Multiple evolutionary origins of bat papillomaviruses

Veterinary Microbiology 165 (2013) 51–60

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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Multiple evolutionary origins of bat papillomaviruses Raquel Garcı´a-Pe´rez a, Marc Gottschling b, Gudrun Wibbelt c, Ignacio G. Bravo a,* a

Infections and Cancer, Catalan Institute of Oncology (ICO)jBellvitge Institute of Biomedical Research (IDIBELL), Barcelona, Spain Ludwig-Maximilians-University Munich, GeoBio-Center, Department Biology, Systematic Botany and Mycology, Germany c Leibniz Institute for Zoo and Wildlife Research, Department of Wildlife Diseases, Berlin, Germany b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 October 2012 Received in revised form 22 December 2012 Accepted 4 January 2013

Infection by papillomaviruses (PVs) has been linked to different types of neoplasias, in both human and non-human hosts. Knowledge about PV diversity is essential to reliably infer the evolutionary history of these pathogens and to elucidate the link between infection and disease. We cloned and sequenced the complete genome of a novel PV, EhelPV1, isolated from hair bulbs from a captive straw-colored fruit bat Eidolon helvum (Pteropodidae, Chiroptera). We also retrieved partial sequences of the E1 and L1 genes from hair bulbs from a captive Indian flying fox Pteropus giganteus (Pteropodidae, Chiroptera). The detected virus (PgigPV1) presumably corresponded to a novel type as well. Maximum likelihood phylogenetic analyses were conducted using a representative collection of 132 PVs. EhelPV1 belonged to the Lambda+Mu-PV crown group and was most closely related to another bat PV, MschPV2. Both fragments of PgigPV1 were placed alongside with EhelPV1. The novel PVs were phylogenetically distant from other previously described bat PVs, namely MrPV1, MschPV1 and RaPV1. We have further characterized the sequence patterns of the E2-binding sites occurring in the upstream regulatory region of Lambda+Mu-PVs. Common fingerprints within this region are shared by certain PVs. However, there is not a sharp correspondence between the repertoire of transcription factor binding sites in the viral regulatory region and host range, tissue tropism or viral life style. Our results reinforce the hypothesis that PVs have undergone an initial radiation prior to the divergence of the mammalian hosts, giving rise to the presentday PV crown groups. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Bat E2 binding sites Evolution Papillomavirus Phylogeny

1. Introduction The aetiological role of certain papillomaviruses (PVs) in a number of human cancers has largely driven PV research and more than 150 human PVs have already been cloned and described. Although humans are the only intensively studied host, it is likely that non-human PVs outnumber by far HPV diversity (Antonsson and Hansson,

* Corresponding author at: Infections and Cancer Laboratory, Research Program in Cancer Epidemiology, Catalan Institute of Oncology (ICO), Avda. Gran Vı´a 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain. E-mail address: [email protected] (I.G. Bravo). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.01.009

2002; Mengual-Chulia´ et al., 2012). PVs from only 51 different mammalian host species and six sauropsid PVs have been isolated and completely sequenced. In fact, the more thorough a given host species is sampled, the larger is the number of known PV genotypes, as it is the case for cows (Hatama et al., 2011), dogs (Lange et al., 2011c), dolphins and porpoises (Gottschling et al., 2011a), horses (Lange et al., 2011b), macaques (Chen et al., 2009), rodents (Schulz et al., 2012), and sheep (Alberti et al., 2012). Some non-human PVs are involved in the development of pathological lesions. Animal PV infection has been likewise linked to dermal and mucosal neoplastic lesions in bats (RaPV1) (Rector et al., 2006a), cats (FcaPV2) (Lange et al., 2009b), dogs (CPV1, CPV3, and CPV7) (Delius et al., 1994; Lange et al., 2009a; Tobler et al., 2006), horses (BPV1,

