Research Article Bovine Papillomavirus in Brazil:...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 270898 7 pageshttpdxdoiorg1011552013270898

Research ArticleBovine Papillomavirus in Brazil Detection of Coinfection ofUnusual Types by a PCR-RFLP Method

R F Carvalho1 S T Sakata12 D N S Giovanni1 E Mori2 P E Brandatildeo2 L JRichtzenhain2 C R Pozzi3 J R P Arcaro3 M S Miranda3 J Mazzuchelli-de-Souza14

T C Melo15 G Comenale1 S L M R Assaf1 W Beccedilak16 and R C Stocco14

1 Laboratorio de Genetica Instituto Butantan Secretaria de Estado da Saude Avenida Vital Brasil 1500 Butantan05503-900 Sao Paulo SP Brazil

2 Laboratorio de Biologia Molecular Aplicada e Sorologia Departamento de Medicina Veterinaria Preventiva e Saude AnimalFaculdade de Medicina Veterinaria e Zootecnia da Universidade de Sao Paulo Avenida Prof Dr Orlando Marques de Paiva87 Cidade Universitaria Butantan 05508-270 Sao Paulo SP Brazil

3 Centro de Pesquisa em Pecuaria do Leite Instituto de Zootecnia Agencia Paulista de Tecnologia dos AgronegociosCAPTA Bovinos Leiteiros Rodovia Luiz de Queiroz km 129 13460-000 Nova Odessa SP Brazil

4 Programa de Pos-Graduacao Interunidades em Biotecnologia Instituto de Ciencias Biomedicas Edifıcio ICB-IIIUniversidade de Sao Paulo Avenida Prof Lineu Prestes 2415 Cidade Universitaria Butantan 05508-900 Sao Paulo SP Brazil

5 Programa de Pos-Graduacao em Biologia Estrutural e Funcional Universidade Federal de Sao PauloRua Botucatu 740 Vila Clementino 04023-900 Sao Paulo SP Brazil

6Departamento de Biologia Universidade Federal da Integracao Latino-Americana (UNILA) Avenida Tancredo Neves 6731Bloco 4 85867-970 Foz do Iguacu PR Brazil

Correspondence should be addressed to R F Carvalho rodsfcuolcombr

Received 26 March 2013 Accepted 16 May 2013

Academic Editor Lubna Nasir

Copyright copy 2013 R F Carvalho et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Bovine papillomavirus (BPV) is recognized as a causal agent of benign and malignant tumors in cattle Thirteen types of BPV arecurrently characterized and classified into three distinct genera associated with different pathological outcomes The describedBPV types as well as other putative ones have been demonstrated by molecular biology methods mainly by the employment ofdegenerated PCR primers Specifically divergences in the nucleotide sequence of the L1 gene are useful for the identification andclassification of new papillomavirus types On the present work a method based on the PCR-RFLP technique andDNA sequencingwas evaluated as a screening tool allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow chronically affected with cutaneous papillomatosisThese findings point to the dissemination of BPVs withunclear pathogenic potential since two relatively rare new described BPV types which were first characterized in Japan were alsodetected in Brazil

1 Introduction

The Bovine papillomavirus (BPV) is recognized as the causalagent of benign and malignant tumors in cattle such as cuta-neous papillomas benign fibroplasias urinary bladder andesophagus cancer causing significant economic losses Thisoncogenic virus has a double-stranded circular DNA genomeof approximately eight kilobases [1]

Currently the Papillomaviridae family is divided into 16genera according to their genomic organization [2 3] Thepapillomavirus (PV) genome codified functional early (E)proteins and structural late (L) proteins expressed at differ-ent stages of the viral cycle The L1 is the most conservedgene within a PV genome and has therefore been used forthe identification of new PVs one PV isolate is recognized asa new type if the complete genome has been cloned and the

2 BioMed Research International

DNA sequence of L1 differs bymore than 10 from the closestknown PV type Differences between 2 and 10 define asubtype and less than 2 a variant [3] Thirteen types ofBPVs are currentlywell characterized and classified into threedistinct generamdashDelta Epsilon and Ximdasheach one associatedwith epithelia lesions of specific histological nature [4]

The BPVs-1 and -2 are classified asDelta papillomaviruses[5] Characteristically these types induce the appearance offibropapillomas associated with the recruitment of the sub-epithelial fibroblasts [6] As far as concerned both types arealso unique in their ability to infect different host species notonly bovines causing the equine sarcoid [7] Lately the ge-nome of a newDelta-BPV type (BPV-13) was fully sequenced[8]

