Journal of General Virology (2008), 89, 158163

DOI 10.1099/vir.0.83334-0

Short Communication
Correspondence Shinichi Hatama hatama@affrc.go.jp

Genomic and phylogenetic analysis of two novel bovine papillomaviruses, BPV-9 and BPV-10
Shinichi Hatama,1 Kiyoko Nobumoto2 and Toru Kanno1
1

Hokkaido Research Station, National Institute of Animal Health, 4 Hitsujigaoka, Toyohira, Sapporo 062-0045, Japan Tokachi Livestock Hygiene Service Center, 59-6 Kisen, Kawanishi, Obihiro 089-1182, Japan

Received 24 July 2007 Accepted 1 October 2007

Eight bovine papillomavirus (BPV) types, BPV-18, have been classified, based on genome nucleotide sequence similarities, in the genera Deltapapillomavirus (BPV-1 and -2), Epsilonpapillomavirus (BPV-5 and -8), Xipapillomavirus (BPV-3, -4 and -6) and an unassigned genus (BPV-7). We report here the complete genome sequence of two new BPV types isolated from separate epithelial squamous papilloma lesions on cattle teats. The genomes are 7303 and 7399 bp in length, respectively, and both have genetic organization and consensus motifs typical of papillomaviruses. A neighbour-joining phylogenetic tree revealed that both viruses cluster with BPV-3, -4 and -6. Nucleotide sequence identities of the BPV L1 major capsid protein of these two new BPVs with BPV-3, their closest relative, are 74.2 and 71.2 %, respectively. These results suggest that both viruses are new BPV types in the genus Xipapillomavirus, and they are designated BPV-9 and BPV-10.

Bovine papillomavirus (BPV) is an aetiological agent associated with several forms of cutaneous and mucosal papillomas (Campo, 2002). Six different types of BPVs have been distinguished on the basis of DNA sequence relatedness. Each BPV is associated with type-specific lesions (Jarrett et al., 1984): BPV-1 and BPV-2 are classified in the genus Deltapapillomavirus and infect the epithelium and dermis, giving rise to fibropapillomas; BPV-3, BPV-4 and BPV-6 are classified in the genus Xipapillomavirus and are strictly epitheliotropic, inducing true epithelial papillomas, and BPV-5 is classified in the genus Epsilonpapillomavirus and infects the epithelium and dermis, inducing both fibropapillomas and true epithelial papillomas of the skin (Bloch et al., 1994; Campo 2002; de Villiers et al., 2004). Recently, 16 putative new BPV types were partially cloned and sequenced from healthy skin swabs (Antonsson & Hansson, 2002; Ogawa et al., 2004). Two of the BPVs were characterized further by cloning and sequencing their complete genomes. Phylogenetic analysis showed that both viruses were new BPV types: one was designated BPV-7 and classified as a member of a new papillomavirus (PV) genus, and the other was designated BPV-8 and classified as a member of the genus Epsilonpapillomavirus (Ogawa et al., 2007; Tomita et al., 2007). However, the tumorigenic potential of these two new BPV types and the other 14 putative new BPV types
The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AB331650 (BPV-9) and AB331651 (BPV-10). Supplementary material is available with the online version of this paper.

has not been elucidated. In a previous report, we identified two putative new BPV types, designated BPV-Type I and BPV-Type II, which may have caused an outbreak of bovine teat papillomatosis in Japan (Maeda et al., 2007). BPV-Type I and II were identified by PCR amplification of a 590 bp DNA fragment within the BPV genome L1 major capsid protein gene (GenBank accession no. AB253592 for type I; no. AB253595 for type II). However, the remaining BPV-Type I and II genome sequences have not been reported. In this study, the complete genome sequences of BPV-Type I and II were determined. These sequencing and phylogenetic analysis results showed that BPV-Type I and II are new BPV types (designated BPV-9 and BPV-10, respectively), classified in the genus Xipapillomavirus. For these studies, 25 separate teat squamous papilloma lesion samples from an epidemic of teat papillomatosis of cattle in Japan (Maeda et al., 2007) and 20 healthy teat skin swab samples were examined for BPV. Total DNAs were extracted from the teat squamous papilloma lesion samples using SepaGene DNA extraction kits (Sankyo Jyunyaku) and from the teat skin swab samples using TRIzol-LS reagent (Invitrogen), according to the manufacturers instructions. The extracted DNAs were dissolved in 50 ml TE buffer and used for PCR amplification. Phylogenetic analysis of PCR amplicons of a fragment of the PV L1 major capsid protein suggested that BPV-Type I and II were closest to Xipapillomavirus (BPV-3, -4 and -6) (Maeda et al., 2007). Therefore, two sets of PCR primers were designed for specific amplification of BPV-Type I and II based on the nucleotide sequences of relatively conserved regions of Xipapillomavirus genomes, such that the two
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BPV-9 and BPV-10