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BPV2, and EcPV2) (Bogaert et al., 2012; Nasir and Campo, 2008; Scase et al., 2010), rodents (McPV2) (Nafz et al., 2008), rabbits (SfPV1) (Giri et al., 1985), and sheep (OvPV3) (Alberti et al., 2012). The incidence of PV infections may thus have significant consequences for certain animal species and could even lead to a conservational and economic negative impact, as has been suggested for manatees (Woodruff et al., 2005) and horses (Hainisch et al., 2012), respectively. Bats are important reservoirs for a number of mammalian viruses with demonstrated or potential consequences for human and/or veterinary health, and recent efforts have focused on the characterization of novel viruses infecting bats (Tse et al., 2012; Wu et al., 2012). The Chiroptera are the second species-richest mammalian order, with over 1300 bat species, but hitherto only four bat PVs have been completely sequenced. RaPV1 has been isolated from the Egyptian fruit bat Rousettus aegyptiacus (Rector et al., 2006a); MrPV1 and MschPV1 have been respectively isolated from a Rickett’s bigfooted bat Myotis ricketti and a Schreiber’s long-fingered bat Miniopterus schreibersii (Wu et al., 2012), while MschPV2 has been obtained from another M. schreibersii individual (Tse et al., 2012). These four viruses are only distantly related, have divergent phylogenetic positions among the Papillomaviridae, and do not share an exclusive common ancestor. Here, we describe the complete sequence of a novel PV (EhelPV1) obtained from hair follicles from a captive straw-colored fruit bat E. helvum. We also report partial sequences from the E1 and L1 genes obtained from hair follicles of an Indian flying fox Pteropus giganteus, which could correspond to a novel PV (PgigPV1). The characterization of novel PVs expands our knowledge of these viruses and contributes to the development of a comprehensive scenario for PV evolution. 2. Methods 2.1. Genome amplification, sequencing and cloning Hair follicles together with hair bulbs were collected from healthy facial skin of a captive adult female straw-colored fruit bat (E. helvum) and of a captive adult female Indian flying fox (P. giganteus). Both animals had died of unrelated causes and were submitted for necropsy to the Leibniz Institute for Zoo and Wildlife Research. They were opportunistically sampled for PV investigations. Genomic DNA extraction and amplification with CP and FAP primers were performed as previously described (Mengual-Chulia´ et al., 2012). Specific primers for long template PCRs were designed based on these preliminary sequences. The complete viral genome was amplified using inverted tail-to-tail primers located within the sequence of L1 using Phusion Hot Start High-Fidelity DNA polymerase (Finnzymes). Both strands of amplicons were sequenced by primer walking using forward and reverse primers, and each position was read at least twice in each direction using the Sanger method. Chromatogram sequencing file inspection and contig edition was

performed using the STADEN Package. Two overlapping fragments were used to clone the complete EhelPV1 genome using the CloneJetTM PCR Cloning Kit (Fermentas). Two clones per overlapping fragment were selected and sequenced to further confirm the PV genome sequence. A description of the primers sequences, amplicon sizes, and PCR conditions is provided in Table S1. A schematic illustration of the strategy followed for the amplification and cloning of the EhelPV1 complete genome is displayed in Fig. S1. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.01.009. 2.2. Genomic and protein sequence annotation The open reading frames (ORFs) existing in EhelPV1 genome were determined with the ORF Finder tool on the NCBI server (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Transcription factor binding sites (TFBS) were predicted using MATCH (Wingender et al., 2000) and were further confirmed using CISTER (Frith et al., 2001). The MEME algorithm (Bailey et al., 2009) was used to identify E2 binding site (E2BS) sequence patterns occurring in the upstream regulatory region (URR) of Lambda+Mu-PVs. Pairwise identities and similarity values for nucleotide and protein sequences were calculated using the EMBOSS Needle software (http://www.ebi.ac.uk/Tools/psa/). 2.3. Phylogenetic analyses Phylogenetic relationships of EhelPV1 and PgigPV1 within Papillomaviridae were examined using a set of 132 PVs covering the currently known PV diversity. This set included representatives of all HPVs species and all nonhuman PVs, except for PVs infecting Rhesus macaque (Macaca mulatta), for which only species-representative MfPVs were considered: MfPV1 representing the Beta-PV species 1 and MfPV2 representing the Beta-PV species 6. A comprehensive description of PVs employed for phylogenetic inference is provided in Table S2. For this selection, the E1, E2, E6, E7, L1, and L2 gene sequences were analyzed. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.01.009. Phylogenetic analyses were conducted as previously described (Gottschling et al., 2011b). Briefly, individual genes were aligned at the amino acid level using MUSCLE and the corresponding nucleotide alignments were filtered with GBLOCKS. Phylogenetic relationships for the individual and concatenated alignments were calculated in a maximum likelihood framework using RAXML v7.2.8 at both levels nucleotide and amino acid. The GTR+G4 nucleotide substitution model and the LG amino acid substitution model have been identified elsewhere as the best suited evolutionary models for the PV genes considered (Bravo et al., 2010; Gottschling et al., 2007). Phylogenies were reconstructed for each alignment considering the corresponding partitions: one per codon position per gene for nucleotide alignments; one per gene

