PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- De Alcântara B.K., Alfieri A.A., Headley S.A., Rodrigues W.B., Otonel R.A.A., Lunardi M. & Alfieri A.F. 2015. Molecular characterization of bovine Deltapapillomavirus (BPV1, 2, and 13) DNA in equine sarcoids. Pesquisa Veterinária Brasileira 35(5):431-436. Laboratory of Animal Virology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Campus Universitário, Cx. Postal 10011, Londrina, PR 86057-970, Brazil. E-mail: alfieri@uel.br Sarcoids are fibroblastic lesions, which are considered as the most common skin tumors of horses; spontaneous regression rarely occurs. The bovine papillomavirus (BPV) types 1 and 2 may be involved in the pathogenesis of sarcoids, and probably the recently described BPV type (BPV13) might be associated with the pathogenesis of this lesion. This study characterized the DNA of BPVs in sarcoids from 15 horses from Brazil by analyzing 20 cutaneous lesions (12 recently collected; 8 from formalin-fixed paraffin-embedded (FFPE) tissues). Histopathology confirmed the proliferative lesions as sarcoids. Three PCRs were performed to amplify papillomavirus (PV) DNA. For screening, the primers IFNR2/IDNT2 were used to amplify a fragment of the PV L1 ORF. The second primer set was complementary to a common sequence of the E5L2 genomic region of BPV1, 2, and 13. The third primer pair (FAP59/FAP64) targeted a fragment of the PVs L1 ORF. The screening and E5L2 PCRs yielded amplicons in all samples evaluated. The FAP amplicons identified BPV1, 2, and 13 only from fresh tissue samples. The phylogenetic analyses of E5L2 resulted in the identification of BPV1, 2, and 13 in 14 (70%), 2 (10%), and 4 (20%) sarcoids, respectively. Two horses demonstrated multiple lesions: the sarcoids of one of these contained only BPV1 DNA and those of the other contained three types of bovine Deltapapillomavirus (BPV1, 2, and 13). This study confirmed the presence of BPV1, 2, and 13 DNA in equine sarcoids. Moreover, these findings represent the first description of three types of BPV diagnosed in the same horse, as well as the first confirmation of BPV1 and 2 in horses from Brazil.

• Papillomavirus Bovine Deltapapillomavirus skin tumor Papilomavírus Deltapapillomavirus bovino tumor de pele

Abstract in Portuguese:

RESUMO.- De Alcântara B.K., Alfieri A.A., Headley S.A., Rodrigues W.B., Otonel R.A.A., Lunardi M. & Alfieri A.F. 2015. Molecular characterization of bovine Deltapapillomavirus (BPV1, 2, and 13) DNA in equine sarcoids. [Caracterização molecular de DNA de Deltapapillomavirus bovino (BPV1, 2 e 13) em sarcoides equinos.] Pesquisa Veterinária Brasileira 35(5):431-436. Laboratory of Animal Virology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Campus Universitário, Cx. Postal 10011, Londrina, PR 86057-970, Brazil. E-mail: alfieri@uel.br Sarcoides são tumores fibroblásticos, considerados os tumores de pele mais comuns em pele de equinos e que raramente apresentam regressão espontânea. Papilomavírus bovino (BPV) tipos 1 e 2 são relacionados com a patogenia do sarcoide e, provavelmente, o BPV tipo 13 (BPV13), recentemente descrito, também pode estar associado com a formação dessa lesão. Neste estudo, 20 amostras de lesões cutâneas, sendo 12 constituídas por tecidos frescos e 8 amostras de tecido fixado em formalina e embebido em parafina, provenientes de 15 cavalos foram utilizadas para a identificação do DNA de BPV. A análise histopatológica (HE) confirmou todas as lesões como sarcoide. Para a amplificação do DNA de papilomavírus (PV) foram realizadas três reações de PCR. Como triagem, os primers IFNR2/IDNT2 foram utilizados para amplificar um fragmento da ORF L1 do PV. O segundo par de primers utilizado é complementar a sequência dos genes E5 e L2 de BPVs 1, 2 e 13. O terceiro par de primers (FAP59/FAP64) utilizado tem o gene L1 como alvo. A primeira e a segunda PCRs permitiram amplificar produtos em todas as amostras avaliadas. Entretanto, na terceira reação, na qual foram utilizados os primers FAP, foi possível amplificar produtos com tamanho molecular esperado somente nas amostras constituídas por tecidos frescos. O sequenciamento de nucleotídeos e as análises filogenéticas realizadas nos fragmentos E5L2 resultaram na identificação de BPV1, 2 e 13 em 14 (70%), 2 (10%) e em 4 (20%) amostras de sarcoides, respectivamente. As amostras de sarcoides de um dos animais continha somente o DNA de BPV1. Entretanto, nas amostras provenientes do segundo cavalo foi possível identificar o DNA de três tipos de Deltapapillomavirus bovino (BPV1, 2 e 13) em lesões distintas. Este estudo ratifica a presença do DNA de BPV1, 2 e 13 em lesões de sarcoides em equinos, além de identificar três tipos de BPVs em um mesmo animal e descrever pela primeira vez no Brasil a presença de BPV1 e 2 nesse tipo de lesão.

