In the last decades, a group of viruses has received great attention due to its relationship with cancer development and its wide distribution throughout the vertebrates: the papillomaviruses. In this article, we aim to review some of the most relevant reports concerning the use of bovines as an experimental model for studies related to papillomaviruses. Moreover, the obtained data contributes to the development of strategies against the clinical consequences of bovine papillomaviruses (BPV) that have led to drastic hazards to the herds. To overcome the problem, the vaccines that we have been developing involve recombinant DNA technology, aiming at prophylactic and therapeutic procedures. It is important to point out that these strategies can be used as models for innovative procedures against HPV, as this virus is the main causal agent of cervical cancer, the second most fatal cancer in women.
Bovine papillomavirus (BPV) is the etiological agent of bovine papillomatosis (BP), infectious disease, characterized by the presence of multiples papillomas that can regress spontaneously or progress to malignances. Although recognized as mutagen, BPV action following cancer initiation remains few explored, since studies about cancer progression and metastasis are based on cell cultures. The lack of attention to in vitro models is a reflection of the papillomavirus replication paradigm, which is dependent of epithelium cell differentiation. Since 2008, we have explored the potential of cell lines derived from BPV-infected neoplasms as model to study the oncogenic process. In this study, we described BPV productive infection in cell lines derived from cutaneous papilloma, fibropapilloma and esophageal carcinoma (EC) in which BPV DNA sequences were previously detected by PCR. Considering that the immunodetection of L1 capsid protein is the main evidence of productive infection, we analyzed the expression of this protein by immunofluorescence and flow cytometry. Results showed the immunodetection of L1 protein in cell lines derived from cutaneous papilloma, fibropapilloma and EC, but not in cells derived from BPV-free normal skin. We also observed the presence of spherical and electron-dense particles, with 41.02-61.94 nm diameter in cytoplasmic vesicles of cells in the sixth passage of cutaneous papilloma, fibropapilloma and EC, being compatible with the expected BPV morphology. Cells derived from BPV-free normal skin, in turn, showed membranous particles up to 75.00 nm not compatible with BPV morphology. These results suggest the BPV productive infection in cells lines derived from BPV-infected neoplasm, reinforcing that these cells are useful models to study the viral biology and pathogenesis.Correspondence to: Rita de Cassia Stocco, Genetics Laboratory, Butantan Institute, São Paulo, 05503-900, Brazil, Tel: +55 11 2627-9701; e-mail: rita. stocco@butantan.gov.br Highlights• Bovine papillomavirus (BPV) cause multiples papillomas that can regress or progress to malignances;• BPV action following cancer initiation remains few explored;• Identification of BPV L1 capsid protein and virion-like particles in cytoplasmic vesicles of cell lines derived from BPVinfected cutaneous papilloma, fibropapilloma and esophageal carcinoma;• Cell lines derived from BPV-infected neoplasm can be considered useful model to study the viral biology and pathology.
MAZZUCHELLI-DE-SOUZA, J. Structural modelling and in silico analysis of E6 protein of the Deltapapillomavirus genus. 2018. (178 f.). Ph. D. these
Atualmente, são descritos 13 tipos de Papilomavírus Bovino (BPV). O tipo 1 (BPV-1) causa fibropapilomas em seu hospedeiro natural (bovinos) e sarcóide em equinos. A papilomatose bovina se caracteriza por lesões na pele e mucosa (verrugas) associadas à expressão de oncoproteínas virais, principalmente E6 e E7. A expressão dessas oncoproteínas em sistema recombinante e sua purificação se torna atraente para estudo, pois possibilita a obtenção de insumos biotecnológicos, como anticorpos e vacinas. Como a melhor forma de prevenção ainda é a intensificação dos controles gerais de manejo há a necessidade do desenvolvimento de novas tecnologias vacinais e de diagnóstico. Dessa forma, os objetivos deste projeto foram a clonagem e expressão do gene E6 do BPV-1, obtendo a proteína alvo e analisar in silico a proteína recombinante E6 do BPV-1. Iniciadores foram sintetizados e utilizados para a amplificação do gene de interesse (E6-1). O amplificado foi purificado e ligado ao vetor de clonagem pCR4-TOPO, sendo clonado em E. coli DH5α. Subsequentemente, foram realizadas a digestão, a ligação do inserto no vetor de expressão pET-28a(+) e subclonagem em E. coli BL21 (DE). Os plasmídeos foram purificados e sequenciados. Foram realizadas a indução da expressão gênica e lise celular para a obtenção da proteína recombinante, a qual foi submetida à eletroforese SDS-PAGE e "Western blot". Foram realizadas análises in silico das sequências gênica e proteica. A sequência amplificada apresentou o tamanho esperado. A clonagem e a matriz correta de leitura foram confirmadas por sequenciamento. As análises in silico apontaram as mutações nas sequências gênica e proteica E6-1 em relação às sequências depositadas no banco de dados. Por homologia, foi realizada a predição tridimensional da estrutura da proteína recombinante. Através de ferramentas de bioinformática foram apontadas as regiões conservadas, antigênicas e as regiões potencialmente capazes de realizar ligações cátion-π. A microscopia eletrônica mostrou a formação de corpúsculos de inclusão. Foram obtidas proteínas purificadas com aproximadamente 15 kDa. Logo, a proteína recombinante E6 do BPV-1 foi obtida e purificada com sucesso possibilitando, juntamente com sua análise por bioinformática, o início de novos experimentos e estudos mais detalhados. Por fim, é visada a obtenção de produtos vacinais e diagnósticos.
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