**7. New BPV types**

malignant carcinomas under the influence of the carcinogenic elements present in bracken

Therefore, although infection by these BPVs plays a central role in the pathogenesis of these cattle neoplasias, the presence of environmental and biological cofactors is essential for the

Although the genomic sequences of approximately 150 HPV types have already been charac‐ terized, at the beginning of the 1980s only six BPV types (BPV1 to 6) had been identified from

Studies performed from the beginning of the 2000s onward to investigate the actual di‐ versity of BPVs have indicated the existence of many BPV types, which is similar to ob‐ servations made regarding the human virus. The first such study employed the generic primer pair FAP59/FAP64 on swabs of healthy skin from 19 species of vertebrates. In six of the 10 analyzed bovines that did not exhibit any clinical sign compatible with BPV in‐ fection, one or two putative new BPV types were detected. These putative new viral

Subsequently, a study aimed at establishing the prevalence of BPV in teat papillomas and teat healthy skin used the primer pairs FAP59/FAP64 and MY09/MY11 to analyze 15 teat papillo‐ mas and 122 swabs of teat healthy skin on cattle from five Japanese prefectures [44]. That study found four previously characterized BPV types (BPV1, 3, 5, and 6), two of the previously identified putative new BPV types (BAA1 and 5), and 11 additional putative new types (named BAPV1 through 10 and BAPV11MY) among the 39 BPV-positive samples. Nevertheless, the putative new types BAA1 and BAPV7 through 10 were detected only in samples of healthy skin. In addition, during one outbreak of mammary papillomatosis that occurred in Japan and affected 560 heifers, the presence of BPV6 was confirmed in the majority of the 16 analyzed samples [45]. The previously described putative new types BAA5 and BAPV1 were also

Although cutaneous papillomatosis poses a serious sanitary problem in beef and, even more so, dairy cattle, studies aimed at identifying the BPV types involved in the occurrence of skin lesions in Brazilian cattle are only sporadically performed. Recently, the detection of BPV1, 2, 6, and 8 in papillomas of cattle from the state of Parana was accomplished using generic FAP primers [46,47]. In another study, the identification of four previously undescribed putative new BPV types, named BPV/BR-UEL 2 through 5, pointed to the occurrence of considerable viral diversity among Brazilian cattle [48]. The genetic characterization of one of these new BPV types, namely, BPV/BR-UEL2, through sequencing of the full L1 gene, confirmed that it

[33,34,35].

development of such lesions [22,36].

cases of bovine cutaneous papillomatosis and cancer [37-42].

122 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

types were named BAA1 through BAA5 [43].

belongs to the genus *Xipapillomavirus* [49] (Figure 4).

**6. Diversity of BPVs**

identified in these animals.

Recently, complementary analysis of several putative new BPV types through sequencing of the full viral genomes allowed the characterization of these new viral types [3]. The first such new type to be characterized was BPV7, which was initially named BAPV6. Because the nt sequence of the BPV7 L1 ORF is more closely related to PVs of the genera *Betapapillomavirus*, *Gammapapillomavirus,* and *Pipapillomavirus*, which include viruses causing skin lesions in human beings and the mucosa of hamsters, this new BPV type constitutes a new and yet unnamed genus [50].

The second recently described BPV type is BPV8, formerly known as BAPV2, which was identified in Japan. The description of this new viral type was performed together with the description of a variant named BPV8-EB, which was detected in a case of cutaneous papillo‐ matosis in a European bison born in Italy [51]. The high degree of similarity observed between the L1 ORF sequences of BPV8 and BPV5 (75%), as well as the results of the phylogenetic analysis, were the basis for classifying this new viral type in the genus *Epsilonpapillomavirus*. In addition, the genomic structures of the early and late regions of these two different members of the genus were almost identical. The only difference exhibited between them was in the E4 ORF, which is present in BPV8 but absent in BPV5.

Recently, two BPV types, namely, BPV9 and 10, were identified from teat papillomas [52]. These new viral types were initially designated BAPV1 and BAA5 [43,44]. Phylogenetic analysis and the greater similarity of the L1 ORFs with BPV3 (74.2% and 71.2%, respectively) allowed the classification of these two new isolates in the genus *Xipapillomavirus* [52].