amplicons for each BPV covered its complete genome. The two primer sets for amplification of the complete BPVType I genome were BPV9A-F1 (forward: 59-TGTCATTAATATTTCAGCAAG-39) and BPV9A-R1 (reverse: 59-TTCATTATAACCACTGTCGTC-39), and BPV9B-F1 (forward: 59-CAAGGGAATTCTGCAGAATTG-39) and BPV9B-R1 (reverse: 59-CCACCTATTCGCAAGCCACT39) (Fig. 1). The two primer sets for amplification of the complete BPV-Type II genome were BPV10A-F1 (forward; 59-CTCTAGGGAGAAAGTTCCTG-39) and BPV10A-R1 (reverse; 59-TCTGACACTCTCGAAAGAGG-39), and BPV10B-F1 (forward; 59-CTCCAAGCCCGTTTCGATGC39) and BPV10B-R1 (reverse; 59-TTMCGCCTACGCTTTGGCGC-39) (Fig. 1). The final volume of the PCR mixture (50 ml) contained 1 ml extracted DNA, 0.25 mM each primer, 200 mM dNTPs, 2.5 mM MgCl2, 5 ml 106 LA Taq buffer and 1.25 units LA Taq DNA polymerase (TaKaRa Bio). PCR was performed by heating to 94 uC for 5 min, thermocycling at 94 uC for 45 s, 55 uC for 30 s and 72 uC for 4 min for 30 cycles, and a final extension at 72 uC for 4 min. Use of these primer sets for long accurate PCR produced two overlapping fragments encompassing the entire BPV-Type I genome and two encompassing the entire BPV-Type II genome. The reaction mixtures were analysed by electrophoresis in 0.8 % agarose gels with ethidium bromide and detected by UV transillumination. Amplicons of the appropriate sizes were excised and purified using QIAquick Gel Extraction Kits (Qiagen). Gelpurified PCR products were cloned into the pCR-XLTOPO vector (Invitrogen). Plasmid clones with inserts of the expected sizes were expanded and purified using QIAprep Spin Midiprep Kits (Qiagen). Initial nucleotide

sequencing of the extracted plasmids was performed with M13 universal primers using an ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI 3130 Genetic Analyzer (Applied Biosystems). Subsequent sequencing was performed by primer walking. Details of the primers used for primer walking are shown in Supplementary Table S1, available with the online version of this paper. Nucleotide sequences were determined for both BPV strands, and the forward and reverse complementary sequences were aligned using GENETYX software (version 13; SDC) for verification. In this way, both complete BPV genome nucleotide sequences were determined. Putative open reading frames (ORFs) and their amino acid sequences were predicted with the National Center for Biotechnology Information (NCBI) ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Peptide motif searches were performed with the TFSEARCH tool of the Parallel Protein Information Analysis system (http://www. cbrc.jp/research/db/TFSEARCHJ.html). Similarity searches were performed with NCBI BLAST software (http://blast. ddbj.nig.ac.jp/top-j.html) and GenBank. Multiple nucleotide sequence alignments were carried out by the neighbourjoining method (Thompson & Neel, 1997) using the CLUSTAL W multiple sequence alignment program (version 1.83), and a phylogenetic tree was constructed using Tree View (version 1.6.6). In these studies, BPV-Type I amplicons were detected in 8 of the 25 (32 %) papilloma lesion samples and in none of the 20 healthy teat skin swab samples. BPV-Type II amplicons were detected in 6 of the 25 (24 %) papilloma lesion samples and in none of the 20 healthy teat skin swab

Fig. 1. Genome organization of BPV-9 (BPVType I) and BPV-10 (BPV-Type II). The circular viral DNAs are represented in their linear form for simplicity. The boxes represent the early (E) and the late (L) ORFs. The PCR-amplified fragments used for molecular cloning and nucleotide sequencing are indicated by bold lines. The long control regions (LCR) encompassing nucleotides 7158323 for BPV-9 (BPV-Type I) and 7006101 for BPV-10 (BPV-Type II) are represented in expanded form below the genome. $, Polyadenylation signals; g, E2-binding sites; e, E1-binding sites; m, TATA box of the viral promoters. http://vir.sgmjournals.org 159

