive specimens is given in parentheses. RTRX7 corresponds to a recently identified isolate in an RTR (HOpfl et al, 1997). Three putative novel genotypes were characterized in psoriasis (PsoXl) and AD (ADX1 and ADX2). A double infection (HPV5 + 36, HPV5 + 38, and HPV9 + 36) was detected in three cases of plaque psoriasis.d Variants were identified by comparison of their nucleotide sequence with those of variants identified in EV (Kawase et al, 1996). Variants char
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