A larger number of BPV types (-3 -4 -6 -9 -10 -11and -12) belong to the Xipapillomavirus genus These virusesare considered exclusively epitheliotropic inducing the for-mation of ldquotrue papillomasrdquowithout the involvement of fibro-blasts [9ndash11] On the other hand the BPVs-5 and -8 have thepotential to induce both fibropapillomas and true papillomasin the course of their infectious cycle being classified intoa third genus Epsilonpapillomavirus [12 13] The BPV-7 re-presents an exception and is classified separately (unsignedgenus) This virus was first isolated from a cutaneous papil-loma lesion and also from healthy teat skin samples [14]

The thirteen described BPV types as well as other putativeones have been demonstrated bymolecular biologymethodssince papillomaviruses are not prone to be replicated or re-covered in cell cultures [15ndash19] According to the guidelinesoutlined by the Papil omavirus Nomenclature Committee(14th International Papillomavirus Conference Quebec CityQC Canada) it has been specified that the amplified se-quences isolated from novel papillomaviruses isolates couldindicate only putative new PV typesmdashinstead of PV typesmdashsince the PCR amplicons represent only part of the L1 gene[15]

Theuse of PCR assayswith degenerated primers followedby sequencing has allowed the identification of several PVtypes in human and other animal hosts [15 20] The PCRprimer FAP set was designed from two relatively conservedregions found in the L1 gene and has been shown to amplifyPVs DNA from both papillomas and healthy tissue of manyanimal species including BPVs in bovines [15 16 20]

Brazil has a cattle herd of approximately 210million beinga major exporter of meat milk and leather BPVs have beenpreviously detected in Brazil [21] but the extent of the impactof BPVs associated diseases both in dairy and cattle herdsneeds further studies Available reports in different regionsof the country indicate a significant diversity of viral typesamong theBrazilian herd implying an evident disease burden[18 22ndash25]Thus the improvement of knowledge concerningthe diagnosis and related clinical aspects of different BPVtypes among the Brazilian herd should be considered inthe development of new sanitary measures aiming to theprevention of BPV infection and its consequences

Unfortunately BPV epidemiological surveys are still lim-ited by the availability of high-throughput diagnostic tech-niques that could discriminate different BPV sequences at thesame time in co infected samples [16 26] In this context

the present work represents an effort to identify BPV typesemploying an alternative screening method based on thePCR-RFLP technique and correlating the histological data ofthe analyzed lesions with the diagnosed viral type

2 Material and Methods

In silico generation of RFLPs the L1 FAP segment digestionprofiles of the BPVs-1 to -13 could be generatedwithNEB cut-ter 20 [27] from all L1 complete nucleotide sequences avail-able in Genbank (httpwwwncbinlmnihgovgenbank)The restriction enzymes sites were chosen both by its pres-ence (or absence) as well as the generated digestion frag-ments sizes in order to differentiate those thirteen differentBPV types Histopathological analysis wart biopsies wereobtained from the trunk of a six-year-old Holstein dairycow chronically affectedwith cutaneous papillomatosis Sam-ples from three different lesions were submitted to macro-scopic histological (hematoxylin and Eosin staining) andmolecular analyses DNA extraction and PCR DNA wasextracted from warts for viral typing (Illustra tissue and cellsgenomic Prep Mini Spin GE Healthcare) and an approx-imately 470 base-pairs L1 gene segment was amplifiedusing the following primer sequences forward FAP59 (51015840-TAA CWG TIG GIC AYC CWT ATT-31015840) reverse FAP64(51015840-CCWATATCWVHCATITCICCATC-31015840) The PCR wereperformedwith slightmodifications of a previously describedprotocol [16] In detail the amplification reactions were per-formed in a Corbett CG1-96 thermocycler (Corbett Life Sci-ence Sydney Australia) with GoTaq Master Mix (PromegaMadison USA) under the following conditions 5min at95∘C followed by 35 cycles of 1min and 30s at 95∘C 2minat 52∘C and 1min and 30s at 72∘C and a final extensionstep of 5min at 72∘C Restriction analysis an aliquot of PCRfragments were submitted to digestion reactions for RFLPanalysis with four different restriction enzymes (DdeI Hinf IHindIII MsLI) following manufacturerrsquos instructions (NewEngland Biolabs Ipswich USA) Cloned BPV-1 and BPV-2 genomes as well as a known typed clinical sample (Mg-19 BPV-2 typed) were used as positive controls PCR-RFLPproducts were analyzed in 20 agarose gel electrophoresisstained with ethidium bromide (05120583gmL) in TAE bufferand visualized under UV light Sequencing An aliquot ofall generated PCR fragments were purified with extractioncolumns (Illustra GFX PCR DNA and Gel Band PurificationKit GE Healthcare) DNA concentration and purity weredetermined in a spectrophotometer (Eppendorf BioPho-tometer Hamburg Germany) and submitted to sequenc-ing reactions three independent sequencing reactions weredone for each PCR fragment in an ABI377 PRISM GeneticAnalyzer (Life Applied Biosystems USA) The quality ofDNA sequenceswas checked and overlapping fragmentswereassembled using the BioEdit package 7090 [28] Assembledsequences with high quality were aligned using ClustalW183 [29] with default gap penalties Homology analyses wereperformed with the NCBI database and BLAST [30] BioEditsoftware was used to identify the equivalent amino acidsequences The sequence alignments were performed using