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for amino acid alignments. The trees were rooted using the sequences of PVs retrieved from Aves and Testudines, as described (Gottschling et al., 2011b). Gene phylogenies at the nucleotide and amino acid level exclusively for Lambda+Mu-PVs were constructed as described above. The individual best-scoring ML trees for each gene were used to create a supernetwork employing SPLITSTREE v4. A phylogenetic network represents the incompatibilities within and between the included data sets. Evolutionary relationships are depicted by edges, where parallel edges symbolize the splits computed from the data. Netted regions correspond to ambiguous phylogenetic signal between individual gene phylogenies. The well-resolved amino acid E1E2L1 phylogeny was used as a scaffold, onto which the partial E1 and L1 sequences of PgigPV1 were introduced through an evolutionary placement algorithm (EPA) (Berger and Stamatakis, 2011) as previously described (Mengual-Chulia´ et al., 2012). FIGTREE v1.3.1 and SPLITSTREE v4 were employed to produce the graphical phylogenetic trees and networks, respectively. 3. Results 3.1. The EhelPV1 genome shows the characteristic features of Papillomaviruses The full-genome of EhelPV1 was obtained from hair bulbs from healthy skin of an adult pteropodid E. helvum. The sequence of the circular genome spanning 7891 bp was deposited in GenBank under the accession number JX123128. Viral DNA extracted from hair bulbs from an adult individual of the pteropodid species P. giganteus was used to amplify and sequence fragments of the E1 and the L1 genes, deposited in Genbank under the accession numbers JX988609 and JX988610, respectively. The genomic organization of the EhelPV1 genome and some of the regulatory elements are displayed in Fig. 1. EhelPV1 showed the classical PV genetic arrangement: six distinct ORFs on the same coding strand and with the same orientation. EhelPV1 contained the typical early proteins

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E6, E7, E1 and E2, the late proteins L2 and L1, plus the non-coding regulatory region between L1 and E6. An intergenic E2–L2 region was not detected, and thus there was no possible space for an E5 ORF. A canonical E4 ORF nested within E2 could likewise not be identified. However, a short sequence of 116 amino acids in length, with remnants of the proline-rich stretches that characterize E4, was detected. This putative E4 protein shared only a low degree of similarity (46%) with HPV4 and HPV95 E4 proteins. Three typical, palindromic E2BS with the consensus sequence ACCG-N4-CGGT were detected in EhelPV1: two of them appeared within the URR at nt positions 7269– 7280 and 7751–7762, while the third one was found within the L2 ORF, at nt position 4580–4591. Two TATA boxes were identified: one was located in the E6 promoter, at the 30 end of the URR (nt 7859), while the second one appeared within the L2 ORF (nt 4486). Three CCAAT boxes were found at nt positions 2991, 3082 and 5007. Up to three polyadenylation signals (AATAAA) were present in EhelPV1 genome sequence: two within the URR at nt positions 7472–7477 and 7481–7486, and one within the L2 ORF at positions 5164–5169. These sites appeared upstream of a CA dinucleotide and a G/T cluster. Additional binding sites for several putative cellular transcription factors involved in the maintenance of PV gene expression were identified (Butz and Hoppe-Seyler, 1993). Five AP-1 binding sites (TGACTCA) and three SP-1 binding sites (GGCGGG) were scattered throughout EhelPV1 genome. At the amino acid level, E6 contained one conserved zinc-binding domain (CX2CX29CX2C). A pRb-binding domain including the LXCXE motif (LNCYE), crucial for the binding of pRb, (Moran, 1993) was present in E7. The E1 ORF coded for the largest EhelPV1 protein and held the conserved ATP-binding site of the ATP-dependant helicase at its carboxy-terminal part: consensus sequence GXXXXGK(T/S), GPPDTGKS in EhelPV1. Both the major and minor capsid proteins (L1 and L2) displayed a series of positively charged arginine and lysine residues at their carboxy terminus (KRRR and KKRKRK respectively), which act as nuclear localization signals.

