1 Department of Molecular Virology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuou-ku, Chiba 260-8670, Japan
2 Division of Virology, Chiba Prefectural Institute of Public Health, 666-2 Nitona-cho, Chuou-ku, Chiba 260-8715, Japan
Correspondence
Yoshimi Tomita
tomita{at}faculty.chiba-u.jp
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this paper are AY300817 (BAPV1), AY300818 (BAPV2), AY300819 (BAPV3), AY426550 (BAPV4), AY426551 (BAPV5), AY426552 (BAPV6), AY426553 (BAPV7), AY426554 (BAPV8), AY426555 (BAPV9), AY426556 (BAPV10), AY300820 (BAPV11MY) and AY555237 (BAPV6MY).
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INTRODUCTION |
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The PCR primer pair FAP59/FAP64 was designed from two relatively conserved regions found in the L1 gene of most HPV types to detect a broad spectrum of HPV types in cutaneous tumours and normal skin (Forslund et al., 1999). Using this primer pair, it was shown that HPV is a divergent group and is found ubiquitously and subclinically in human healthy skin, suggesting a commensal nature of these viruses (Antonsson et al., 2000
, 2003a
, b
). The FAP59/FAP64 PCR has also been shown to amplify papillomavirus (PV) DNA in the healthy skin of many animal species including cows, in which five putative BPV types (BAA1 to -5) have been detected in forehead skin (Antonsson & Hansson, 2002
). The PCR primer pair MY09/MY11 was originally designed to detect mucosal HPV types (Manos et al., 1989
) and has been reported to amplify the L1 gene of most genital HPV types (Bernard et al., 1994
; Chan et al., 1995
; Kado et al., 2001
), as well as some animal PV types, including BPV-1, -3, -5 and -6 (Chan et al., 1995
).
In this study, we showed the prevalence of BPVs and putative BPVs in teat papillomas and healthy teat skin by PCR using the primer pairs FAP59/FAP64 and MY09/MY11, and also detected 11 putative new BPV types.
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METHODS |
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DNA extraction.
The frozen specimens were homogenized under liquid nitrogen. DNA was extracted from papilloma specimens and swab samples using the High Pure PCR template preparation kit (Roche Diagnostics). Purified DNAs were eluted from the column in a volume of 200 µl and 2·5 µl was used for each PCR.
PCR.
DNA samples were amplified using two PCR primer pairs: FAP59 (forward; 5'-TAACWGTIGGICAYCCWTATT-3') and FAP64 (reverse; 5'-CCWATATCWVHCATITCICCATC-3') (Forslund et al., 1999); and MY11 (forward; 5'-GCMCAGGGWCATAAYAATGG-3') (Manos et al., 1989
) and MY09 (reverse; 5'-CGTCCMARRGGAWACTGATC-3'). PCR was carried out in a volume of 50 µl containing 2·5 µl DNA sample, 0·25 µM of each primer, 200 µM dNTP mixture, 5 µl 10x EX Taq buffer and 1·25 U EX Taq polymerase (Takara Shuzo). For the primer pair FAP59/FAP64, after an initial denaturation at 94 °C for 10 min, the PCR consisted of 45 cycles of 1·5 min at 94 °C, 1·5 min at 50 °C and 1·5 min at 72 °C, followed by extension at 72 °C for 5 min. For the primer pair MY09/MY11, after an initial denaturation at 95 °C for 5 min, the PCR consisted of 35 cycles of 30 s at 95 °C, 30 s at 55 °C and 1 min at 72 °C, followed by extension at 72 °C for 5 min. The mixed-primer PCR using FAP59 and MY09 was carried out under the same conditions used for the primer pair FAP59/FAP64. Five microlitres of the amplified material was analysed by electrophoresis on a 1·5 % agarose gel in TBE buffer (Sambrook & Russell, 2001
), followed by staining with ethidium bromide for 30 min. PV DNA-specific bands were identified by size determination under UV light.
Sequence analysis.
The PCR products were purified using the High Pure PCR products purification kit (Roche Diagnostics) and direct sequencing was carried out using the ABI PRISM Big Dye Terminator cycle sequencing kit (Applied Biosystems) using an ABI Genetic Analyzer 310 (Applied Biosystems) with forward and reverse primers. Some PCR products were cloned using the TOPO TA Cloning kit (Invitrogen). Six to ten clones from each PV-positive sample were isolated and sequenced. The forward and reverse complementary sequences were aligned using SEQED computer software (version 1.0.3). The DNA sequences were compared with all sequences in GenBank through the BLAST server (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/blast/). The percentage similarity of the PV sequences determined in this work in comparison with previously determined PV sequences was estimated using GENETYX computer software (version 11). The partial sequences of the BPV-6 L1 gene were determined after PCR amplification using FAP59/FAP64 and MY09/MY11.
The guidelines from the Papillomavirus Nomenclature Committee 1995 (14th International Papillomavirus Conference, Quebec City, Quebec, Canada) were followed to identify putative new PV types. An isolate was defined as a new PV type if the sequence of its L1 gene displayed less than 90 % similarity with the L1 genes of all types already known. The isolated novel sequences were called putative new PV types instead of PV types, since the PCR products represent only part of the L1 gene (de Villiers, 2001; Antonsson & Hansson, 2002
). In this study, the putative new PV types that were amplified using primers FAP59/FAP64 were designated BAPV1 to -10. The DNA sequences of the putative new PV types that were amplified using primers MY09/MY11 were designated BAPV6MY and BAPV11MY.
Alignment of amino acid sequences and phylogenetic analysis of DNA sequences.
Sequences of 9698 aa from FAP59/FAP64 PCR-amplified DNA segments corresponding to nt 55285821 of BPV-3 and 96 aa sequences of MY09/MY11 PCR-amplified DNA segments corresponding to nt 64466733 of BPV-3 were selected and used for the alignment. The CLUSTAL X multiple alignment program (version 1.83) (Thompson et al., 1997) was used for these alignments. Phylogenetic analysis was based on the neighbour-joining method. The DNA sequences of the HPV and animal PV types were selected by similarity searches with the DNA sequence corresponding to nt 54845933 and nt 62936753 of BPV-3. The former region was used for phylogenetic analysis of BAA1 to -5 and BAPV1 to -10 and the latter region was used for phylogenetic analysis of BAPV6MY and BAPV11MY. The phylogenetic tree was constructed using TreeView version 1.6.6.
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RESULTS |
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DISCUSSION |
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Recently, it has been reported that a large number of putative HPV types and putative animal PV types have been isolated by PCR from the healthy skin of humans and other animals (Antonsson & Hansson, 2002; Antonsson et al., 2000
, 2003a
, b
; Astori et al., 1998
; Boxman et al., 1997
). These reports showed latent or subclinical infection of skin PV and a commensal nature of PV. BPV-1 to -6 were originally isolated from bovine papillomas and these have been thought to be a causal agent of papillomatosis (Campo, 2002
; Jarrett et al., 1984
). In this study, however, genome DNA of BPV-1, -3 and -6, BAA1 and -5 and most putative new BPV was detected in swab samples of healthy teat skin on which papilloma was not apparent, suggesting latent or subclinical infections of these BPV types. In addition, the potential to induce tumours caused by BAPV2 and -4, which are relatively close to BPV-5 in the phylogenetic analysis, remains to be elucidated.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 4 March 2004;
accepted 1 April 2004.
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