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1 Department of Medical Microbiology, Malmö University Hospital, Lund University, SE-20502 Malmö, Sweden
2 3-3-35 Yatsushiro-Shi, Kumamoto 830, Japan
3 Department of Oncology, Bangabandhu Sheikh Mujib Medical University, Shahbagh, Dhaka, Bangladesh
Correspondence
Bengt Göran Hansson
bengt-goran.hansson{at}mikrobiol.mas.lu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank accession numbers of new sequences reported in this paper are AY009879, AY009880, AY009883, AY009884, AY009887, AY040275–AY040280, AY040282–AY040284, AF440445–AF440450 and AF542099–AF542103.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Epidermodysplasia verruciformis (EV) is a rare hereditary skin disease manifesting as multiple warts. A broad variety of HPV types, which have been referred to as EV HPVs, e.g. HPV-5, -8, -9, -12, -14, -15, -17 and -19–25 (Kremsdorf et al., 1984; Orth et al., 1978), has been found in these patients. Some skin HPV types, mainly HPV-5 and HPV-8, have been found in skin cancer lesions, for example in EV patients (Jablonska & Majewski, 1994, Orth et al., 1979) and in immunosuppressed patients (Bens et al., 1998; Berkhout et al., 2000; de Villiers et al., 1997), and have therefore been suggested to be high-risk HPVs for these patients. However, in recent years it has been found that skin HPV types are widely spread among humans, giving rise to subclinical infections generally without causing warts or other lesions (Antonsson et al., 2000; Astori et al., 1998; Boxman et al., 1997).
In previous reports, we have shown that normal skin of healthy Swedish adults and children is infected with a great number of different HPV types and putative types, and that colonization of the skin with HPV starts very early in life (Antonsson et al., 2000, 2003). In the present study, we have collected and analysed skin samples from healthy individuals from three continents in order to investigate whether there are any major differences in skin HPV prevalence or genotype distribution between temperate, subtropical and tropical geographical areas or, alternatively, between Caucasian and non-Caucasian population groups.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Samples.
Skin swab samples were collected with pre-wetted (0·9 % NaCl solution) cotton-tipped swabs (Bio Hospital, Kopparberg, Sweden), which were drawn back and forth five times over the forehead skin within an area of 5x10 cm and then suspended in 1 ml 0·9 % NaCl solution. The samples from Sweden were kept at 4 °C for a maximum of 24 h before being analysed, while the specimens from Bangladesh, Japan, Zambia and Ethiopia were kept at room temperature for a maximum of 7 days until they reached the laboratory and were analysed.
PCR and HPV type determination.
A PCR test with the primer pair FAP59/FAP64 was used for detection of skin HPV DNA, as previously described (Forslund et al., 1999). The protocol was followed as described except for the MgCl2 concentration, which was modified to 3·5 mM. The PCR products were cloned into the pCR-script SK(+) cloning vector (Stratagene). Between two and four clones per sample were sequenced with both forward and reverse primers (BigDye; Applied Biosystems) and the sequences obtained were compared with available sequences in the GenBank database using the BLAST server (http://www.ncbi.nlm.nih.gov/blast/).
The PCR products obtained spanned the HPV L1 gene from nt 6044 to 6480 (numbering relative to HPV 20). Since full-length L1 sequences were not obtained, the new HPV isolates detected in this study were designated putative HPV types. The guidelines from the Papillomavirus Nomenclature Committee 1995 (14th International Papillomavirus Conference, Quebec City, Quebec, Canada) were followed in defining a new putative HPV type (de Villiers, 2001).
All samples were also analysed for human DNA by PCR, targeting the L1 repetitive sequence (Deragon et al., 1990).
Statístical analysis.
The chi-square test was used to compare the prevalence of HPV DNA and human DNA among the different countries.
Nucleotide sequence accession numbers.
Sequences of FA46, FA48–49, FA52, FA56–60, FA62–64, FA73–78 and FA93–97 have been submitted to GenBank with the following accession numbers; FA46, AY009880; FA48, AY009879; FA49, AY009884; FA52, AY009883; FA56, AY040275; FA57, AY040276; FA58, AY040277; FA59, AY040278; FA60·1, AY040279; FA60·2, AY040280; FA62, AY040282; FA63, AY040283; FA64, AY040284; FA73, AF440445; FA74, AF440446; FA75, AF440447; FA76, AF44048; FA78, AF440450; FA93, AF542099; FA94, AF542100; FA95, AF542101; FA96, AF542102; FA97, AF542103.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). One sample from a male contained a subtype of FA20 (92 % sequence homology). It was not possible to determine the HPV type of two samples collected from males, despite several attempts.