BioMed Research International 3

Table 1 L1 FAP segment digestion profiles of the BPVs-1 to -13 for Dde I Hinf I MsL I and HindIII

L1 FAP Fragment DdeI Hinf I MsLI HindIII

BPV-1 475 bp264 329 301 475159 146 17452

BPV-2 475 bp 316 329 475 475159 146

BPV-3 473 bp319 473 373 473154 87

13

BPV-4 469 bp324 469 469 230145 182

87

BPV-5 469 bp469 254 469 469

14570

BPV-6 472 bp 321 384 373 472151 88 99

BPV-7 484 bp403 271 484 48481 119

94

BPV-8 469 bp 469 317 469 322152 147

BPV-9 469 bp 316 344 469 469153 125

BPV-10 472 bp319 381 373 47290 91 9963

BPV-11 475 bp 420 407 370 47555 68 105

BPV-12 469 bp 351 317 469 469118 152

BPV-13 475 bp316 329 475 475109 14650

Reference SequencesBPV-1 NC 0015221 BPV-1 X023461 BPV-2 M202191 BPV-2 X017681BPV-3 AF4861841 BPV-3 AJ6202071 BPV-3 NC 0041971 BPV-4 X058171BPV-5 AF4574651 BPV-5 NC 0041951 BPV-6 AJ6202081 BPV-7 DQ2177931BPV-7 NC 0076121 BPV-8 EB DQ0989171 BPV-8 NC 0097521 BPV-9 AB3316501BPV-10 AB3316511 BPV-11 AB543507 BPV-12 JF834523 BPV-13 JQ798171

the MEGA 50 software [31] using full alignment and 2000total replications on the bootstrap in order to ensure a higherlevel of confidence to our analysis [32] Phylogenetic Analysisphylogenetic relationship comparing nucleotide sequenceswas performed with MEGA Neighbor-joining trees weredrawn using TreeView version 166 [33] Nucleotide andamino acid sequences from other BPV types and of a HPV-16

were retrieved from the GenBank (httpwwwncbinlmnihgov) for comparison with the obtained sequences here

Ethics Statement The protocols used in this study wereapproved by the Ethical Committee for Animal Experimen-tation of the Instituto de Zootecnia (Protocol no 109 on July06th 2009) assigned by the President of this Committee Allefforts were made to minimize animal suffering


IZ-1214

(a)

IZ-1214

(b)

100120583m100120583m100120583m

IZ1 IZ2 IZ3

(c)

Figure 1 (a) Analyzed animal IZ-1214 with disseminated cutaneous papillomatosis (b) Gross aspect of the papilloma lesions with acauliflower-like appearance (c) Histological preparation (hematoxylin and eosin staining or HE) of the collected lesions (IZ1 IZ2 and IZ3)indicating characteristic hyperkeratosis acanthosis and papillomatosis with dermis proliferation in HE preparation (100x)

IZ2100120583m

(a)

IZ2

(b)

Figure 2 (a) Histopathology of a wart biopsy detailed aspect of the IZ2 lesion exhibiting characteristic hyperkeratosis acanthosis and dermalproliferation indicated by arrows (100x) (b) Presence of koilocytosis