Abstract in English:

ABSTRACT.- De Alcântara B.K., Alfieri A.A., Rodrigues W.B., Otonel R.A.A., Lunardi M., Headley S.A. & Alfieri A.F. 2014. Identification of canine papillomavirus type 1 (CPV1) DNA in dogs with cutaneous papillomatosis. Pesquisa Veterinária Brasileira 34(12):1223-1226. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Campus Universitário, Cx. Postal 10011, Londrina. PR 86057-970, Brazil. E-mail: alfieri@uel.br Canine oral papillomavirus (COPV), also known as Canine Papillomavirus type 1 (CPV1), induces papillomas at the mucous membranes of the oral cavity and at the haired skin of dogs. The classification of Papillomavirus (PV) types is based on the L1 capsid protein and nucleotide sequence; so far, 14 CPV types have been described in several countries, but the molecular characterization of CPV in Brazil is lacking. This study investigated the presence of the PV in seven papillomas from four mixed breed dogs from Londrina/PR, Southern Brazil, by partial sequencing of the L1 gene. Seven exophytic cutaneous lesions were surgically removed and processed for histopathological and molecular characterization. Histopathology confirmed the lesions as viral papillomas due to typical histological features. Polymerase Chain Reaction (PCR) assay using the FAP59 and FAP64 primers targeted the L1 gene followed by sequence analysis of the amplicons identified CPV1 in all evaluated papilloma samples. This study represents the first description of CPV1 DNA associated with canine papillomatosis in Brazil.

• Papillomatosis canine papillomavirus papilomatose papillomavirus canino

Abstract in Portuguese:

RESUMO.- De Alcântara B.K., Alfieri A.A., Rodrigues W.B., Otonel R.A.A., Lunardi M., Headley S.A. & Alfieri A.F. 2014. Identification of canine papillomavirus type 1 (CPV1) DNA in dogs with cutaneous papillomatosis. [Identificação do DNA de Papillomavirus canino tipo 1 em cães com papilomas cutâneos no Brasil.] Pesquisa Veterinária Brasileira 34(12):1223-1226. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Campus Universitário, Cx. Postal 10011, Londrina. PR 86057-970, Brazil. E-mail: alfieri@uel.br O papilomavírus oral canino (COPV), também denominado Papillomavirus canino tipo 1 (CPV1), tem a capacidade de induzir papilomas na mucosa da cavidade oral e também em pele de cães. A classificação dos tipos de papilomavírus (PV) é baseada na proteína L1 do capsídeo e na sequência de nucleotídeos que a codifica. Atualmente são descritos 14 tipos de CPV, no entanto, ainda faltam estudos moleculares relacionados à identificação dos tipos de CPV no Brasil. O objetivo deste estudo foi investigar a presença de PV em fragmentos de papilomas obtidos de quatro cães sem raça definida, provenientes de Londrina/PR, região sul do Brasil, e definir o tipo viral por meio da análise da sequência parcial de nucleotídeos do gene L1. Sete lesões cutâneas foram cirurgicamente removidas e processadas ​​para a caracterização histopatológica e molecular. O exame histopatológico confirmou as lesões como papilomas. Foi realizada reação em cadeia de polimerase (PCR), utilizando os primers FAP59 FAP64 para a amplificação parcial do gene L1, seguida por análise das sequências dos produtos amplificados, que confirmou a presença do CPV1 em todas as amostras avaliadas. Este estudo representa a primeira identificação do DNA de CPV1 associado com papilomatose canina no Brasil.