S. Hatama, K. Nobumoto and T. Kanno

samples. These results suggest that BPV-Type I and II are associated with teat papilloma lesions. Complete genome sequences of BPV-Type I and II were obtained from the cloned overlapping amplicons, which were isolated from separate epithelial squamous papilloma lesions of teats. BPV-Type I and II genomes were 7303 and 7399 bp with G+C contents of 44.56 and 43.53 %, respectively. All of the putative BPV-Type I and II ORFs were located on the same strand, and both genome organizations were similar to the known PVs. Both BPV genomes contained four early (E1, E2, E5 and E7) and two late (L1 and L2) ORFs, separated by a long control region (LCR) (Fig. 1). In addition, ORF E4 was found in the early region of the BPV-Type II genome, but not in the BPVType I genome (Fig. 1). LCR is the region between the early and late ORFs and contains several PV replication regulatory elements. Virusencoded E1 and E2 proteins are essential for efficient initiation of viral DNA replication and bind cooperatively to adjacent sites in the virus replication origin in the LCR. PVs usually have one E1-binding site (E1BS: TTATTGTTGTTAACAAT) flanked by two E2-binding sites (E2BS: ACCG-N4-CGGT) for binding of an E1/E2 complex. PVs additionally have one or two other E2BS in the LCR. The BPV-Type I LCR had two typical E2BS, one modified E2BS (ACCT-N4-CGGT) and one modified E1BS (TAACAA) at nt 171182, 262273, 247258 and 216221, respectively (Fig. 2a). The BPV-Type II LCR had one typical E2BS, two modified E2BS (ACCA-N4-CGGT and ACCG-N4-AGGT) and one modified E1BS (TAACAA) at nt 73397350, 1526, 3142 and 73837388, respectively, corresponding to the sites in BPV-Type I (Fig. 2a). Some other important elements were found to be conserved in both BPV genomes. At the 59 end of the LCR, there were polyadenylation (polyA) sites (AATAAA) for late mRNA at nt 72317236 in BPV-Type I and nt 70797084 and 72487253 in BPV-Type II (Fig. 2a). TATA boxes [TATA(A/T)A(A/T)] were at the 39 end of the LCR, at nt 279283 in BPV-Type I and nt 4650 in BPV-Type II (Fig. 2a). However, a second non-coding-region was not found in either the BPV-Type I or II genome, although it has been identified in certain artiodactyla and carnivore PVs; e.g. European elk PV (EEPV), deer PV (DPV), reindeer PV (RPV), canine oral PV and Felis domesticus PV (Groff & Lancaster, 1985; Delius et al., 1994: Tachezy et al., 2002; Rector et al., 2005). The early region of the PV genome encodes viral regulatory proteins necessary for initiation of virus replication. Five ORFs (E1, E2, E4, E6 and E7) are present in almost all PV genomes. Furthermore, BPV-1 and -2 contain ORF E5 (localized in the early and late regions of the genome) (Schiller et al., 1986), and BPV-3, -4 and -6 contain ORF E5 (localized in the E6 region in the genome) but lack ORF E6 (Jackson et al., 1996; Morgan & Campo, 2000). Since BPV-Type I and II genomes contained ORF E5 in the E6 region and lacked ORF E6, they were similar to BPV-3, -4
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and -6. This genetic organization is characteristic of Xipapillomavirus, suggesting that BPV-Type I and II are members of the genus Xipapillomavirus. Motifs in PV ORFs E7 and E5 that are characteristic of the Xipapillomavirus were also found in BPV-Type I and II. The BPV-Type I and II E7 ORF contained two conserved motifs, retinoblastoma tumour-suppressor protein-binding domain (pRbBD: LxCxE) and zinc-binding domain [ZnBD: CxxC(x)29CxxC] (Fig. 2b), that are responsible for immortalization and transformation of host cells and are found in most PVs, including BPV-3, -4 and -6 (Chan et al., 2001; Dahiya et al., 2000; Dick & Dyson, 2002). However, the pRbBD motif is not found in ORF E7 of some artiodactyla PVs, including BPV-1, BPV-2, BPV-5, EEPV and DPV (Narechania et al., 2004). All the viruses with an E7 lacking the pRb-binding domain are fibropapillomaviruses, which may have biological significance. The E5 protein of BPV-Type I and II had a hydrophobic transmembrane domain consisting of 23 aa and a hydrophilic domain that included the conserved C-terminal amino acids LxGWD (Fig. 2c). Similar motifs were reported in the E5 ORF of BPV-1 and BPV-2. Based on the biological function of the BPV-1 E5 protein, these motifs might be responsible for downregulation of major histocompatibility complex (MHC) class-I and thus evasion of the host immune response (Ashrafi et al., 2002; Marchetti et al., 2002, 2006). ORF E1 of both BPVType I and II contained a conserved ATP-binding site (GPPDTGKS) in its ATP-dependent helicase C-terminal part (Titolo et al., 1999). However, apparent differences between BPV-Type I and II were seen in ORF E4. ORF E4 was found in the BPV-Type II genome, completely overlapping ORF E2 but in a different reading frame, and was not found in the BPV-Type I genome. E4 protein is usually translated from spliced E1^E4 transcripts and contains the first five amino acids of the E1 protein fused to the E4 coding sequence (Doorbar et al., 1990). E1^E4 protein is abundantly expressed in the cytoplasm of terminally differentiated epithelial cells in the productive phase of the viral life cycle (Doorbar et al., 1997). E1^E4 has also been implicated in inducing apoptosis (Raj et al., 2004), causing G2 arrest in the cell cycle (Davy et al., 2005; Nakahara et al., 2002) and aiding successful virus replication (Roberts et al., 2003) or viral egress (Bryan & Brown, 2000; Wang et al., 2004). However, the role of E1^E4 in the PV life cycle is still speculative. The FAP59/FAP64 region in the L1 ORF is generally used for detection and classification of PV types (Forslund et al., 1999). Therefore, a molecular phylogenetic analysis was carried out using nucleotide sequence alignment of the FAP59/FAP64 region of BPV-Type I, BPV-Type II and the other eight BPV types. The results (Fig. 3) showed that BPV-Type I and II are members of the genus Xipapillomavirus closely related to BPV-3, BPV-4 and BPV-6. Pairwise sequence identities of the entire L1 ORF between BPV-Type I, BPV-Type II and the other BPV types are shown in Supplementary Table S2, available with the
Journal of General Virology 89