Fig. 1. Linear representation of the genomic arrangement of EhelPV1. Open reading frames are represented to scale and indicated in boxes. Information on molecular mass in kilodaltons and theoretical isoelectric point of the predicted proteins, as well as positions of the corresponding start and stop codons is included. Location of different cis regulatory elements is depicted (see legend).

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Fig. 2. Best-known maximum likelihood phylogenetic tree for the E1E2L1 gene concatenate both at the nucleotide and amino acid level. Numbers above the branches indicate the ML bootstraps support values (nucleotide/amino acid). Bootstraps support values above 95 are represented with an asterisk (*). Bootstraps support values below 50 are indicated with a dash (–). Color code highlights the four PV crown groups: red, Alpha+Omikron; green, Beta+Xi;

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3.2. EhelPV1 is a member of the Lambda+Mu-PV crown group We studied the evolutionary relationships of EhelPV1 and PgigPV1 among Papillomaviridae. The E1E2L1 gene concatenation yielded well-supported phylogenies both at the nucleotide and amino acid level, with the majority of nodes showing branch bootstrap support values above 90 (88–81%, respectively) (Fig. 2). PVs segregated into four major, well-supported PV crown groups (Alpha+Omikron-, Beta+Xi-, Delta+Zeta- and Lambda+Mu-PVs) plus 20 PVs (RaPV1, OvPV3, FdPV2, PmPV1, MnPV1, ZcPV1, TmPV1, MsPV1, CPV3, CPV4, CPV5, CPV8, CPV9, CPV10, CPV11), whose accurate evolutionary relationships could not be discerned. EhelPV1 was consistently placed within the monophyletic Lambda+Mu-PV crown group. However, the precise relationships between EhelPV1 and MschPV2, EdPV1, and HPV41 could not be completely resolved because of low statistical support. The EPA placed the partial sequences of the E1 and L1 genes of PgigPV1 along with EhelPV1, with the maximum posterior likelihood score value (1.0) (Fig. S2). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.01.009. Pairwise alignments at the nucleotide and amino acid levels were calculated for each of the six EhelPV1 ORFs and for the ORFs of a selected group of PV types: all members of the Lamba+Mu-PV crown group and all the previously isolated bat PVs. Alignment scores are provided in Table 1. EhelPV1 was only distantly related to the three bat PVs MrPV1, MschPV1, and RaPV1, as was also evident from their inferred phylogenetic positions in Fig. 2. The relationships within the Lambda+Mu-PV crown group were further analyzed constructing an evolutionary supernetwork as a complement to phylogenetic trees. To this purpose, we used the six best scoring ML trees that corresponded to each gene and combined them into the network shown in Fig. 3. The netted regions in the network represent the alternative evolutionary trajectories that the branching events among different taxa may have followed according to the individual phylogenetic reconstructions. The results show that deep relationships among members of this crown group were often inconsistent for different genes, evidenced in the central netted region. Further, the relative position of CPV1 among Lambda-PVs could not be inferred with certainty. 3.3. E2 binding sites in Lambda+Mu-PVs We analyzed the conservation and diversity of the E2BS for the Lambda+Mu-PV crown group. The consensus E2BS sequences and occurrences within the URR of Lambda+Mu-PVs are depicted in Fig. 4. We identified up to six different versions of the canonical E2 binding sequence.

