Dentified as molecular markers to differentiate VACV from other OPV members or to discriminate between the Brazilian VACV groups. The viral growth factor gene (vgf) has been a marker that is screened by PCR because it is conserved among OPV members and duplicated in most OPV genomes [20]. However, vgf sequencing only allows for VACV species identification but not for VACV strain differentiation. Other conserved genes had been sequenced from BrazilianFigure 1. Map of the outbreak region and lesions found on cattle. (A) Overview map of Title Loaded From File Brazil (left) and the Minas Gerais State (right). The red circle highlights Resplendor County where the VACV DMTV-2005 strain was isolated. The expanded map shows the average milk production (liters) in each county. Resplendor is an important milk production region in the Minas Gerais State (governmental source/2006). (B) Lesions caused by DMTV infection. VACV-like lesions on the teats and udder of an infected dairy cow (left) and of the calf’s oral mucosa (right) are as 18325633 shown. doi:10.1371/journal.pone.0050413.gC23L Gene as a Brazilian Vaccinia virus MarkerVACV because vgf is not remarkably Title Loaded From File different between VACV groups 1 and 2 [20]. For the past few years, A56R has been the most widely used gene marker in genetic analyses for Brazilian VACV strain differentiation. Damaso et al. (2000) demonstrated that VACV Cantagalo strain (Group 1) has an 18-nucleotide (nt) deletion in A56R. Most of the new VACV isolates were analyzed for this gene. With the discovery of new VACV isolates, A56R proved 24272870 to be even more interesting because some Group 2 isolates do not have this particular 18-nt deletion. Other molecular markers have been discovered in the past few years, such as the serine protease inhibitor-3 gene (K2L) and the A-type inclusion body gene (ATI), among others [11,21,22]. These approaches are important, not only for sample identification but also to infer ancestry and to investigate hypothetical correlation of each sample or group with its unique epidemiological and biological features. In this study, we described the isolation of a VACV that has been characterized by sequencing, along with five genes analyses thatincluded traditional and new, conserved and variable genetic markers. Analysis of C23L, the gene that encodes the CCchemokine-binding protein, revealed not only a new Brazilian VACV genetic marker but also a possible premature stop-codon mutation shared by Group 1 VACV strains.Materials and Methods Ethic Statement?This study was approved by the Comite de Etica em ^ Experimentacao Animal – CETEA. In Brazil, it is not necessary to have specific ethics approval for collection of clinical samples from domestic cattle; all collection procedures were performed by veterinarians of a Brazilian Surveillance Institute (Instituto Mineiro de Agropecuaria – IMA), following institutional recom?mendations (http://www.ima.mg.gov.br). Mice were sacrificed with anesthesia (ketamine + xilasin), followed by cervical dislocation.Figure 2. Infectivity assays of Brazilian VACV strains. (A) Body weight loss and (B) survival rates of BALB/c mice that were infected with VACVBR. Groups of mice (n = 4) were inoculated intranasally with 106 PFU of VACV. Weight and clinical signs of the mice were monitored daily (until day 14 p.i.), and animals that lost more than 25 of their initial weight were euthanized. The control group was inoculated with PBS. The error bars represent standard deviations. Both the weight loss and surviv.Dentified as molecular markers to differentiate VACV from other OPV members or to discriminate between the Brazilian VACV groups. The viral growth factor gene (vgf) has been a marker that is screened by PCR because it is conserved among OPV members and duplicated in most OPV genomes [20]. However, vgf sequencing only allows for VACV species identification but not for VACV strain differentiation. Other conserved genes had been sequenced from BrazilianFigure 1. Map of the outbreak region and lesions found on cattle. (A) Overview map of Brazil (left) and the Minas Gerais State (right). The red circle highlights Resplendor County where the VACV DMTV-2005 strain was isolated. The expanded map shows the average milk production (liters) in each county. Resplendor is an important milk production region in the Minas Gerais State (governmental source/2006). (B) Lesions caused by DMTV infection. VACV-like lesions on the teats and udder of an infected dairy cow (left) and of the calf’s oral mucosa (right) are as 18325633 shown. doi:10.1371/journal.pone.0050413.gC23L Gene as a Brazilian Vaccinia virus MarkerVACV because vgf is not remarkably different between VACV groups 1 and 2 [20]. For the past few years, A56R has been the most widely used gene marker in genetic analyses for Brazilian VACV strain differentiation. Damaso et al. (2000) demonstrated that VACV Cantagalo strain (Group 1) has an 18-nucleotide (nt) deletion in A56R. Most of the new VACV isolates were analyzed for this gene. With the discovery of new VACV isolates, A56R proved 24272870 to be even more interesting because some Group 2 isolates do not have this particular 18-nt deletion. Other molecular markers have been discovered in the past few years, such as the serine protease inhibitor-3 gene (K2L) and the A-type inclusion body gene (ATI), among others [11,21,22]. These approaches are important, not only for sample identification but also to infer ancestry and to investigate hypothetical correlation of each sample or group with its unique epidemiological and biological features. In this study, we described the isolation of a VACV that has been characterized by sequencing, along with five genes analyses thatincluded traditional and new, conserved and variable genetic markers. Analysis of C23L, the gene that encodes the CCchemokine-binding protein, revealed not only a new Brazilian VACV genetic marker but also a possible premature stop-codon mutation shared by Group 1 VACV strains.Materials and Methods Ethic Statement?This study was approved by the Comite de Etica em ^ Experimentacao Animal – CETEA. In Brazil, it is not necessary to have specific ethics approval for collection of clinical samples from domestic cattle; all collection procedures were performed by veterinarians of a Brazilian Surveillance Institute (Instituto Mineiro de Agropecuaria – IMA), following institutional recom?mendations (http://www.ima.mg.gov.br). Mice were sacrificed with anesthesia (ketamine + xilasin), followed by cervical dislocation.Figure 2. Infectivity assays of Brazilian VACV strains. (A) Body weight loss and (B) survival rates of BALB/c mice that were infected with VACVBR. Groups of mice (n = 4) were inoculated intranasally with 106 PFU of VACV. Weight and clinical signs of the mice were monitored daily (until day 14 p.i.), and animals that lost more than 25 of their initial weight were euthanized. The control group was inoculated with PBS. The error bars represent standard deviations. Both the weight loss and surviv.