Hatama [53] assessed the viral genotypes present in 167 skin warts in Japanese herds through polymerase chain reaction (PCR), cloning, and sequencing. A total of 124 of the assessed lesions tested positive for BPV using PCR. Three putative new BPV types, and eight previously described BPV types (BPV1, 2, 3, 4, 5, 6, 9, and 10) were identified in the partial sequences obtained from sequencing the PCR products. The characterization of the full sequence of one of the new BPV types (BPV11) and the comparison of its L1 gene nt sequence to other members of this viral family allowed its classification in the genus *Xipapillomavirus* [53].

The complete genome sequence of an isolate identified from an epithelial tongue lesion in a Japanese bovine was recently obtained, and this isolate was named BPV12 [54]. Comparison of the BPV12 L1 gene nt sequence to other viral types isolated from cattle suggested that it should also be classified in the genus *Xipapillomavirus*.

Recently, the sequencing of the complete genome of the putative new viral type BPV-BR-UEL4, which was isolated from a skin papilloma on a cow from a herd in southern Brazil,was performed by subjecting the viral genome to rolling circle amplification (RCA), PCR, the subsequent cloning of two long amplicons, and sequencing by means of primer walking. Phylogenetic analysis based on the L1 ORF nt sequences of 45 PVs distributed among 17 genera, including the previously sequenced BPV types and PVs identified from different artiodactyl species, showed that the new viral type, named BPV13, belongs to the genus *Deltapapillomavirus*, which is generally dominated by artiodactyl PVs and also includes BPV1 and 2 (Figure 5). As previously reported for BPV1 and 2, the putative E7 protein of BPV13 does not contain a retinoblastoma tumor suppressor-binding domain. Additionally, the BPV13 E5 ORF also encodes a small transforming protein (Figure 6) [55]. The combination of these two different biological aspects has been recognized as a distinct marker for fibropapilloma development. This pathogenic mechanism appears to be unique among delta-PVs [56].

**Figure 5.** Phylogenetic tree based on L1 ORF nt sequences. In addition to 14 genera where animal PVs are classified, the genera *Deltapapillomavirus*, *Epsilonpapillomavirus*, and *Xipapillomavirus*, which contain BPVs, are indicated in the tree. Additionally, the six species classified within the *Deltapapillomavirus* genus are shown. The numbers at the inter‐

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125

nal nodes represent the bootstrap support values determined in 1000 replications. Source: [55]

sequence of the BPV7 L1 ORF is more closely related to PVs of the genera *Betapapillomavirus*, *Gammapapillomavirus,* and *Pipapillomavirus*, which include viruses causing skin lesions in human beings and the mucosa of hamsters, this new BPV type constitutes a new and yet

The second recently described BPV type is BPV8, formerly known as BAPV2, which was identified in Japan. The description of this new viral type was performed together with the description of a variant named BPV8-EB, which was detected in a case of cutaneous papillo‐ matosis in a European bison born in Italy [51]. The high degree of similarity observed between the L1 ORF sequences of BPV8 and BPV5 (75%), as well as the results of the phylogenetic analysis, were the basis for classifying this new viral type in the genus *Epsilonpapillomavirus*. In addition, the genomic structures of the early and late regions of these two different members of the genus were almost identical. The only difference exhibited between them was in the E4

Recently, two BPV types, namely, BPV9 and 10, were identified from teat papillomas [52]. These new viral types were initially designated BAPV1 and BAA5 [43,44]. Phylogenetic analysis and the greater similarity of the L1 ORFs with BPV3 (74.2% and 71.2%, respectively)

Hatama [53] assessed the viral genotypes present in 167 skin warts in Japanese herds through polymerase chain reaction (PCR), cloning, and sequencing. A total of 124 of the assessed lesions tested positive for BPV using PCR. Three putative new BPV types, and eight previously described BPV types (BPV1, 2, 3, 4, 5, 6, 9, and 10) were identified in the partial sequences obtained from sequencing the PCR products. The characterization of the full sequence of one of the new BPV types (BPV11) and the comparison of its L1 gene nt sequence to other members

The complete genome sequence of an isolate identified from an epithelial tongue lesion in a Japanese bovine was recently obtained, and this isolate was named BPV12 [54]. Comparison of the BPV12 L1 gene nt sequence to other viral types isolated from cattle suggested that it