Abstract in English:

ABSTRACT.- Dezen S., Otonel R.A.A., Alfieri A.F., Lunardi M. & Alfieri A.A. 2013. [Bovine viral diarrhea virus (BVDV) infection profile in a high production dairy herd with vaccination program against BVDV.] Perfil da infecção pelo vírus da diarreia viral bovina (BVDV) em um rebanho bovino leiteiro de alta produção e com programa de vacinação contra o BVDV. Pesquisa Veterinária Brasileira 33(2):141-147. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Cx. Postal 6001, Londrina, PR 86051-990, Brazil. E-mail: alfieri@uel.br The profile of bovine viral diarrhea virus (BVDV) infection was studies in a high production dairy herd selected based on a history of reproductive failures and regular vaccination against BVDV. Virus identification was performed by RT-PCR and serological profile was determined by virus-neutralization (VN). Initially, 100% (n=692) of the animals in the herd were monitored for identification of an active infection by RT-PCR in sera. Four months later, all positive animals (n=29) were retested by RT-PCR, along with newly born animals (n=72), or those that had reproductive failures (n=36) in the interval. The RT-PCR assay identified 27 transiently infected animals and three persistently infected (PI). Serology performed only in positive animals in the first RT-PCR and in cows with reproductive failures between the first and second RT-PCR analysis, showed large variation VN antibody titers and seroconversion in most animals. Increases in VN titers were demonstrated, with variation between 3 and 8 log2, indicating virus circulation within the herd. Virus circulation in the vaccinated herd evaluated in this study was likely responsible for reproductive failures observed in cows with low VN titers and for fetal infections. These results demonstrate that control of BVDV infection by regular vaccination in dairy cattle herds with PI animals represents a great challenge for the prophylaxis of this infection.

• BVDV bovine viral diarrhea virus vírus da diarreia viral bovina

Abstract in Portuguese:

RESUMO.- Dezen S., Otonel R.A.A., Alfieri A.F., Lunardi M. & Alfieri A.A. 2013. [Bovine viral diarrhea virus (BVDV) infection profile in a high production dairy herd with vaccination program against BVDV.] Perfil da infecção pelo vírus da diarreia viral bovina (BVDV) em um rebanho bovino leiteiro de alta produção e com programa de vacinação contra o BVDV. Pesquisa Veterinária Brasileira 33(2):141-147. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Cx. Postal 6001, Londrina, PR 86051-990, Brazil. E-mail: alfieri@uel.br A infecção pelo vírus da diarreia viral bovina (BVDV) foi avaliada em um rebanho bovino leiteiro de alta produção com histórico de problemas reprodutivos e de vacinação regular contra o BVDV. A identificação do vírus foi realizada por RT-PCR em soro sanguíneo e o perfil sorológico por vírus-neutralização. Inicialmente, 100% (n=692) dos animais do rebanho foram avaliados com relação à presença de infecção ativa pelo BVDV por meio da RT-PCR. Quatro meses após, todos os animais positivos (n=29) na primeira avaliação foram avaliados novamente pela RT-PCR, assim como todos os animais que nasceram (n=72) e os que apresentaram problemas reprodutivos (n=36) no intervalo entre a primeira e a segunda colheita de sangue. Os resultados finais do estudo possibilitaram identificar 27 animais transitoriamente infectados e três animais persistentemente infectados (PI). A sorologia, realizada apenas nos animais positivos na primeira avaliação pela RT-PCR e nas vacas que apresentaram problemas reprodutivos entre a primeira e a segunda RT-PCR, demonstrou grande flutuação nos títulos de anticorpos neutralizantes, além de soroconversão na maioria dos animais. Foram identificados aumentos nos títulos de anticorpos neutralizantes que variaram entre 3 e 8 log2, indicando infecção ativa no rebanho. A circulação viral no rebanho avaliado foi responsável pela expressão de sinais clínicos da esfera reprodutiva em animais com baixo título de anticorpos e consequente falha na proteção fetal. Os resultados demonstram que o controle da infecção pelo BVDV apenas por meio da vacinação regular em rebanhos com animais PI pode não ser eficaz na profilaxia dessa virose.