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Sweden
Thirty-five (70 %) of the 50 Swedish samples were HPV DNA-positive, 20 from men and 15 from women. Five of the samples tested negative for human DNA and two of these were HPV DNA positive. HPV type determination of the positive specimens revealed 34 previously described HPV types or putative types and five new putative types, FA73–76 and FA78 (Table 1).
Zambia
Twenty-one (42 %), 14 men and 7 women, of the 50 Zambian individuals were HPV DNA positive. Twenty-nine of the specimens tested negative for human DNA by PCR, out of which 16 were HPV DNA positive. HPV type determination revealed two new putative types (FA93 and FA94) together with 11 previously described skin HPV types or putative types (Table 1). Two subtypes, one of FA27 (92 % sequence homology) and another of HPV-20 (91 % sequence homology) were found in samples from two males.
Ethiopia
Twenty-six (52 %) of the 50 Ethiopian samples were HPV DNA positive, 11 from men and 15 from women. Thirty-one of the samples tested negative for human DNA and 17 of these were HPV DNA positive. The HPV types were determined in the HPV DNA-positive specimens and 25 previously described HPV types or putative types plus three new putative HPV types (FA95–FA97) were found (Table 1). Two putative subtypes were detected, one of HPV-37 (92 % sequence homology) and another of FA25 (92 % sequence homology). Three samples could not be HPV type determined; one was collected from a male and two from females.
Summary
The HPV DNA prevalence found in the Zambian samples was significantly lower than that of the samples collected in Sweden (P<0·01) and in Bangladesh (P<0·05).
All of the putative HPV types that have not been reported previously, together with the types most closely related to them, are presented in Table 2.
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). Only one HPV type, HPV-5, was detected in samples from all five countries and HPV-5 was found as the single type or together with one or more other HPV types or putative types in 16 of the 248 samples analysed (6·5 %). HPV-47 and FA25 were also common and were detected in samples from all countries except Zambia. Fifty-three (39 %) of the 137 HPV type-determined samples contained two or more HPV types or putative types: eight samples from Bangladesh, 14 from Japan, 18 from Sweden, four from Zambia and nine from Ethiopia.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2003) also applies to people from other parts of the world. The quality of the cellular human DNA as determined by PCR amplification of the human L1 repetitive DNA sequence varied significantly (P<0·001) between the samples collected from the different countries, ranging from five negative Swedish samples to 29 in Zambia and 31 in Ethiopia and Japan. The Zambian, Ethiopian and Japanese samples had been handled without a cold chain, which might have caused the observed decrease in the number of samples positive in the human DNA test. However, even in samples that were negative in the human DNA PCR test, we were able to detect HPV DNA at as high a prevalence as in the human DNA-positive samples, indicating that the viral DNA contained in the virions was protected against DNA degradation, contrary to the human cellular DNA in the samples.
Altogether, 88 different HPV types or putative types were found and of these 38 were detected in at least two countries. However, the majority of the HPV types and putative types were seen in one of the countries only. Furthermore, of the 137 HPV type-determined specimens, 53 (39 %) contained two or more HPV types or putative types each.
It is worth noting that five different subtypes of previously described HPV types or putative types were found in specimens from Bangladesh, Zambia and Ethiopia. In our previous studies spanning some 1000 HPV type determinations by DNA sequencing, we have only been confronted with putative HPV subtypes twice (Antonsson et al., 2000), which involved 90–98 % sequence homology with a previously described HPV type. However, the two putative types that were found previously had a sequence homology close to 98 %, while the sequence homology of the subtypes detected in this study was between 91 and 92 %.
The findings in this and previous studies together emphasize the ubiquity and impressive multiplicity of genotypes amongst the skin papillomaviruses, both in humans (Antonsson et al., 2000, 2003) and in animals (Antonsson & Hansson, 2002). Adapted to their hosts presumably over hundreds of millions of years, these viruses are the first viral skin commensals to be described.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Antonsson, A., Forslund, O., Ekberg, H., Sterner, G. & Hansson, B. G. (2000). The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses. J Virol 74, 11636–11641.
Antonsson, A., Karanfilovska, S., Lindqvist, P. & Hansson, B.G. (2003). General acquisition of human skin papillomavirus infections occurs in early infancy. J Clin Microbiol (in press).
Astori, G., Lavergne, D., Benton, C., Hockmayr, B., Egawa, K., Garbe, C. & de Villiers, E. M. (1998). Human papillomaviruses are commonly found in normal skin of immunocompetent hosts. J Investig Dermatol 110, 752–755.[CrossRef][Medline]
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Received 18 September 2002;
accepted 10 March 2003.
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