Recently, the sequencing of the complete genome of the putative new viral type BPV-BR-UEL4, which was isolated from a skin papilloma on a cow from a herd in southern Brazil,was performed by subjecting the viral genome to rolling circle amplification (RCA), PCR, the subsequent cloning of two long amplicons, and sequencing by means of primer walking. Phylogenetic analysis based on the L1 ORF nt sequences of 45 PVs distributed among 17 genera, including the previously sequenced BPV types and PVs identified from different artiodactyl species, showed that the new viral type, named BPV13, belongs to the genus *Deltapapillomavirus*, which is generally dominated by artiodactyl PVs and also includes BPV1 and 2 (Figure 5). As previously reported for BPV1 and 2, the putative E7 protein of BPV13 does not contain a retinoblastoma tumor suppressor-binding domain. Additionally, the BPV13 E5 ORF also encodes a small transforming protein (Figure 6) [55]. The combination of these two different biological aspects has been recognized as a distinct marker for fibropapilloma development. This pathogenic mechanism appears to be unique among delta-PVs [56].

allowed the classification of these two new isolates in the genus *Xipapillomavirus* [52].

of this viral family allowed its classification in the genus *Xipapillomavirus* [53].

unnamed genus [50].

ORF, which is present in BPV8 but absent in BPV5.

124 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

should also be classified in the genus *Xipapillomavirus*.

**Figure 5.** Phylogenetic tree based on L1 ORF nt sequences. In addition to 14 genera where animal PVs are classified, the genera *Deltapapillomavirus*, *Epsilonpapillomavirus*, and *Xipapillomavirus*, which contain BPVs, are indicated in the tree. Additionally, the six species classified within the *Deltapapillomavirus* genus are shown. The numbers at the inter‐ nal nodes represent the bootstrap support values determined in 1000 replications. Source: [55]

**9. Vaccines against BPV**

tablished lesions [76].

production of vaccines [34].

to be highly efficient [78,79].

induced papillomas [83].

Immunity against BPV is considered to be type-specific, and the immune status of the in‐ fected animals is considered to be the crucial factor for clinical progression. Whereas hu‐ moral immunity prevents new infections, cellular immunity (possibly mediated by T lymphocytes) is associated with the spontaneous and immune-mediated regression of es‐

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The finding that epitopes that induce the production of neutralizing antibodies are present in the structural proteins L1 and L2 explains the success of the use of these proteins in the

The recent availability of VLP-based immunogens against HPV that are able to protect mainly against infection by HPV16 and 18 has allowed the development of the first vaccine against one of the main human neoplasias, *i.e.,* cervical cancer [77]. The data that have been collected since the implementation of the HPV vaccine are quite encouraging, and these vaccines seem

In addition, preventive vaccines have been developed for cattle that are mainly against BPV2 and 4. These viral types were selected because they represent the cutaneous and mucous BPVs, respectively, and are associated with the development of cancer in cattle [80]. A vaccine prepared with the BPV2 L1 capsid protein produced as a beta-galactosidase fusion protein in Escherichia coli induced the production of neutralizing antibodies and was able to prevent infection [81]. A similar effect was achieved using an E. coli derived BVP1 L1 protein, which

VLPs produced from the L1 or L1 and L2 genes from BPV4 have also proven to be highly immunogenic and produce powerful prophylactic vaccines. The prevention of infection during challenge with BPV4 through vaccination with L1 VLPs has shown that L1 promotes the production of neutralizing antibodies [34]. Vaccination with VLPs produced from BPV4 L1 and L2 proteins in insect cells also efficiently prevented the development of experimentally

Because BPV does not grow in conventional cell cultures for the production of killed or attenuated live vaccines, protein expression systems, such as yeast and insect cells, have been used to produce VLP vaccines. However, the use of these systems is expensive. Recently, as has been described for other papillomaviruses (e.g., HPV16), a candidate vaccine against BPV1 consisting of L1 VLPs produced *in planta* elicited a strong and specific immune response, which demonstrated its potential as a future vaccine that could be produced at a lower cost [84].

Because the viral life cycle and the progression from benign to malignant lesion are similar in humans and animals, animal PVs and their natural hosts have represented good models for the study of HPV [30,85]. In addition, animal PVs, particularly BPV1 and 4, SfPV1, and CPV1, have also served as models for vaccines against PVs, and observation of the induction of protective immunity through the use of VLP-based vaccines in their corresponding host

species has opened the way for the implementation of VLPs in HPV vaccines [86,87].

protected calves against post-vaccine challenge with a homologous virus [82].

**Figure 6.** A diagram of the genomic organisation of BPV13. The three main regions characteristic of PV genomes are shown as grey rectangles above the ruler. The viral genome is represented as linear, and ORFs are shown as white rectangles. The numbers below each ORF indicate the nt positions of the start to stop codons and the corresponding molecular mass (in parentheses) for each putative viral protein. Source: [55].