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Five of these versions corresponded to changes that break the palindromic nature of the E2BS. Regarding the bat PVs in this crown group, EhelPV1 and MschPV1, the E2BS in the URR correspond to the canonical one. Interestingly, none the E2BS found in the closely related HPV41 and EdPV1 corresponded to the canonical one. 4. Discussion We communicate here the genetic characterization of EhelPV1, the fifth PV isolated from hosts belonging to the Chiroptera. EhelPV1 presents the characteristic PV genomic organization containing four early genes, two late genes, and a non-coding region. Several nucleotide and amino acid consensus motifs known to play a role in the PV life cycle are also present in EhelPV1 genome (Fig. 1). We also report partial sequences from the E1 and L1 genes from a second novel bat PV, PgigPV1. The L1 ORF is classically used for PV classification (de Villiers et al., 2004), but the revised guidelines for PV taxonomy have recommended interpreting raw similarity data on the light of phylogeny, genome organization, biology, and pathogenicity (Bernard et al., 2010), with the global aim of integrating evolution within taxonomy (Bravo et al., 2010). In the case of EhelPV1, the percentage of nucleotide identities in the L1 gene is not enough to determine whether it belongs to a particular genus within the Lambda+Mu-PV crown group. Originally, the taxonomic category ‘‘genus’’ had been defined for PVs sharing L1 nucleotide identities above 60% (de Villiers et al., 2004). Following this criterion, EhelPV1 would belong either to the Lambda-, Kappa-, Nu-, or Sigma-PVs, since its L1 gene presents nucleotide identities above 60% when compared to different members of these genera. However, in the reconstructed phylogenetic trees, EhelPV1 cannot be ascribed to any of these taxa. Our phylogenetic analyses in Figs. 2 and 3 show instead that both EhelPV1 and MschPV2 share a common ancestor with EdPV1 and HPV41, indicating that they should be accommodated into two different PV genera within the Lambda+Mu-PV crown group. It is a matter of debate whether the current PV naming scheme, based on Greek letters, using only two letters to identify each host and tailored for human PVs, will be able to accommodate the vast diversity of animal PVs while remaining informative and structured (Bravo et al., 2010, 2011; Van Doorslaer et al., 2011). Novel PVs are currently named using the first letter of the host’s generic names and epithets (Bernard et al., 2010), but there is obviously not enough information in this letter combination to describe the expected large PV diversity (Antonsson and Hansson, 2002; Mengual-Chulia´ et al., 2012). A taxonomic update has disambiguated certain naming conflicts (Bernard et al., 2010), but the descriptions of new PVs have generated synonyms such as MsPV1 used for viruses retrieved from a diamond python, Morelia

blue, Delta+Zeta; ochre, Lambda+Mu. Papillomaviruses, whose detailed phylogenetic relationships could not be disentangled, are labeled in black. The scale bar indicates the genetic distance in substitutions per site. Silhouettes represent the host infected by the corresponding viruses. Generic PV classification is included. Bat PVs branches are highlighted with a black spot. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Table 1 Pairwise nucleotide identities (amino acid similarities) of the different EhelPV1 ORFs compared to the ORFs of all PV types within the Lamba+Mu-PV crowngroup and to the ORFs of other bat PVs, MrPV1, MschPV1 and RaPV1.

