Isolamento e identificação do papilomavírus bovino em grupo experimental de bovinos para obtenção de um banco de vírus.

Abstract

Araldi RP. Isolation and identification of Bovine papillomavirus in experimental group of cattle in order to obtain a virus bank. [dissertation (Masters thesis in Biotechnology)]. Sao Paulo: Instituto de Ciencias Biomedicas, Universidade de Sao Paulo; 2014. The bovine papillomavirus (BPV) is the etiological agent of bovine papillomatosis, infectious disease that causes significant economic losses to livestock. It is characterized by the presence of papillomas that regress spontaneously, but can persist and progress to malignancy. Currently, there are 13 types of BPVs described in the literature and 32 putative new types of BPVs. The study aimed to isolate viral particles of BPV from skin papillomas, previously identified and typified. Also, we performed the differential diagnosis by histopathology and immunohistochemistry (IHC) and, evaluate the clastogenic potential of the virus in situ. 77 cutaneous papilloma samples of 27 cattle of Simmental breed were surgically removed. The extracted DNA was subjected to PCR using Delta-Epsilon and Xi primers. The bands were purified and sequenced. The sequences were analyzed using bioinformatics and compared to the GenBank database, by BLASTn tool. A representative sample from each animal was subjected to histopathological and IHC analysis. The evaluation of clastogenicity was performed using the comet assay (CA) method for tissues. The viral typing showed a prevalence of BPV-2 in 81.81% of the samples, followed by BPV-5, viruses associated with the development of fibropapillomas. It was also detected the presence of the putative new virus type BR/UEL-2 in one sample, showing the diversity of circulating virus types in Brazil. Histopathological analysis revealed the presence of acanthosis, koilocytosis, hypergranulosis and hyperkeratosis. It was possible to detect the presence of transformed fibroblasts, which is associated with the combination of E5, E6, and E7 proteins, suggesting a pathway of infection through blood. By IHC, it was detected the labelling of L1in corneal and basal layer, near the transformed fibroblasts, labelling indicating productive infection at other sites less differentiated. An expressive E7 labelling was observed in all layers of the epithelium and stroma, demonstrating the viral activity in fibroblasts. The CA analysis showed the clastogenic action of BPV-2, 5 and 9, although no statistically significant difference between viral types was observed. The findings support the oncogenic action of BPV in establishing of favorable microenvironment for malignant transformation. Virus isolation was performed by ultracentrifugation in single density of cesium chloride, using a new method, which is less laborious than those already described, involving fewer steps of centrifugation, allowing the isolation of entire particles of BPV-2, composing the virus stock of Laboratory of Genetics of Butantan Institute. The new proposed methodology can be used with equal success in isolation of other papillomaviruses. Keyword: Veterinary oncology. Oncogenic virus. Papovaviridae, Animal histopathology. Immunohistochemistry. Mutagenesis.