Abstract in English:

ABSTRACT.- Claus M.P., Lunardi M., Alfieri A.F., Sartori D., Fungaro M.H.P & Alfieri A.A. 2009. Identification of the recently described new type of bovine papillomavirus (BPV-8) in a Brazilian beef cattle herd. Pesquisa Veterinária Brasileira 29(1):25-28. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Cx. Postal 6001, Campus Universitário, Londrina, PR 86051-990, Brazil. E-mail: alfieri@uel.br Bovine papillomavirus type 8 (BPV-8) was first detected and described in teat warts as well as in healthy teat skin from cattle raised in Japan. The entire viral genome was sequenced in 2007. Additionally, a variant of BPV-8, BPV-8-EB, was also identified from papillomatous lesions of a European bison in Slovakia. In Brazil, despite the relatively common occurrence of BPV infections, the identification and determination of viral types present in cattle is still sporadic. The aim of this study is to report the occurrence of the recently described BPV-8 in Brazil. The virus was identified in a skin warts obtained from a beef cattle herd located in Parana state, southern Brazil. The papilloma had a macular, non-verrucous gross aspect and was located on the dorsal thorax of a cow. Polymerase chain reaction (PCR) was performed using generic primers for partial amplification of L1 gene. The obtained amplicon (480bp) was cloned and two selected clones were sequenced. The nucleotide sequence was compared to existing papillomaviral genomic sequences, identifying the virus as BPV type 8. This study represents the first report of BPV-8 occurrence in Brazil, what suggests its presence among Brazilian cattle.

• Bovine bovine papillomavirus type 8 BPV-8 cutaneous papillomatosis molecular analysis

Abstract in Portuguese:

ABSTRACT.- Claus M.P., Lunardi M., Alfieri A.F., Sartori D., Fungaro M.H.P & Alfieri A.A. 2009. Identification of the recently described new type of bovine papillomavirus (BPV-8) in a Brazilian beef cattle herd. Pesquisa Veterinária Brasileira 29(1):25-28. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Cx. Postal 6001, Campus Universitário, Londrina, PR 86051-990, Brazil. E-mail: alfieri@uel.br Bovine papillomavirus type 8 (BPV-8) was first detected and described in teat warts as well as in healthy teat skin from cattle raised in Japan. The entire viral genome was sequenced in 2007. Additionally, a variant of BPV-8, BPV-8-EB, was also identified from papillomatous lesions of a European bison in Slovakia. In Brazil, despite the relatively common occurrence of BPV infections, the identification and determination of viral types present in cattle is still sporadic. The aim of this study is to report the occurrence of the recently described BPV-8 in Brazil. The virus was identified in a skin warts obtained from a beef cattle herd located in Parana state, southern Brazil. The papilloma had a macular, non-verrucous gross aspect and was located on the dorsal thorax of a cow. Polymerase chain reaction (PCR) was performed using generic primers for partial amplification of L1 gene. The obtained amplicon (480bp) was cloned and two selected clones were sequenced. The nucleotide sequence was compared to existing papillomaviral genomic sequences, identifying the virus as BPV type 8. This study represents the first report of BPV-8 occurrence in Brazil, what suggests its presence among Brazilian cattle.