HOST ORDER

Chiroptera

HOST SPECIES Eidolon helvum Miniopterus schreibersii Rouseus aegypacus Myos ricke Miniopterus schreibersii Canis lupus familiaris Canis lupus familiaris Felis domescus

PV GENUS

PV TYPE

Unclassified

EhelPV1

Unclassified

MschPV1

Psi

RaPV

Omikron*

MrPV1

Unclassified

CPV1 CPV6 FcaPV1

Lynx rufus Carnivora

Puma concolor Procyon lotor Panthera leo persica

LrPV1 Lambda PcPV1 PlPV1 PlpPV1

Uncia uncia

Lagomorpha

Primates

Rodena

Oryctolagus cuniculus Sylvilagus floridanus Homo sapiens Homo sapiens Homo sapiens Erethizon dorsatum

MschPV2

UuPV1 OcPV1 Kappa SfPV1 HPV1 Mu HPV63 Nu

HPV41

Sigma

EdPV1

ORF E6

E7

E1

E2

L2

L1

47.5 (39.7) 41.9 (44.4) 32.7 (39.5) 46.7 (46.9) 49.0 (45.3) 50.7 (50.0) 44.1 (46.3) 44.9 (49.6) 45.8 (44.7) 46.8 (44.7) 49.0 (46.6) 49.0 (48.3) 36.0 (28.2) 28.9 (25.0) 49.0 (52.5) 45.1 (47.9) 43.8 (38.4) 43.7 (38.7)

40.1 (44.1) 47.9 (47.3)

53.2 (55.3) 50.4 (52.5) 52.3 (50.0) 52.9 (55.3) 54.7 (57.7) 54.9 (60.2) 53.8 (60.0) 54.5 (57.5) 54.4 (57.9) 56.3 (56.9) 54.3 (57.9) 53.1 (57.4) 55.3 (57.8) 54.5 (58.1) 55.6 (56.2) 54.9 (57.2) 51.4 (51.7) 53.2 (52.2)

46.2 (45.7) 46.5 (47.6) 48.3 (49.4) 49.2 (56.2) 48.8 (53.5) 52.1 (56.2) 51.6 (55.2) 50.3 (56.5) 52.4 (56.1) 49.3 (54.8) 49.6 (56.4) 51.4 (56.3) 48.9 (58.0) 50.3 (55.9) 51.5 (57.2) 51.8 (56.8) 47.1 (51.2) 50.4 (48.5)

43.5 (40.0) 42.3 (40.9) 46.2 (40.6) 46.3 (42.7) 49.0 (50.5) 47.6 (50.6) 46.7 (48.5) 45.9 (48.2) 44.7 (50.8) 47.5 (34.6) 46.7 (49.4) 47.8 (49.2) 48.4 (48.9) 47.8 (46.6) 47.6 (53.3) 48.8 (50.3) 49.1 (50.0) 47.6 (47.5)

52.1 (61.3) 52.7 (62.4) 56.4 (67.0) 60.0 (69.9) 61.0 (69.8) 58.9 (69.7) 57.5 (68.4) 61.1 (71.3) 60.2 (71.5) 58.8 (72.0) 61.2 (72.5) 59.2 (71.4) 60.3 (72.0) 57.9 (70.8) 60.5 (71.1) 59.6 (70.3) 52.0 (62.4) 60.2 (70.9)

spilota (Lange et al., 2011a), and from a Schreiber’s longfingered bat, M. schreibersii (Wu et al., 2012). Further, a second PV isolated also from M. schreibersii has been named MscPV1 (Tse et al., 2012). Here, we have tentatively named the described novel PVs using four letters to identify the host, corresponding to the first letter of the generic name and the first three of the epithet: EhelPV1 and PgigPV1. To prevent confusion between the PVs infecting the snake and the common bent-wing bats, we have renamed the M. schreibersii PVs using four letters and different numbers following the date of publication: MschPV1 (Wu et al., 2012) and MschPV2 (Tse et al., 2012). We are aware that no PV nomenclature based on a few letters of the host’s name will avoid all possible confusion between hosts. Further, no naming scheme based on the name of the host in