3 Results

In silico analyses of the restrictionsites in FAP L1 fragmentsof 20 deposited BPVs sequences (from BPV-1 to BPV-13)revealed no intratype variation associated with the relativecut positions for the four enzymes employed (Table 1)Macroscopic and histological evaluations identified the col-lected lesions as cutaneous fibrous papillomas (Figures 1and 2) From all the three lesions studied two- (IZ1 andIZ3) RFLP profiling suggests the presence of BPV-11 On theother hand IZ2 sample gene rated a profile which couldnot match with any of the thirteen characterized viral types(Figure 3 Table 1) In accordance with these results DNAsequencing and BLAST analysis of IZ2 indicated a rareputative type (BAPV-3) originally described in Japan [16]Furthermore the DNA sequencing of other two samples con-firms them as the recently described BPV-11 [10] These se-quences were deposited in GenBank (access numbersHQ435675 and HQ612180) The phylogenetic reconstructionusing this partial genomic sequence allowed its comparison

with other sequences of Papillomaviridae family members(Figure 4)

4 Discussion

Originally the recently characterized BPV-11 was describedwith the employment of aXipapillomavirus consensus primer[10] Here the same type using the FAP generic primer couldbe detected indicating this set as an effective alternative forthe identification of BPVs Other than that we described thesimultaneous presence of two BPV types in three differentwart samples obtained from a chronically affected animalwith disseminated papillomatosis

The typed BPV-11 and BAPV-3 samples have a fibrousaspect with a similar fibropapilloma histology that is notcommonly associated with the exclusive infection of the ker-atinocytes commonly attributed to theXi BPVs In a previousreport [10] Hatama and others discussed the ldquouncertainnature of BPV-11 tumorigenicityrdquo since BPV-11 was firstdiagnosed in a fibropapilloma lesion in which the BPV-1 was


M Mg19 IZ1 IZ2 IZ3 BPV-1 BPV-2

Dde I

(a)


Hinf I

(b)


MsL I

(c)


Hind III

(d)

Figure 3 RFLP performed for restriction enzymes DdeI Hinf I MsLI and HindIII As BPV-1 and BPV-2 are the commonly associated withfibropapillomas their L1 FAP amplicons were generated and digested as positive controls IZ1 IZ2 IZ3 amplicons are from three differentlesions of the same animal (IZ-1214) Mg-19 sample is from another bovine clinically affected with cutaneous papillomatosis Molecularmarker 100 bp ladder (New England Biolabs Ipswich UK)

4 10

9

3

IZ02

6

12

HPV-16

7

5

8

1

2

13

IZ0111

005

Figure 4 Neighbor-joining phylogenic tree constructed with 2000bootstrap replications using partial L1 nucleotide sequences indi-cating the putative type BAPV-3 in IZ2 sample originally describedin Japan (accession AY300819) The analysis of the other twosamples IZ1 and IZ3 demonstrated the presence of an identicalsequence in both lesions sharing 99 percent of similarity withthe recently described BPV-11 type (accession AB5435071) As IZ3sequence is identical to IZ1 it will not be shown here

also detected Despite the eventual limitations of the diagnos-tic methods employed we could detect the same viral type inat least two different cauliflower-like lesions with a suggestivefibrous core In addition to Hatamarsquos report our data alsolink the BPV-11 with cutaneous papillomas indicating it asa pathogenic type Furthermore the putative type BAPV-3described by Ogawa and others [16] was detected just oncefrom a skin papilloma sample without histological descrip-tion being genetically associated with the BPV-3 -4 and-6 or Xi BPVs

The L1 gene has taxonomical relevance due to its highdegree of conservation which can be accessed with the use ofgeneric primer sets In accordance with this the restrictionsites located in FAP fragments appears to be maintainedwithout intratype variations for the restriction enzymes em-ployed indicating that phylogenetic studies comparing BPVsand other members of the Papillomaviridae family are possi-ble using a relatively short DNA sequence

Since the early nineties methods based on PCR-RFLPanalysis within the L1 gene of the human papillomavirus(HPV) have been used for viral typing and infection diagnosisfrom a variety of sources including cervical samples freshandparaffin-embedded tissues [34 35] Particularly the PCR-RFLP method is useful to identify coinfections due to itssensitivity and specificity [36]


5 Conclusion

As an easy rapid and cost-effective assay the PCR-RFLP re-presents a less-laborious approach than DNA cloning and se-quencing being an alternative as a first-line screening testboth for the diagnosis of an already classified virus type asto indicating the needing of DNA sequencing due to mixedandor unknown digestion profiles In a previous survey inParana State Brazil Claus and others [26] suggest that theoccurrence ofmultiple ormixedBPVs infectionmay bewide-spread throughout Brazilian cattle herds and may occur inother Brazilian geographical regions In agreement our find-ings support these results and reinforce the notion thatmulti-ple papillomaviruses infections with a significant pathogenicpotential can be as frequent in cattle as in human hosts [26]