Abstract in English:

ABSTRACT.- Claus M.P., Vivian D., Lunardi M., Alfieri A.F. & Alfieri A.A. 2007. [Phylogenetic analysis of bovine papillomavirus associated with skin warts in cattle herds from the state of Paraná.] Análise filogenética do papilomavírus bovino associado a lesões cutâneas em rebanhos do Estado do Paraná. Pesquisa Veterinária Brasileira 27(7):314-318. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Cx. Postal 6001, Campus Universitário, Londrina, PR 86051-990, Brazil. E-mail: alfieri@uel.br Bovine papillomavirus (BPV) infection causes hyperplastic lesions in the cutaneous epithelium of cattle. Six types of BPV were classified in two sub-groups, being correlated to the anatomical regions of the infection and morphologic characteristics of the lesions. The present study was carried out to identify the types of BPV present in skin warts of cattle from the state of Paraná, Brazil. The generic primers FAP59 and FAP64 were used for amplification of a 478 bp fragment of BPV L1 gene in nine cutaneous papilloma samples obtained from six animals in four herds. In all papillomas examined, a product with the expected molecular size was amplified. Phylogenetic analysis of the PCR products identified BPV-2 in three samples, BPV-1 in one, and BPV-6 in five papillomas. BPV-6 was detected in cutaneous papillomas of the teat and in other body parts as well. In one animal, from which more than one sample was collected, a concomitant infection by BPV-1 and BPV-2 was identified. The five positive BPV-6 samples showed a nucleotide identity of 100% with the sequence of the reference strain available in GenBank. However, differences among BPV-2 and BPV-1 Brazilian samples and the respective reference sequences deposited in GenBank were observed. Molecular comparison of the two BPV-2 strains identified showed the involvement of two viral variants. This study revealed the diversity of BPV types circulating in the state of Paraná.

• Bovine papillomavirus cutaneous papillomatosis phylogeny polymerase chain reaction

Abstract in Portuguese:

ABSTRACT.- Claus M.P., Vivian D., Lunardi M., Alfieri A.F. & Alfieri A.A. 2007. [Phylogenetic analysis of bovine papillomavirus associated with skin warts in cattle herds from the state of Paraná.] Análise filogenética do papilomavírus bovino associado a lesões cutâneas em rebanhos do Estado do Paraná. Pesquisa Veterinária Brasileira 27(7):314-318. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Cx. Postal 6001, Campus Universitário, Londrina, PR 86051-990, Brazil. E-mail: alfieri@uel.br Bovine papillomavirus (BPV) infection causes hyperplastic lesions in the cutaneous epithelium of cattle. Six types of BPV were classified in two sub-groups, being correlated to the anatomical regions of the infection and morphologic characteristics of the lesions. The present study was carried out to identify the types of BPV present in skin warts of cattle from the state of Paraná, Brazil. The generic primers FAP59 and FAP64 were used for amplification of a 478 bp fragment of BPV L1 gene in nine cutaneous papilloma samples obtained from six animals in four herds. In all papillomas examined, a product with the expected molecular size was amplified. Phylogenetic analysis of the PCR products identified BPV-2 in three samples, BPV-1 in one, and BPV-6 in five papillomas. BPV-6 was detected in cutaneous papillomas of the teat and in other body parts as well. In one animal, from which more than one sample was collected, a concomitant infection by BPV-1 and BPV-2 was identified. The five positive BPV-6 samples showed a nucleotide identity of 100% with the sequence of the reference strain available in GenBank. However, differences among BPV-2 and BPV-1 Brazilian samples and the respective reference sequences deposited in GenBank were observed. Molecular comparison of the two BPV-2 strains identified showed the involvement of two viral variants. This study revealed the diversity of BPV types circulating in the state of Paraná.