40.4 (33.7) 54.7 (52.5) 58.7 (54.5) 52.1 (51.5) 51.0 (47.9) 53.2 (50.0) 51.9 (54.1) 53.0 (54.6) 56.4 (50.0) 49.6 (49.0) 51.5 (46.1) 51.0 (50.0) 54.9 (57.0) 37.1 (28.9) 38.4 (28.7)

which the virus was first retrieved will ever account for possible broad host range (Bravo et al., 2010). We suggest nevertheless that a naming alternative, such as the one proposed here should be considered for future PV taxonomy. The DNAs of the novel PVs EhelPV1 and PgigPV1 have been retrieved from hair bulbs from healthy skin of captive individuals of two pteropodid species, namely E. helvum and P. giganteus. Both viruses are close relatives (Fig. S2) and belong in the same PV crown group together with MschPV2, which has been obtained from a rectal swab from a wild individual of the vespertilionid species M. schreibersii (Tse et al., 2012). The three bat PVs in the Lambda+Mu-PV crown group are only distantly related to previously characterized bat PVs, namely MrPV1, MschPV1, and RaPV1 (Fig. 2). These bat PVs have been

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Fig. 3. Lambda+Mu-PVs supernetwork constructed from the best-known maximum likelihood trees of each individual nucleotide PV gene (E6, E7, E1, E2, L2, and L1). Color code represents the different orders of the hosts. Specific PVs tropisms and outcome of the corresponding infections are indicated in the inset. PV genera are specified in gray. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4. Consensus E2 binding site sequence for Lambda+Mu-PVs. The motif is represented as a sequence LOGO (position-specific probability matrix where the probability of each possible letter appearing at each possible position is specified). Error bars indicate small-sample correction. The E2BS occurrence within the URR of all Lambda+Mu-PVs is depicted. Sequence variations are indicated in the legend. Nucleotide changes are highlighted in bold and underlined.

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retrieved from different tissues and locations: RaPV1 has been isolated from a basosquamous carcinoma present in the wing of an Egyptian fruit bat (Rector et al., 2006a), while MrPV1 and MschPV1 have been found in pharyngeal and anal swabs from the vespertilionids M. ricketty and M. schreibersii (Wu et al., 2012). Bat PVs are thus not monophyletic, but instead appear scattered throughout the PV phylogeny: (i) RaPV1 and MschPV1 are close to root, orphan PVs; (ii) MrPV1 clusters together with UmPV1, retrieved from a lesion in the tongue of a polar bear (Stevens et al., 2008), and belongs to the Alpha+Omikron-PV crown group, which gathers essentially mucosotropic PVs infecting also primates and cetaceans (Gottschling et al., 2011a); (iii) MschPV2, EhelPV1 and PgigPV1 belong to the Lambda+Mu-PV crown group, which spans PVs infecting primates, rodents, and lagomorphs (Euarchontoglires) as well as carnivores and chiropterans (Laurasiatheria). The diverse phylogenetic positions of these PVs imply the existence of several bat PV lineages, nesting with different PV lineages. This distribution supports the hypothesis of a series of duplication events in the ancestral PV lineage, prior to the diversification of the mammalian hosts (Gottschling et al., 2007, 2011b). Thus, the different bat PV lineages appear already present before the time of the origin of bats, which molecular studies date back to the Eocene, ca. 50 million years ago (Teeling et al., 2005). The phylogenetic tree for the E1E2L1 concatenated genes in Fig. 2 shows a high support for the Lambda+MuPV crown group, in agreement with previous descriptions (Bravo et al., 2010; Gottschling et al., 2007, 2011b). However, the phylogenetic relationships among the genera in this crown group are not clear-cut defined, as they differ for nucleotide and amino acid reconstructions. Further, the evolutionary network in Fig. 3 reconstructed for the Lambda+Mu-PV crown group using the bestknown trees for the six conserved ORFs illustrates different plausible evolutionary trajectories (Huson and Bryant, 2006). Differences in topology for the different gene phylogenies generate the central netted region of the supernetwork. In the Lambda-PVs, the basal reticulations highlight the paraphyly of canine PVs. This is particularly significant, since Lambda-PVs have been long considered one of the hallmarks of PV-host coevolution (Rector et al., 2007). We can conclude that the Lambda+Mu-PV crown group is clearly monophyletic, albeit diverse. This diversity is reflected not only in the variety of hosts but also in the different tropisms and outcomes of the infection, as illustrated in Fig. 3. Members of the Lambda+Mu-PV crown group infect different hosts within Laurasiatheria and Euarchontoglires. Some of these viruses display cutaneous tropism while others infect mucosa, and some were retrieved from asymptomatic infections, while others were associated with benign lesions or with malignant neoplasias. The description of novel PVs that expands the sampling of human and animal PVs in this crown group will help us understand the origin of such variety. One of the most variable segments of the PV genome is the URR, highly divergent both in composition and length. Differences in the presence of regulatory sites may