To the best of our knowledge the restriction map em-ployed here is the first to be produced specifically for thescreening and typing of BPVs Our findings also point to theubiquity of BPVs dissemination since two relatively rare newdescribed BPV types which were first characterized in Japanwere also detected in Brazil

Acknowledgments

The authors thank the Ministerio de Ciencia Tecnologia eInovacaoConselho Nacional de Desenvolvimento Cientıficoe Tecnologico (CNPq Proc 4025392011-7) Coordenacao deAperfeicoamento Pessoal de Nıvel Superior (CAPES) andFundacao do Desenvolvimento Administrativo (PAP-FUNDAP) for the financial support and Nadia Jorge BerrielCarolina da Paz Sabino and Jim Hesson (httpwwwAca-demicEnglishSolutionscom) for the editorial support

References

[1] M S Campo ldquoPapillomavirus and disease in humans and ani-malsrdquo Veterinary and Comparative Oncology vol 1 pp 3ndash142003

[2] S-Y Chan H Delius A L Halpern and H-U Bernard ldquoAnal-ysis of genomic sequences of 95 papillomavirus types unitingtyping phylogeny and taxonomyrdquo Journal of Virology vol 69no 5 pp 3074ndash3083 1995

[3] E-M de Villiers C Fauquet T R Broker H-U Bernard andH Zur Hausen ldquoClassification of papillomavirusesrdquo Virologyvol 324 no 1 pp 17ndash27 2004

[4] G Borzacchiello and F Roperto ldquoBovine papillomaviruses pa-pillomas and cancer in cattlerdquo Veterinary Research vol 39 no5 article 45 2008

[5] M S Campo W F H Jarrett R Barron B W OrsquoNeil andK T Smith ldquoAssociation of bovine papillomavirus type 2 andbracken fernwith bladder cancer in cattlerdquoCancer Research vol52 no 24 pp 6898ndash6904 1992

[6] F Jelınek and R Tachezy ldquoCutaneous papillomatosis in cattlerdquoJournal of Comparative Pathology vol 132 no 1 pp 70ndash81 2005

[7] L Nasir and M S Campo ldquoBovine papillomaviruses their rolein the aetiology of cutaneous tumours of bovids and equidsrdquoVeterinary Dermatology vol 19 no 5 pp 243ndash254 2008

[8] M Lunardi A A Alfieri R A Otonel et al ldquoGenetic char-acterization of a novel bovine papillomavirus member of the

Deltapapillomavirus genusrdquo Veterinary Microbiology vol 162no 1 pp 207ndash213 2012

[9] W F H Jarrett M S Campo B W OrsquoNeil H M Laird andLW Coggins ldquoA novel bovine papillomavirus (BPV-6) causingtrue epithelial papillomas of the mammary gland skin a mem-ber of a proposed new BPV subgrouprdquo Virology vol 136 no 2pp 255ndash264 1984

[10] S Hatama R Ishihara Y Ueda T Kanno and I UchidaldquoDetection of a novel bovine papillomavirus type 11 (BPV-11)using Xipapillomavirus consensus polymerase chain reactionprimersrdquoArchives of Virology vol 156 no 7 pp 1281ndash1285 2011

[11] W Zhu J Dong E Shimizu et al ldquoCharacterization of novelbovine papillomavirus type 12 (BPV-12) causing epithelial papil-lomardquo Archives of Virology vol 157 no 1 pp 85ndash91 2012

[12] N Bloch R H Sutton and P B Spradbrow ldquoBovine cutaneouspapillomas associated with bovine papillomavirus type 5rdquoArchives of Virology vol 138 no 3-4 pp 373ndash377 1994

[13] Y Tomita I Literak T Ogawa Z Jin and H Shirasawa ldquoCom-plete genomes and phylogenetic positions of bovine papillo-mavirus type 8 and a variant type from a European bisonrdquoVirusGenes vol 35 no 2 pp 243ndash249 2007

[14] T Ogawa Y Tomita M Okada and H Shirasawa ldquoCompletegenome and phylogenetic position of bovine papillomavirustype 7rdquo Journal of General Virology vol 88 no 7 pp 1934ndash19382007

[15] A Antonsson and B G Hansson ldquoHealthy skin ofmany animalspecies harbors papillomaviruses which are closely related totheir human counterpartsrdquo Journal of Virology vol 76 no 24pp 12537ndash12542 2002

[16] T Ogawa Y Tomita M Okada et al ldquoBroad-spectrum detec-tion of papillomaviruses in bovine teat papillomas and healthyteat skinrdquo Journal of General Virology vol 85 no 8 pp 2191ndash2197 2004

