Molecular characterization of Bovine leukemia virus (BLV) isolates from Brazil using the env gene sequences revealed a high conservation of this gene. In most cases the substitutions corresponded to silent transitions. In addition, cystein residues, potential glycosylation sites, neutralization domains and other critical residues involved with the envelope structural domains and viral infectivity were conserved. Most of the substitutions found in the aminoacid sequences of the gp51 protein were localized in the G and H epitopes. Using the SIFT software, it was predicted that they should not alter the protein functions. Phylogenetic analyses showed that partial or complete env gene sequences grouped in three or four phylogenetic clusters, respectively. The sequences from the Brazilian isolates had similar mutation rates as compared to samples from other countries, and belonged to at least two phylogenetic clusters.

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Analysis of the partial bovine leukaemia virus (BLV) env gp51 gene sequences obtained from three BLV strains isolated in three different regions of Brazil was carried out. The Brazilian BLV env gp51 sequences were compared with seven other corresponding sequences of BLV strains isolated in different countries and with consensus sequence as well. The obtained data point on qualitative and quantitative differences among the analysed strains as far as the occurrence of single point mutations is concerned. Two Brazilian strains show significantly higher mutation rate than other analysed strains. Amino acid analysis did not show, however, any substantial changes of the primary protein structure coded by well conserved region of BLV env gp51 gene. Based on the obtained data, the putative dendogram image of possible phylogenetic relations among the studied BLV strains is presented as well.

Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot‐and‐mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT‐ddPCR) using one‐step and two‐step approaches. Analytical sensitivity and specificity were done in parallel with real‐time PCR, RT‐qPCR (one‐step and two‐step) for comparison of sensitivity and specificity of both methods. In the standardization of RT‐ddPCR, the double‐quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one‐step techniques showed superior performance than two‐step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT‐ddPCR and RT‐qPCR using the one‐step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT‐PCR had a better performance than RT‐PCR when swine serum pools were tested. According to the results, the one‐step RT‐ddPCR and RT‐qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT‐ddPCR), being a useful tool for vesicular diseases control programs.