M2 Phosphorylation and Latency Figure 5. Activation of Vav proteins is necessary to the establishment of normal levels of latency in spleen. Balb/c mice were intranasally infected with viruses of the indicated viral genotypes and latent load quantified by ex-vivo reactivation. Each point represents the infectious center titre from an individual mouse. Horizontal bars indicate arithmetic means. The dashed line indicates the limit of detection of the assay. doi:10.1371/journal.pone.0001654.g005 the presence of large clusters of latently infected cells. This pattern reflects the cessation of the virus driven GC B cell proliferation (Fig. 6A, panels f,j; and Fig. 5B). At later periods (day 50), infection became confined to a reduced number of cells scattered within secondary follicles, a phenotype that correlates with the mainte- nance phase of latent infection (Fig. 6A, panels k,o). The kinetics and pattern of infection in the spleens of mice inoculated with vM2FS was as previously reported [17]. Thus, after infection with this virus, maximal numbers of large clusters of infected GC B cells were reached only at day 21 post-infection (Fig. 6A, panel g). High levels of latent infection were still observed at day 50 post-infection (Fig. 6A, panel l), where almost 100% of the miRNA-positive follicles presented with large clusters of infected cells (Fig. 6B). In the case of infections by vM2Y and vM2P2, there was a reduced number of miRNA positive follicles at day 14 post-infection (Fig. 6A, panels c,d). This deficit reflected both a decreased number of infected follicles as well as a low number of positive follicles with large clusters of latently infected cells when compared Table 1. Frequency of genome-positive GC B cellsa Reciprocal frequencyb of viral DNA+ GC B cells (95% CI) %Cellsc Total no. of cellsd No. of viral DNA- positive cellse 3 (2–6) 61 (39–142) 23 (15–53) 26 (17–60) 6 (4–13) 79 (51–175) 6 (4–12) 23 (14–58) 10 (7–21) 51 (32–119) 148 (92–380) 8 (6–17) 128 (77–381) 256 (169–527) 78 (52–162) 5.7 5.7 7.8 8.9 6.8 6.0 6.0 4.3 4.1 4.9 2.1 4.9 1.5 2.6 1.7 1.16107 1.16107 1.66107 1.86107 1.46107 1.26107 1.26107 8.66106 8.26106 9.86106 4.26106 9.86106 3.06106 5.26106 3.46106 3,666,667 180,328 608,696 692,308 2,333,333 151,899 2,000,000 373,914 820,000 192,157 28,378 1,225,000 23,437 20,233 43,897 Days p.i. 14 21 50 Virus WT M2FS M2Y M2P2 M2Y-R WT M2FS M2Y M2P2 M2Y-R WT M2FS M2Y M2P2 M2Y-R aData were obtained from pools of at least five spleens. bFrequencies were calculated by limiting-dilution analysis with 95% confidence intervals (CI). cThe percentage of GC B cells from total spleen was estimated by FACS analysis. dThe total number of cells was estimated from the percentage of the total spleen, based on an estimate of 26108 cells/spleen. eThe number of latently infected cells was based on the frequency of latency within GB B cells and the estimated total number of cells. doi:10.1371/journal.pone.0001654.t001 PLoS ONE | www.plosone.org 7 February 2008 | Volume 3 | Issue 2 | e1654