[17] Y Maeda T Shibahara Y Wada et al ldquoAn outbreak of teatpapillomatosis in cattle caused by bovine papilloma virus (BPV)type 6 and unclassified BPVsrdquo Veterinary Microbiology vol 121no 3-4 pp 242ndash248 2007

[18] M P Claus M Lunardi A F Alfieri L M Ferracin M H PFungaro and A A Alfieri ldquoIdentification of unreported puta-tive new bovine papillomavirus types in Brazilian cattle herdsrdquoVeterinary Microbiology vol 132 no 3-4 pp 396ndash401 2008

[19] S Hatama K Nobumoto and T Kanno ldquoGenomic and phylo-genetic analysis of two novel bovine papillomaviruses BPV-9and BPV-10rdquo Journal of General Virology vol 89 no 1 pp 158ndash163 2008

[20] O Forslund A Antonsson P Nordin B Stenquist and B GHansson ldquoA broad range of human papillomavirus types de-tected with a general PCR method suitable for analysis of cuta-neous tumours and normal skinrdquo Journal of General Virologyvol 80 no 9 pp 2437ndash2443 1999

[21] R C StoccoDos Santos C J Lindsey O P Ferraz et al ldquoBovinepapillomavirus transmission and chromosomal aberrations anexperimental modelrdquo Journal of General Virology vol 79 no 9pp 2127ndash2135 1998

[22] S R Wosiacki M P Claus A F Alfieri and A A AlfierildquoBovine papillomavirus type 2 detection in the urinary bladderof cattle with chronic enzootic haematuriardquoMemorias do Insti-tuto Oswaldo Cruz vol 101 no 6 pp 635ndash638 2006

[23] A Yaguiu C Carvalho A C Freitas et al ldquoPapillomatosis incattle in situ detection of Bovine Papillomavirus DNA se-quences in reproductive tissuesrdquo Brazilian Journal of Morpho-logical Sciences vol 23 no 3 pp 129ndash136 2006


[24] NDiniz T CMelo J F Santos et al ldquoSimultaneous presence ofbovine papillomavirus in blood and in short-term lymphocytecultures from dairy cattle in Pernambuco Brazilrdquo Genetics andMolecular Research vol 8 no 4 pp 1474ndash1480 2009

[25] M Lunardi M P Claus A A Alfieri and M H P FungaroldquoPhylogenetic position of an uncharacterized Brazilian strainof bovine papillomavirus in the genus Xipapillomavirus basedon sequencing of the L1 open reading framerdquo Genetics andMolecular Biology vol 33 no 4 pp 745ndash749 2010

[26] M P Claus M Lunardi A A Alfieri et al ldquoMultiple bovinepapillomavirus infections associated with cutaneous papillo-matosis in Brazilian cattle herdsrdquo Brazilian Archives of Biologyand Technology vol 52 pp 93ndash98 2009

[27] T Vincze J Posfai and R J Roberts ldquoNEBcutter a program tocleave DNA with restriction enzymesrdquo Nucleic Acids Researchvol 31 no 13 pp 3688ndash3691 2003

[28] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acid Symposium Series vol 41 pp 95ndash98 1999

[29] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[30] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[31] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[32] B Efron EHalloran and SHolmes ldquoBootstrap confidence lev-els for phylogenetic treesrdquo Proceedings of the National Academyof Sciences of the United States of America vol 93 no 23 pp13429ndash13434 1996

[33] R Dmpage ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996

[34] O Lungu T C Wright Jr and S Silverstein ldquoTyping of humanpapillomaviruses by polymerase chain reaction amplificationwith L1 consensus primers and RFLP analysisrdquo Molecular andCellular Probes vol 6 no 2 pp 145ndash152 1992

[35] G Astori A Arzese C Pipan E-M De Villiers and GA Botta ldquoCharacterization of a putative new HPV genomicsequence from a cervical lesion using L1 consensus primers andrestriction fragment length polymorphismrdquoVirus Research vol50 no 1 pp 57ndash63 1997

[36] R J Nobre L P de Almeida and T C Martins ldquoCompletegenotyping of mucosal human papillomavirus using a restric-tion fragment length polymorphism analysis and an originaltyping algorithmrdquo Journal of Clinical Virology vol 42 no 1 pp13ndash21 2008

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


DNA sequence of L1 differs bymore than 10 from the closestknown PV type Differences between 2 and 10 define asubtype and less than 2 a variant [3] Thirteen types ofBPVs are currentlywell characterized and classified into threedistinct generamdashDelta Epsilon and Ximdasheach one associatedwith epithelia lesions of specific histological nature [4]