BPV-9 and BPV-10

Fig. 2. (a) Aligned LCR nucleotide sequences of BPV-9 (BPV-Type I), BPV-10 (BPV-Type II) and one of the closest related species, BPV-4. Numbers represent nucleotide positions in the LCR. Polyadenylation signals (polyA) are underlined. One E1-binding site (E1BS) and two E2-binding sites (E2BS) are shown in the open boxes. TATA boxes (TATA) are shown in the shaded box. (b) Aligned amino acid sequences of the E7 protein of BPV-9 (BPVType I), BPV-10 (BPV-Type II) and BPV-4. pRb, Retinoblastoma tumour-suppressor protein-binding domain; ZnBD, zinc-binding domain. (c) Aligned amino acid sequences of the E5 (formerly E8) protein of BPV-9 (BPVType I), BPV-10 (BPV-Type II) and BPV-4. The LxGWD motif is shown in an open box. The shaded box denotes a transmembrane domain.

online version of this paper. The entire L1 ORF of BPVType I and II had 71.974.2 % and 70.471.2 % sequence identity, respectively, with Xipapillomavirus BPV-3, -4 and -6. The L1 ORF of the other BPV types had sequence identities of 55.757.8 % and 56.457.6 % with BPV-Type I and II, respectively. The International PV Workshop in Quebec in 1995 defined an isolate as a new PV type if the complete genome of the PV had been cloned and the DNA sequence of the L1 ORF differed by more than 10 % from the closest known PV type (de Villiers et al., 2004). Different PV genera share less than 60 % nucleotide sequence identity in the L1 ORF. Based on this criterion, our results indicate that both BPV-Type I and II
http://vir.sgmjournals.org

are new PV types in the genus Xipapillomavirus and, therefore, they are designated BPV-9 and BPV-10, respectively. It is noteworthy that the two new BPVs isolated and characterized in this study are easily isolated from teat papilloma lesions, but not from healthy teat swabs. Although further study is needed to determine if BPV-9 and BPV-10 are specifically associated in the aetiology of teat papillomas and the current disease epidemic in Japan, our results provide important new information for establishment of effective molecular diagnostic techniques and biological control measures.
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S. Hatama, K. Nobumoto and T. Kanno

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Acknowledgements
We would like to thank Ms. R. Ishihara, Hokkaido Research Station, National Institute of Animal Health, for technical help with genetic analysis. This work was partially supported by the National Institute of Animal Health, Grant-in-Aid for Specially Weighted Research.

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