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  • Brazilian Vaccinia virus strains used in this study are shown in Table 1. The Vaccinia virus, strain Western Reserve (VACV-WR), kindly provided by Dr. C. Jungwirth (Universität Würzburg, Germany), was used as a highly virulent reference strain in murine model by intranasal infection [23], [32]. Lister Butantan strain (LST-BUT), kindly provided by Instituto Butantan (São Paulo, Brazil) is a vaccine strain and it was used as a low virulence reference strain [27], [32]. All virus stocks were grown in Vero cells (ATCC CCL-81) cultivated in Eagle's minimum essential medium (MEM) supplemented with 5% of fetal calf serum (Cultilab, Brazil) and antibiotics, at 37°C in 5% CO2 atmosphere. Viruses were purified on sucrose gradients as described elsewhere [33]. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0003043.t001 Brazilian Vaccinia virus strains used in this study. *BAV, BeAn58058; SAV, SpAn232 virus; VBH, Belo Horizonte virus; ARAV, Araçatuba virus; GP1V Guarani P1 virus; GP2V: Guarani P2 virus; VACV-WR, Vaccinia virus Western Reserve. For all animal experimentation, four-weeks-old male BALB/c mice were used. Mice were housed in filter-top micro isolator cages and provided with commercial mouse feed and water ad libitum. All the animal experimentation was carried out in accordance with regulations and guidelines of Committee of Ethics for Animal Experimentation from Universidade Federal de Minas Gerais/Brazil. BALB/c mice were anesthetized by intraperitonial injection of ketamine and xilazine (3.2 mg and 0.16 mg/mice, in phosphate buffered saline (PBS) 0.9%, respectively) and inoculated by intranasal route. Mice belong to negative control group were inoculated with 10 µl of PBS [32]. Survival rate and lethal dose (LD50) in mice: For the survival rate analysis, mice were infected with 106 PFU (plaque forming units) of BR-VACV BeAn58058 (BAV), SpAn232 virus (SAV), Belo Horizonte virus (SAV), Araçatuba virus (SAV), Guarani P1 virus (GP1V), Guarani P2 virus (GP2V), Passatempo virus (PSTV) and with the control strains VACV-WR and LST-BUT. To establish the lethal dose of 50% of the animals (LD50) mice were infected with virus titers ranging from 103 to 108 PFU of BAV, SAV, VBH, GP1V and control strain in a total volume of 10 µl which was dropped by intranasal route. For both experiments, mice were daily observed during 20 days post inoculation (d.p.i.) or until their death. Mice that survived until day 20 p.i. were euthanized. Vaccinia virus tropism in mice: Groups of four, BALB/c mice were infected with 106 PFU of each purified virus strain in a total volume of 10 µl which was dropped by intranasal route. Mice were daily weighted and those ones that had lost more than 25% of their initial body weight were euthanized with an overdose of anesthetics (ketamine+xilazine). Those animals had their organs (trachea, lungs, heart, kidneys, liver, brain and spleen) asseptically removed after euthanasia. Those mice that survived were euthanized on day 12 p.i. and their organs and blood were also collected. In order to compare the replication of all VACV strains in lungs, (a main site of viral replication in this model), a virus multiplication kinetic was made at days 1, 3 and 5 post intranasal infection with 106 PFU of BAV, SAV, VBH, GP1V, ARAV GP2V, PSTV, and with the control strains VACV-WR and LST-BUT. The organs were macerated in MEM (Gibco, USA) and centrifuged at 2000×g for 3 min, 4°C. Supernatant fluids from macerated organs were collected and virus titer (PFU/g) was determined by plaque forming assay in Vero cell culture [34]. Blood samples were collected from BALB/c mice just before euthanasia. The serum was heated at 56°C for 30 min and used in virus neutralization assay as described previously [28]. The neutralization titer was expressed as the reciprocal of the highest dilution that exhibited 50% of plaque formation inhibition, when compared to cells infected with VACV WR (positive control). Fragments of lungs were fixed in 10% neutral-buffered formalin and routinely processed for paraffin embedding. Sections (5 µm) were cut on a standard rotary microtome and placed on glass slides for staining with hematoxylin and eosin (H&E). Other unstained sections were used for immunohistochemical analysis to localize viral antigen. Histological sections were examined by an experienced pathologist and classified in mild, moderate or severe according to lesion extent and severity. For immunohistochemistry analysis, deparaffinized lung sections were washed in PBS for 15 min at room temperature. Antigen retrieval was performed by incubating the slides in a specific solution (Target Retrieval Solution, S1700, Dako Corporation, USA) at 98°C for 20 min. Endogenous peroxidase activity was abolished by incubation with 3.5% H2O2 for 30 min, which was followed by incubation with 1∶20 normal goat serum (NGS) and 2% bovine serum albumin (BSA) for 30 min each at room temperature in humid chamber. Primary rabbit anti-H3L protein from VACV [35] was used at the dilution 1∶10.000, and the antibody incubation was performed overnight at 4°C in humid chamber. Secondary biotinylated antibody was goat anti-rabbit, followed by streptavidin-peroxidase complexes (LSAB2 system – HRP, DAKO, USA). The reaction was visualized by incubating the section with 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) and counter staining with hematoxylin. Tissues from non-infected animals were used as negative controls. Technical control was performed by the omission of the primary antibody. The sections were examined by Olympus BX51 microscope and digital images were acquired for documentation. For viral DNA detection, a semi-nested PCR for vaccinia growth factor (vgf) gene amplification was used (unpublished data). In the first reaction, 2 µl of clarified tissue samples were used as template without previous DNA extraction, added to 18 µl of the PCR reaction mixture. PCR products from the second round of amplification were fractionated by electrophoresis on 8% PAGE and silver stained. Significance variations were calculated using the Student's t-test (p≤0.05: statistical significance). Analysis of significant differences on weight curves and survival were done, respectively, by Wilcoxon paired test (p≤0.05: statistical significance) and the log-rank test (p≤0.05: statistical significance). Data were analyzed for individual mice. Statistical analysis was performed using GraphPad Prism Software, version 3.0. (GraphPad Software, Inc., San Diego, CA).
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