The BPVs-1 and -2 are classified asDelta papillomaviruses[5] Characteristically these types induce the appearance offibropapillomas associated with the recruitment of the sub-epithelial fibroblasts [6] As far as concerned both types arealso unique in their ability to infect different host species notonly bovines causing the equine sarcoid [7] Lately the ge-nome of a newDelta-BPV type (BPV-13) was fully sequenced[8]

A larger number of BPV types (-3 -4 -6 -9 -10 -11and -12) belong to the Xipapillomavirus genus These virusesare considered exclusively epitheliotropic inducing the for-mation of ldquotrue papillomasrdquowithout the involvement of fibro-blasts [9ndash11] On the other hand the BPVs-5 and -8 have thepotential to induce both fibropapillomas and true papillomasin the course of their infectious cycle being classified intoa third genus Epsilonpapillomavirus [12 13] The BPV-7 re-presents an exception and is classified separately (unsignedgenus) This virus was first isolated from a cutaneous papil-loma lesion and also from healthy teat skin samples [14]

The thirteen described BPV types as well as other putativeones have been demonstrated bymolecular biologymethodssince papillomaviruses are not prone to be replicated or re-covered in cell cultures [15ndash19] According to the guidelinesoutlined by the Papil omavirus Nomenclature Committee(14th International Papillomavirus Conference Quebec CityQC Canada) it has been specified that the amplified se-quences isolated from novel papillomaviruses isolates couldindicate only putative new PV typesmdashinstead of PV typesmdashsince the PCR amplicons represent only part of the L1 gene[15]

Theuse of PCR assayswith degenerated primers followedby sequencing has allowed the identification of several PVtypes in human and other animal hosts [15 20] The PCRprimer FAP set was designed from two relatively conservedregions found in the L1 gene and has been shown to amplifyPVs DNA from both papillomas and healthy tissue of manyanimal species including BPVs in bovines [15 16 20]

Brazil has a cattle herd of approximately 210million beinga major exporter of meat milk and leather BPVs have beenpreviously detected in Brazil [21] but the extent of the impactof BPVs associated diseases both in dairy and cattle herdsneeds further studies Available reports in different regionsof the country indicate a significant diversity of viral typesamong theBrazilian herd implying an evident disease burden[18 22ndash25]Thus the improvement of knowledge concerningthe diagnosis and related clinical aspects of different BPVtypes among the Brazilian herd should be considered inthe development of new sanitary measures aiming to theprevention of BPV infection and its consequences

Unfortunately BPV epidemiological surveys are still lim-ited by the availability of high-throughput diagnostic tech-niques that could discriminate different BPV sequences at thesame time in co infected samples [16 26] In this context

the present work represents an effort to identify BPV typesemploying an alternative screening method based on thePCR-RFLP technique and correlating the histological data ofthe analyzed lesions with the diagnosed viral type

2 Material and Methods

In silico generation of RFLPs the L1 FAP segment digestionprofiles of the BPVs-1 to -13 could be generatedwithNEB cut-ter 20 [27] from all L1 complete nucleotide sequences avail-able in Genbank (httpwwwncbinlmnihgovgenbank)The restriction enzymes sites were chosen both by its pres-ence (or absence) as well as the generated digestion frag-ments sizes in order to differentiate those thirteen differentBPV types Histopathological analysis wart biopsies wereobtained from the trunk of a six-year-old Holstein dairycow chronically affectedwith cutaneous papillomatosis Sam-ples from three different lesions were submitted to macro-scopic histological (hematoxylin and Eosin staining) andmolecular analyses DNA extraction and PCR DNA wasextracted from warts for viral typing (Illustra tissue and cellsgenomic Prep Mini Spin GE Healthcare) and an approx-imately 470 base-pairs L1 gene segment was amplifiedusing the following primer sequences forward FAP59 (51015840-TAA CWG TIG GIC AYC CWT ATT-31015840) reverse FAP64(51015840-CCWATATCWVHCATITCICCATC-31015840) The PCR wereperformedwith slightmodifications of a previously describedprotocol [16] In detail the amplification reactions were per-formed in a Corbett CG1-96 thermocycler (Corbett Life Sci-ence Sydney Australia) with GoTaq Master Mix (PromegaMadison USA) under the following conditions 5min at95∘C followed by 35 cycles of 1min and 30s at 95∘C 2minat 52∘C and 1min and 30s at 72∘C and a final extensionstep of 5min at 72∘C Restriction analysis an aliquot of PCRfragments were submitted to digestion reactions for RFLPanalysis with four different restriction enzymes (DdeI Hinf IHindIII MsLI) following manufacturerrsquos instructions (NewEngland Biolabs Ipswich USA) Cloned BPV-1 and BPV-2 genomes as well as a known typed clinical sample (Mg-19 BPV-2 typed) were used as positive controls PCR-RFLPproducts were analyzed in 20 agarose gel electrophoresisstained with ethidium bromide (05120583gmL) in TAE bufferand visualized under UV light Sequencing An aliquot ofall generated PCR fragments were purified with extractioncolumns (Illustra GFX PCR DNA and Gel Band PurificationKit GE Healthcare) DNA concentration and purity weredetermined in a spectrophotometer (Eppendorf BioPho-tometer Hamburg Germany) and submitted to sequenc-ing reactions three independent sequencing reactions weredone for each PCR fragment in an ABI377 PRISM GeneticAnalyzer (Life Applied Biosystems USA) The quality ofDNA sequenceswas checked and overlapping fragmentswereassembled using the BioEdit package 7090 [28] Assembledsequences with high quality were aligned using ClustalW183 [29] with default gap penalties Homology analyses wereperformed with the NCBI database and BLAST [30] BioEditsoftware was used to identify the equivalent amino acidsequences The sequence alignments were performed using




