Detection of different types of papillomavirus and co-infection in cattle in the State of Goiás-Brazil

Bovine papilomavírus (BPVs) is the etiological agent of bovine papillomatosis, a disease that triggers warts throughout the skin, udder, roofs, genitalia and in more severe cases can develop extensive papillomas, cause neoplasia in the digestive tract and bladder, cause losses in productivity and losses to livestock. In Brazil, the occurrence of BPV infection is relatively common, but the identification of viral types is still sporadic. The present study is a research report that aimed to describe the occurrence of BPV infections in dairy cattle affected by papillomatosis, based on the nucleotide sequences of the ORF L1, the most conserved sequence. Twenty-five samples of cutaneous wart from nine cattle clinically diagnosed as cutaneous papillomatosis were analyzed in the state of Goiás, central-western Brazil. Amplification was obtained in 11 samples (papilloma) from different cattle. PCR reactions followed by sequencing revealed the presence of BPV-1 in 60%, BPV-5 in 40%, and BPV-14 in 20% of the samples analyzed. The presence of coinfection was verified in 60% of the amplified samples. These data suggest that several types of BPV can infect a lesion simultaneously and demonstrate the possibility that BPV infection in epithelial tissue can occur without restriction to one or two viral types, demonstrating the region's genetic diversity. As far as we know, this is the first registry of typification of BPVs of the central-western region of Brazil. This analysis provides important information for bovine papillomavirus (BPV) research in Brazil.


Introduction
Papillomatosis is an infectious disease which has the etiologic agent papilloma virus (PV), characterized by the presence of hyperproliferative lesions (papillomas), which can progress to malignancy (Daudt et al., 2018). The bovine papilloma virus (BPV) is easily disseminated in the herd and to date, there is no vaccine or truly effective treatment against the etiological agent (Módolo et al., 2017). Still, it promotes significant losses in the herd, generating economic loss for the producer and the meat, milk and leather industries (Módolo et al., 2017).
In horses, the delta papillomavirus (δPV) is associated with lesions known as sarcoids, characterized by fibroblast proliferation and hyperplasia or dysplasia (Nasir & Brandt, 2013). These types of injury are aggressive, rarely regress and often recur after therapy (Lunardi et al., 2013). It is notable that δPV 14 infection has been related to similar feline lesions (Munday et al., 2015).
The δPVs were also observed in cutaneous fibropapillomas and buffalo bladder tumors (Roperto et al., 2013). Bovine δPVs are thought to cause cutaneous sarcoids in African lions (Orbel et al., 2010), domestic cats (Munday et al., 2015), cape mountain zebras, giraffes and sable antelope (Williams, 2011). The types of bovine δPVs were also detected clinically in normal skin samples from many wild non-bovine ruminants (Savini, 2016) and in peripheral blood samples from sheep (Roperto et al., 2018).
The identification of viral types in the region and their prevalence are necessary to characterize the epidemiology of circulating viral types, which once identified, allows directing the development of containment and prophylactic measures (Claus et al., 2009). The association of BPV 14 with aggressive lesions and interspecies infection calls attention in a scenario with lack of information of its prevalence.
Aiming at this, the present study aimed to identify BPVs found in cattle herds in the State of Goiás -Brazil, this study represents the first case of BPV 14 in the State of Goiás, aiming to contribute to the knowledge of the diversity of PVs that affect the animals and to the generation of effective prophylactic or therapeutic vaccines.

Methodology
In this article was used the methodology according Koche (2011).

Collection of samples
The cattle selected for the study came from beef and dairy farms in Midwest Brazil where cutaneous papilomatosis occurs. We examined a total of 9 animals male and female Girolanda cattle, aged 9-24 months, in the city of Goiânia -Goiás, Brazil. The lesions were variously located on the head, nose or around the eyes and dispersed around the body. Multiple samples were obtained from animals with several skin lesions to assess co-infection, resulting in the collection of 25 cutaneous lesions, clinically classified as cauliflower, flat and peduncle. These were obtained from different anatomical parts of the animal.
The collection was characterized by the tricotomy around the papillomas, with 10% iodine asepsis at the anesthetized site, using Lidocaine hydrochloride and Epinephrine, and then the papillomas were removed and stored in a falcon tube and identified with the cattle number and location. papilloma. The excision site was sutured, and 10% iodine was applied over the lesion. All procedures were performed according to the Ethics Committee on the Use of Animals of the Federal University of Goias (Protocol Number 049-14).