account for the molecular substrate underlying different behavior patterns and different transforming abilities among related PVs (Garcı´a-Vallve´ et al., 2006; Sichero et al., 2012). The E2 protein plays an essential role in PV life cycle, since it regulates viral gene expression, and along with the E1 protein is required for viral DNA replication (Blakaj et al., 2009). E2 typically recognizes and binds to the consensus sequence ACCG-N4-CGGT located in the URR (Li et al., 1989). The interaction between E2 proteins and its binding sites on DNA has been conserved throughout PV evolution, and sequence variation has been linked to differential binding affinity (Blakaj et al., 2009). However, these types of studies have focused on human PVs (Sichero et al., 2012). Here, we have addressed an in silico study of the genome location and 12-bp-long nucleotide sequence that may constitute a binding site for the E2 proteins in Lambda+Mu-PVs. Six different versions of the canonical E2BS have been identified within the URR of Lambda+Mu-PVs (Fig. 4). The diversity of the E2BS patterns even in closely related PVs is noteworthy. The repertoire of E2BS in the PVs retrieved from lion (Panthera leo persica), puma (Puma concolor), leopard (Uncia uncia) and bobcat (Lynx rufus) are very similar, but they differ significantly from those found in FcaPV1, retrieved from domestic cat (Felis domesticus). The URR in SfPV1, retrieved from cottontail rabbit (Sylvilagus floridanus), shows a remarkable increase in the number of E2BS compared to OcPV1, retrieved from European rabbit (Oryctolagus cuniculus). For human PVs, the E2BS present in the URR of HPV1, HPV41 and HPV63 are very divergent. Given the importance of the regulatory sites in the viral life cycle, our results indicate that the diversity of E2BS repertoire might be connected with viral adaptation during realization of novel ecological niches and with the outcome of the viral infection for the members of the Labmda+Mu-PV crown group. Additional informative patterns of regulatory elements in the URR might be detected as new Lambda+Mu-PVs are characterized and other TFBS are considered. 5. Conclusion EhelPV1 and PgigPV1 belong to the Lambda+Mu-PV crown group, which includes PVs infecting members of the orders Carnivora, Chiroptera, Lagomorpha, Primates and Rodentia. The description of novel PVs within this crown group is a prerequisite to disentangle the complex evolutionary mechanisms that have shaped this highly diverse clade. Finally, functional knowledge about the repertoire and genomic location of TFBS may provide useful information and alternative tools to guide the study of recent PV evolution, tropism and differential outcome of PV infections. Authors’ contributions GW obtained and identified the animal samples; MG performed the initial laboratory experiments; RGP performed laboratory experiments, analyzed the data and drafted the manuscript; IGB conceived the study, analyzed the data and drafted the manuscript. All authors have

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contributed to, read, and approved the final version of the manuscript.

Acknowledgements RGP was the recipient of grants from the Red Tema´tica de Investigacio´n Cooperativa en Ca´ncer (RTICC) and ‘‘La Caixa’’ Foundation. IGB was funded by the disappeared Spanish Ministry for Science and Innovation (MICINN) grant CGL2010-16713, and is a member of the Red Tema´tica de Investigacio´n Cooperativa en Ca´ncer (RTICC).

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