using an unpaired t test. P,0.05 was considered statistically significant.Results Infection with MCMV Smith strain successfully mounts anti-MCMV IgG seroconversionRecipient mice were either infected with MCMV Smith strain or mock treated as described in Materials and Methods. MCMV infection was well tolerated and no MCMV-related death occurred during the observation period of 25 weeks. MCMV treated animals showed no difference in weights and clinical scores when compared to mock infected (Fig. 1A). Prior to subsequent transplant, animals were analyzed for MCMV seroconversion by ELISA in order to ensure successful MCMV infection. As shown in figure 1B, anti MCMV IgG antibodies were detected in all mice treated with the virus, and as expected, none of the mock treated animals was tested IgG positive. None of the animals was clinically sick at this time point and accordingly considered to be latently infected.Chimerism analysis after allogeneic HCT using D2Mit265 gene polymorphismTo exclude differences in engraftment of allogeneic recipients accounting for the observed differences between groups, we next tested for splenic donor chimerism in survivors at day +100, by analyzing for D2Mit265 as described in Materials and Methods. The amplified D2Mit265 gene product in BALB/c mice is 139 bp of size, where as it is 103 bp in B10.D2 animals. As depicted in figure 2C, mixing studies of BALB/c and B10.D2 DNA show absence of BALB/c 139 bp product size at a ratio of 20 (BALB/c): 80 (B10.D2), and absence of B10.D2 product size at a ratio of 100:0, respectively. As demonstrated in figure 2D , syngeneic recipients showed as expected a product at 139 bp only. BALB/cFigure 1. Weight change after MCMV infection and MCMV serology testing. MCMV infection was done by intraperitoneal injection of 36104 PFU purified Smith strain in naive BALB/c mice and another set of mice were mock infected as control. (A) Weight change was monitored following infection for 25 weeks; n per group = 28 (MCMV) and 24 (mock); Data are presented as mean. (B) 25 weeks following infection, animals were analyzed for anti-MCMV IgG inhibitor seropositivity as indicator for MCMV infection. Data shown present the index value with 1 determined as positive, data points for individual mice are shown. doi:10.1371/journal.pone.0061841.gCMV and GVHDFigure 2. Survival, clinical GVHD and engraftment following HCT. (A+B) Animals were transplanted as described in Materials and Methods, and survival and clinical GVHD scores were monitored for 100 days (n = 6 for syngeneic control group; n = 9 for the MCMV treated syngeneic group, n = 18 for allogeneic control group and n = 19 for the MCMV treated allogeneic group). Data are combined from two identical experiments. (*p,0.005,**p,0.001). (C ) Detection of gene D2Mit265 PCR products for BALB/c (139 bp) and B10.D2 (103 bp) was used to determine donor cell chimerism in the spleen. doi:10.1371/journal.pone.0061841.grecipients receiving B10.D2 donor cells demonstrated at least 80 donor chimerism, consistent with successful donor cell engraftment.of the MCMV allogeneic group (figure 3). On the basis of.Ed, mortality was still low (8.3 ). In 1516647 contrast, allogeneic MCMV infected animals showed increased mortality (26.3 ) along with significantly increased clinical GVHD scores (figure 2A ).Statistical analysisAll values are expressed as the mean 6 SEM. Survival curves were plotted and compared by log-rank analysis. Statistical comparisons between groups were completed using an unpaired t test. P,0.05 was considered statistically significant.Results Infection with MCMV Smith strain successfully mounts anti-MCMV IgG seroconversionRecipient mice were either infected with MCMV Smith strain or mock treated as described in Materials and Methods. MCMV infection was well tolerated and no MCMV-related death occurred during the observation period of 25 weeks. MCMV treated animals showed no difference in weights and clinical scores when compared to mock infected (Fig. 1A). Prior to 
subsequent transplant, animals were analyzed for MCMV seroconversion by ELISA in order to ensure successful MCMV infection. As shown in figure 1B, anti MCMV IgG antibodies were detected in all mice treated with the virus, and as expected, none of the mock treated animals was tested IgG positive. None of the animals was clinically sick at this time point and accordingly considered to be latently infected.Chimerism analysis after allogeneic HCT using D2Mit265 gene polymorphismTo exclude differences in engraftment of allogeneic recipients accounting for the observed differences between groups, we next tested for splenic donor chimerism in survivors at day +100, by analyzing for D2Mit265 as described in Materials and Methods. The amplified D2Mit265 gene product in BALB/c mice is 139 bp of size, where as it is 103 bp in B10.D2 animals. As depicted in figure 2C, mixing studies of BALB/c and B10.D2 DNA show absence of BALB/c 139 bp product size at a ratio of 20 (BALB/c): 80 (B10.D2), and absence of B10.D2 product size at a ratio of 100:0, respectively. As demonstrated in figure 2D , syngeneic recipients showed as expected a product at 139 bp only. BALB/cFigure 1. Weight change after MCMV infection and MCMV serology testing. MCMV infection was done by intraperitoneal injection of 36104 PFU purified Smith strain in naive BALB/c mice and another set of mice were mock infected as control. (A) Weight change was monitored following infection for 25 weeks; n per group = 28 (MCMV) and 24 (mock); Data are presented as mean. (B) 25 weeks following infection, animals were analyzed for anti-MCMV IgG seropositivity as indicator for MCMV infection. Data shown present the index value with 1 determined as positive, data points for individual mice are shown. doi:10.1371/journal.pone.0061841.gCMV and GVHDFigure 2. Survival, clinical GVHD and engraftment following HCT. (A+B) Animals were transplanted as described in Materials and Methods, and survival and clinical GVHD scores were monitored for 100 days (n = 6 for syngeneic control group; n = 9 for the MCMV treated syngeneic group, n = 18 for allogeneic control group and n = 19 for the MCMV treated allogeneic group). Data are combined from two identical experiments. (*p,0.005,**p,0.001). (C ) Detection of gene D2Mit265 PCR products for BALB/c (139 bp) and B10.D2 (103 bp) was used to determine donor cell chimerism in the spleen. doi:10.1371/journal.pone.0061841.grecipients receiving B10.D2 donor cells demonstrated at least 80 donor chimerism, consistent with successful donor cell engraftment.of the MCMV allogeneic group (figure 3). On the basis of.