Hed novel genetic markers of Brazilian strains groups (C23L). A56R, B5R, C23L and C11R were amplified with primers as Madrasin site described elsewhere [11,20,28]. The H5R primer pair used in this study was as follows: H5R F: ATTATCGCGATATCCGTTAA and H5R R: AGAGTTTACCATCTTTAT. The chemistry and thermal conditions of these PCR reactions were similar to those used for the vgf nested-PCR; however, the annealing temperatures of the specific primers were changed. The PCR fragments obtained from this study were sequenced in both orientations and in triplicates (Mega-BACE 1000 sequencer) (GE Healthcare, Buckinghamshire, UK). The sequences were aligned with previously published OPV sequences from GenBank using the ClustalW method, and the alignments were checked manually with the MEGA version 4.0 software (Arizona State University, Phoenix, AZ, USA). Accession numbers for the analyzed sequences can be found in their respective figures. MaximumFigure 4. Phylogenetic trees generated from OPV nucleotide sequences, including 18325633 the Brazilian VACV strains. Phylogenetic analyses of B5R (A) and A56R (B) sequences showed that the Brazilian VACV strains are split into two different branches (Groups 1 and 2), which do not cluster directly with the vaccine strains (vaccine strains: those with no asterisk; and VACV-WR, a laboratorial strain). The DMTV-2005 isolate is depicted as a black dot. The Brazilian VACV strains are depicted with asterisks. Maximum likehood trees were reconstructed using different datasets containing sequences from the genes listed above, using MEGA 4.0. Using ModelTest, server [35] the nucleotide substitution model of Tamura 1992 was selected as the best one fitting the data. Rates of variation among sites were estimated for each Microcystin-LR manufacturer dataset and two discrete Gamma categories were used to model evolutionary rate differences among sites and the reliability of branching patterns was tested through 1000 bootstrap sampling. doi:10.1371/journal.pone.0050413.gC23L Gene as a Brazilian Vaccinia virus MarkerC23L Gene as a Brazilian Vaccinia virus MarkerFigure 5. Nucleotide sequence alignment of C23L coding DNA sequences. Sequences were retrieved from GenBank and aligned using ClustalW method as implemented in the MEGA 4.0 program. The DMTV-2005 isolate showed a 10-nt deletion that leads to an early stop-codon and most likely results in non-functional protein production. The same deletion was observed in the Brazilian VACV Group 1 strains, including DMTV-2005. Nevertheless, the Brazilian Group 2 strains had no deletion in C23L. The deletion site is highlighted with a red box. doi:10.1371/journal.pone.0050413.glikehood trees were reconstructed using different datasets containing sequences from the genes listed above, using MEGA 4.0. Using ModelTest, server [29] the nucleotide substitution model of Tamura 1992 was selected as the best one fitting the data. Rates of variation among sites were estimated for each dataset and two discrete Gamma categories were used to model evolutionary rate differences among sites and the reliability of branching patterns was tested through 1000 bootstrap sampling.Results and DiscussionTypical pox-like CPEs were observed in BSC-40 cells that were inoculated with supernatants from the exanthematic clinical samples, such as cell-cell dissociation and migration due to alterations in microtubules, actin and intermediate filaments that culminated in plaque formation in the cell monolayer. Both samples that were collected from infected dairy.Hed novel genetic markers of Brazilian strains groups (C23L). A56R, B5R, C23L and C11R were amplified with primers as described elsewhere [11,20,28]. The H5R primer pair used in this study was as follows: H5R F: ATTATCGCGATATCCGTTAA and H5R R: AGAGTTTACCATCTTTAT. The chemistry and thermal conditions of these PCR reactions were similar to those used for the vgf nested-PCR; however, the annealing temperatures of the specific primers were changed. The PCR fragments obtained from this study were sequenced in both orientations and in triplicates (Mega-BACE 1000 sequencer) (GE Healthcare, Buckinghamshire, UK). The sequences were aligned with previously published OPV sequences from GenBank using the ClustalW method, and the alignments were checked manually with the MEGA version 4.0 software (Arizona State University, Phoenix, AZ, USA). Accession numbers for the analyzed sequences can be found in their respective figures. MaximumFigure 4. Phylogenetic trees generated from OPV nucleotide sequences, including 18325633 the Brazilian VACV strains. Phylogenetic analyses of B5R (A) and A56R (B) sequences showed that the Brazilian VACV strains are split into two different branches (Groups 1 and 2), which do not cluster directly with the vaccine strains (vaccine strains: those with no asterisk; and VACV-WR, a laboratorial strain). The DMTV-2005 isolate is depicted as a black dot. The Brazilian VACV strains are depicted with asterisks. Maximum likehood trees were reconstructed using different datasets containing sequences from the genes listed above, using MEGA 4.0. Using ModelTest, server [35] the nucleotide substitution model of Tamura 1992 was selected as the best one fitting the data. Rates of variation among sites were estimated for each dataset and two discrete Gamma categories were used to model evolutionary rate differences among sites and the reliability of branching patterns was tested through 1000 bootstrap sampling. doi:10.1371/journal.pone.0050413.gC23L Gene as a Brazilian Vaccinia virus MarkerC23L Gene as a Brazilian Vaccinia virus MarkerFigure 5. Nucleotide sequence alignment of C23L coding DNA sequences. Sequences were retrieved from GenBank and aligned using ClustalW method as implemented in the MEGA 4.0 program. The DMTV-2005 isolate showed a 10-nt deletion that leads to an early stop-codon and most likely results in non-functional protein production. The same deletion was observed in the Brazilian VACV Group 1 strains, including DMTV-2005. Nevertheless, the Brazilian Group 2 strains had no deletion in C23L. The deletion site is highlighted with a red box. doi:10.1371/journal.pone.0050413.glikehood trees were reconstructed using different datasets containing sequences from the genes listed above, using MEGA 4.0. Using ModelTest, server [29] the nucleotide substitution model of Tamura 1992 was selected as the best one fitting the data. Rates of variation among sites were estimated for each dataset and two discrete Gamma categories were used to model evolutionary rate differences among sites and the reliability of branching patterns was tested through 1000 bootstrap sampling.Results and DiscussionTypical pox-like CPEs were observed in BSC-40 cells that were inoculated with supernatants from the exanthematic clinical samples, such as cell-cell dissociation and migration due to alterations in microtubules, actin and intermediate filaments that culminated in plaque formation in the cell monolayer. Both samples that were collected from infected dairy.
