Phylogenetic position of an uncharacterized Brazilian strain of bovine papillomavirus in the genus Xipapillomavirus based on sequencing of the L1 open reading frame

The use of PCR assays with degenerate primers has suggested the existence of numerous as yet uncharacterized bovine papillomaviruses (BPV). Despite the endemic nature of BPV infections, the identification of BPV types in Brazilian cattle is still only sporadic. However, in a recent analysis of a partial segment of the L1 gene, we observed notable diversity among the BPV types detected. The aim of this study was to determine the phylogenetic position of the previously identified wild strain BPV/BR-UEL2 detected in the state of Paraná in Brazil. Since previous analysis of the partial L1 sequence had shown that this strain was most closely related to BPV type 4, genus-specific primers were designed. Phylogenetic analysis using complete L1 ORF sequences revealed that BPV/BR-UEL2 was related to BPV types classified in the genus Xipapillomavirus and shared the highest L1 nucleotide sequence similarity with BPV type 4 (78 percent). This finding suggests that BPV/BR-UEL2 should be classified as a potential new type of BPV in the genus Xipapillomavirus.

Identification of the recently described new type of bovine papillomavirus (BPV-8) in a Brazilian beef cattle herd: [revision] / Identificação do novo tipo de papilomavírus bovino (BPV-8) recentemente descrito num rebanho bovino no Brasil: [revisão]

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.

A primeira descrição do papilomavírus bovino tipo 8 (BPV-8) foi realizada em amostras de papilomas de teto e de pele saudável de tetos de bovinos no Japão. Em 2007, a seqüência genômica completa do BPV-8 foi determinada. Ainda em 2007, uma variante do BPV-8 (BPV-8-EB) foi identificada em lesões papilomatosas de um bisão europeu na Eslováquia. No Brasil, apesar da infecção pelo BPV ser comumente observada em bovinos, a determinação dos tipos virais associados com a infecção ainda é esporádica. Este estudo tem o objetivo de relatar a ocorrência do BPV-8 no país. A amostra clínica foi obtida em um rebanho de corte do estado do Paraná, região sul do Brasil. O papiloma cutâneo, de aspecto macular e não-verrucoso, estava localizado na região dorsal torácica do animal. A identificação do vírus foi realizada pela reação em cadeia da polimerase (PCR) utilizando primers genéricos para a amplificação parcial do gene L1. O produto amplificado, com aproximadamente 480 pb, foi clonado e os fragmentos presentes em dois clones foram seqüenciados. A comparação da seqüência de nucleotídeos com a de outros papilomavírus demonstrou 100 por cento de identidade com o BPV-8. Assim, esta é a primeira descrição da ocorrência do BPV-8 no Brasil, o que sugere a sua presença nos rebanhos bovinos brasileiros.

Análise filogenética de papilomavírus bovino associado com lesões cutâneas em rebanhos do Estado do Paraná / Phylogenetic analysis of bovine papillomavirus associated with skin warts in cattle herds from the state of Paraná

A infecção pelo papilomavírus bovino (BPV) causa lesões hiperplásicas no epitélio cutâneo dos animais. De acordo com a localização e as características morfológicas das lesões, os seis tipos de BPV são classificados em dois sub-grupos. O objetivo desse trabalho foi identificar os tipos de BPV presentes em lesões cutâneas em bovinos de rebanhos do Estado do Paraná. Os primers degenerados FAP59 e FAP64 foram utilizados para a amplificação de um fragmento com 478 pb do gene L1 do BPV bovino em nove amostras de papilomas cutâneos obtidos de seis animais provenientes de quatro rebanhos bovinos do Estado. Em todas as amostras foi possível a amplificação de um produto com a massa molecular esperada. Por meio da análise filogenética das seqüências dos amplicons foi possível identificar o BPV-2 em três amostras, o BPV-1 em uma e o BPV-6 em cinco amostras de papilomas. O BPV-6 foi encontrado tanto em papilomas localizados no teto quanto em outras partes do corpo. Em um dos animais, do qual foram colhidas mais de uma amostra, foi detectada infecção concomitante do BPV-1 com o BPV-2. As cinco amostras positivas para o BPV-6 apresentaram 100 por cento de identidade de nucleotídeos com a amostra padrão disponível no GenBank. No entanto, foram identificadas diferenças entre as amostras do BPV-2 e BPV-1 e aquelas depositadas neste banco de dados. Esse estudo demonstrou a diversidade de tipos do BPV circulantes em rebanhos do Estado do Paraná.