BPV-1 475 bp264 329 301 475159 146 17452

BPV-2 475 bp 316 329 475 475159 146

BPV-3 473 bp319 473 373 473154 87

13

BPV-4 469 bp324 469 469 230145 182

87

BPV-5 469 bp469 254 469 469

14570

BPV-6 472 bp 321 384 373 472151 88 99

BPV-7 484 bp403 271 484 48481 119

94

BPV-8 469 bp 469 317 469 322152 147

BPV-9 469 bp 316 344 469 469153 125

BPV-10 472 bp319 381 373 47290 91 9963

BPV-11 475 bp 420 407 370 47555 68 105

BPV-12 469 bp 351 317 469 469118 152

BPV-13 475 bp316 329 475 475109 14650






IZ-1214

(a)

IZ-1214

(b)

100120583m100120583m100120583m

IZ1 IZ2 IZ3

(c)


IZ2100120583m

(a)

IZ2

(b)


3 Results



4 Discussion





Dde I

(a)


Hinf I

(b)


MsL I

(c)


Hind III

(d)


4 10

9

3

IZ02

6

12

HPV-16

7

5

8

1

2

13

IZ0111

005






5 Conclusion



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















BPV-1 475 bp264 329 301 475159 146 17452

BPV-2 475 bp 316 329 475 475159 146

BPV-3 473 bp319 473 373 473154 87

13

BPV-4 469 bp324 469 469 230145 182

87

BPV-5 469 bp469 254 469 469

14570

BPV-6 472 bp 321 384 373 472151 88 99

BPV-7 484 bp403 271 484 48481 119

94

BPV-8 469 bp 469 317 469 322152 147

BPV-9 469 bp 316 344 469 469153 125

BPV-10 472 bp319 381 373 47290 91 9963

BPV-11 475 bp 420 407 370 47555 68 105

BPV-12 469 bp 351 317 469 469118 152

BPV-13 475 bp316 329 475 475109 14650






IZ-1214

(a)

IZ-1214

(b)

100120583m100120583m100120583m

IZ1 IZ2 IZ3

(c)


IZ2100120583m

(a)

IZ2

(b)


3 Results



4 Discussion





Dde I

(a)


Hinf I

(b)


MsL I

(c)


Hind III

(d)


4 10

9

3

IZ02

6

12

HPV-16

7

5

8

1

2

13

IZ0111

005






5 Conclusion



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IZ-1214

(a)

IZ-1214

(b)

100120583m100120583m100120583m

IZ1 IZ2 IZ3

(c)


IZ2100120583m

(a)

IZ2

(b)


3 Results



4 Discussion





Dde I

(a)


Hinf I

(b)


MsL I

(c)


Hind III

(d)


4 10

9

3

IZ02

6

12

HPV-16

7

5

8

1

2

13

IZ0111

005






5 Conclusion



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Dde I

(a)


Hinf I

(b)


MsL I

(c)


Hind III

(d)


4 10

9

3

IZ02

6

12

HPV-16

7

5

8

1

2

13

IZ0111

005






5 Conclusion



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 Conclusion



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Bovine Papillomavirus in Brazil:...

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