Extraction of Nucleic Acid
DNA extraction from bovine tissue samples was performed according to the Genomic DNA Purification kit protocol (Wizard, Promega Corporation, USA). DNA quality was verified by PCR of the bovine β-globin gene, as described by Freitas et al. (2003).

β-globin Detection
Each DNA sample was tested for identification of the bovine β-globin protein, with the Forward oligonucleotides: 5'-AACCTCTTTGTTCACAACCAG-3' and Reverse 5'-CAGATGCTTAACCCACTGAGG-3' which amplifies a fragment of 450pb. The PCR reaction was performed according to the protocol of the 2x PCR-Mix LGC Biotechnology kit® (Labtrade & LGCBio, Brasil). All data related to the PCR reaction and parameters for amplification of the oligonucleotides for β-globin are summarized in Table 1 and Table 2. β-globin was used to check the quality of the extracted DNA (Freitas et al., 2003).  , the primers were previously tested for cross-amplification according to protocol of Silva et al. (2011), in each assay were tested up to five samples to avoid contamination. We subjected 3 μl DNA to thermocycling in a 25 μl reaction mixture containing 2.5U Taq DNA Polymerase, 3.5 mM dNTP mix, 10 pmol of each primer, 1.5 mM MgCl2, and 10X PCR buffer. Thermal cycling conditions were denaturation for 10 min at 96°C, then 50 sec at 94°C, followed by 40 cycles of 51°C for 55 sec, and 50 sec at 72°C. In addition, the reaction tubes were kept for a further 10 min at 72°C for final extensions (Roperto et al., 2018). PCR amplified DNA samples were analyzed by 1% (w / v) agarose gel electrophoresis, dissolved in 1x TAE and stained with ethidium bromide (0.2 μg / ml). After the electrophoresis process, the DNA bands were visualized under low intensity ultraviolet irradiation. The material was analyzed by comparison, having as parameter, the size of the molecular marker bands 1 Kb DNA Ladder promega® (Promega Corporation, USA). Samples amplified with BPV-specific nucleotides were sequenced in order to confirm the results of standard PCR. The sequencing was performed by the company Ludwing Biotecnologia, located in the city of Alvorada -RS, applying Sanger's methodology for sequencing (Sanger & Coulson, 1975).

Sequence alignment and phylogenetic analysis
The nucleotide sequences were aligned using BioEdit Sequence Alignment Editor software version 7.0.9.0 (Ibis Therapeutics Carlsbad, CA, EUA) and analyzed in the database of the National Center of Biotechnology -NCBI applying the BLAST tool, with the objective of evaluating the identity and similarity between the sequences obtained in this study and those deposited in the NCBI database for correlation of statistical significance (Altschul, 1997).
For phylogenetic proximity analysis, the sequences were aligned with non-redundant homologous nucleotides, obtained from the "GenBank" Database (Table 4). For characterization of each viral type, the gene coding for L1 protein was used. Sequences were chosen where the probability values were below (Evalue -22). Phylogenetic relationships of the partial nucleotide sequences of the viral types found in this study were performed and the phylogenetic trees were constructed through the Neighbor-joining program, a method developed by Saitou and Nei (1978). A later bootstrap analysis was performed to test the reliability of the tree (Felsenstein, 1985).

Viral type ID Gene Bank
* obtained by sequencing Source: Authors.
Trees were constructed by multiple sequence alignments using the ClustalX program (Thompson, 1997). The size of the branches was estimated with 1000 bootstrapped replicates and the percentage of times in all species are indicated as a monophyletic group. The maximum likelihood phylogenetic tree based on the first and second position of the codon was constructed in Jalview (Waterhouse, 2009).