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 percent 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.

Análise filogenética de papilomavírus bovino associado com lesões cutâneas em rebanhos do Estado do Paraná / Phylogenetic analysis of bovine papillomavirus associated with skin warts in cattle herds from the state of Paraná

A infecção pelo papilomavírus bovino (BPV) causa lesões hiperplásicas no epitélio cutâneo dos animais. De acordo com a localização e as características morfológicas das lesões, os seis tipos de BPV são classificados em dois sub-grupos. O objetivo desse trabalho foi identificar os tipos de BPV presentes em lesões cutâneas em bovinos de rebanhos do Estado do Paraná. Os primers degenerados FAP59 e FAP64 foram utilizados para a amplificação de um fragmento com 478 pb do gene L1 do BPV bovino em nove amostras de papilomas cutâneos obtidos de seis animais provenientes de quatro rebanhos bovinos do Estado. Em todas as amostras foi possível a amplificação de um produto com a massa molecular esperada. Por meio da análise filogenética das seqüências dos amplicons foi possível identificar o BPV-2 em três amostras, o BPV-1 em uma e o BPV-6 em cinco amostras de papilomas. O BPV-6 foi encontrado tanto em papilomas localizados no teto quanto em outras partes do corpo. Em um dos animais, do qual foram colhidas mais de uma amostra, foi detectada infecção concomitante do BPV-1 com o BPV-2. As cinco amostras positivas para o BPV-6 apresentaram 100 por cento de identidade de nucleotídeos com a amostra padrão disponível no GenBank. No entanto, foram identificadas diferenças entre as amostras do BPV-2 e BPV-1 e aquelas depositadas neste banco de dados. Esse estudo demonstrou a diversidade de tipos do BPV circulantes em rebanhos do Estado do Paraná.

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 percent 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 type 2 detection in the urinary bladder of cattle with chronic enzootic haematuria

The bovine papillomavirus type 2 (BPV-2) involvement in the aetiology of chronic enzootic haematuria associated to bracken fern ingestion has been suggested for a long time. However, a few reports have shown the presence of the BPV-2 in urinary bladder tumors of cattle. The aim of this study was to investigate the presence of the BPV-2 infection in the urinary bladder of cattle with chronic enzootic haematuria in Brazilian cattle herds. Sixty-two urinary bladders were collected from adult cattle in beef herds from the north region of the state of Paraná, Brazil. According to clinical and pathological finds the specimens were distributed in three groups: the group A was constituted by 22 urinary bladders with macroscopic lesions collected at necropsy of cattle with clinical signs of chronic enzootic haematuria; the group B by 30 urinary bladders with macroscopic lesions collected in a slaughterhouse of cows coming from bracken fern-endemic geographical region; and the group C (control) by 10 urinary bladders without macroscopic lesions collected from asymptomatic cattle in a bracken fern-free geographical region. By a semi-nested polymerase chain reaction (PCR) assay, with an internal control, a fragment of the BPV-2 L1 gene with 386 bp length was amplified in 36 (58 percent) urinary bladder. The rate of BPV-2 positive urinary bladders was 50 percent (11/22) for group A, 80 percent (24/30) for group B, and 10 percent (1/10) for group C (control). The rate of the positive results found in groups A and B that included urinary bladder samples with macroscopic lesions was 67 percent (35/52) and the detection of the BPV-2 in both groups was significantly higher (P < 0.05) than in the control group. RFLP with Rsa I and Hae III enzymes evaluated the specificity of the BPV-2 amplicons. The PCR internal control that amplified a 626 bp fragment of the ND5 gene of the bovine mitochondrial genome was amplified in all analyzed samples and excluded false-negatives or invalid results in the semi-nested PCR...