Results
Only samples from catthle A.2, A.3, A.4, A.6 and A.9 presented 450 bp of amplified product corresponding to the expected sequence of the β-globin gene confirming the viability of the DNA sample for PCR.
Samples obtained with the oligonucleotides for BPVs -9, 10, 12 and 13 were not successful in sequencing (Table 5). As a result, they were not included in phylogenetic analysis.
Phylogeny was also performed between the sequenced viral types for phylogenetic analysis, the CLUSTAL X program was used and type 14 (BPV 14) and 1 (BPV 1) were shown to be phylogenetic, whereas viral type 5 (BPV 5) presented phylogenetic distance (Fig. 1). Viral type 9 (BPV-9) grouped into a separate branch, thus, this type viral is considered phylogenetically distant. Maximum Likelihood tree of bovine papillomavirus, which comprises 3 BPV types, based on partial sequences of L1 ORF. Two groups of viruses are distinguished, which forms the previously described genera (Deltapapillomavirus and Epsilon papillomavirus). Source: Authors.
To configure the classification of BPV 1, BPV 5 and BPV 14, a survey of the nucleotides of the L1 sequence was carried out and a phylogenetic tree constructed.  (Table 4).

Discussion
In the present research, the lesions were classified mainly as cauliflower, the choice of specific oligonucleotides for viral typing aimed at a more accurate viral identification with target gene annealing, since the use of specific primers for the identification of BPV is more sensitive in viral detection than the consensus primers  PCR results demonstrated the occurrence of seven viral species present in the pushed cattle (BPV 1,5,9,10,12,13 and 14), with the most prevalent BPV 1 present in 60% of the amplified samples, Batista et al. (2013) relates the high prevalence of BPV 1 with a presence of this viral type in the bovine cutaneous lesion in Brazil. Coinfections were verified in 60% of the samples with four types of viral lesions, demonstrating a high degree of adaptation of the virus to its host. The occurrence of BPVs (1, 2, 6, and 8) was reported in the northern region of southern Brazil and it was noted that the occurrence of multiple infections does not generate cross-immunity from one viral type to the other and propose that treatment for papillomatosis requires a broad spectrum of coverage to be better (Claus et al., 2009 (Roperto et al., 2015. The similarity or difference observed in the phylogeny can be attributed to the gender classification of the respective viral types. Viral types 1 and 14 belong to the same genus, the Deltapapillomavirus, demonstrated approximation in branches 14 and 1. The papillomavirus type 5 belongs to the genus Epsilonpapillomavirus, thus justifying the phylogenetic distance between these viral types, verified in the histogram (Fig. 1) (de Villlers et al., 2004).
Molecular phylogeny data reinforce the idea that certain viral types are quite different between them. Evidence of nucleotide distancing between viral types suggests that there may have been mutations that induced molecular diversity with the capacity to originate 24 viral types. However, it should be noted that although this distancing occurs in the genome, the ORF L1 has similar distribution in all papillomaviruses (de Villlers et al., 2004). This was observed in Fig. 1 and Fig. 2, confirming this statement.
The ORF L1 of BPV 1 and BPV 14 used in the tree construction suggest the phylogenetic proximity with the other Deltapapillomavirus, BPV type, 2 and 13, which cause typical cutaneous fibropapilloma in bovine (Lunardi, 2013, Munday, 2015 and are commonly associated with interspecies infection (Brandt, 2016;Rector, 2013) as equine and feline sarcoid and BPV 5 L1 ORF showed phylogenetic proximity to BPV 8 also classified as Epsilonpapillomavirus (Savini et al., 2016).

Conclusion
Despite the high frequency of lesions, BPV genotyping is still sporadic. We emphasize the importance of understanding the diversity and epidemiology of BPV in order to target prevention strategies. Most reports of the prevalence of BPV types are from Brazil and Japan, where Brazil is one of the largest producers of meat and milk in the world and where several types have been described, but as far as we know, this is the first BPV typification record of the Brazilian midwest region. The identification of multiple BPV infections can contribute to the understanding of the epidemiological, clinical and immunological characteristics of papillomatosis in cattle. Additional molecular epidemiological investigations on the incidence and diversity of BPV infection in cattle will help to establishing a more accurate view of the distribution of this virus.
More studies must be carried out to indicate the prevalence of two types of BPV circulating in all of Brazil. Studies associating anatomic region of lesions, macroscopic characteristics and types or genus BPV are important studies to be considered.