Loss of M2 expression <i>in vivo</i> correlates with reduced serum IL-10 at the onset of latency.

<p>The reduction in serum IL-10 is independent of the route of infection and correlates with a significant reduction in reactivation from latency in the absence of M2. Groups of four to five mice were infected with 1000 pfu of M2 null mutant (MHV68/M2.Stop) or the M2 marker rescue virus (MHV68/M2.MR) via intranasal inoculation or 100 pfu via intraperitoneal inoculation (or naïve mice). On days 14 or 15 post-infection, mice were injected with biotin labeled anti-IL-10 antibody i.p. Twenty-four hours later, serum was collected for analysis of IL-10 levels (A and B), and splenocytes were recovered for determinations of the frequency of viral latency and reactivation (C and D). Data shown for intranasal inoculations is representative of two independent experiments, 4–5 mice per group, with IL-10 levels measured in individual mice and splenocytes pooled for viral latency assays (as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000039#s4" target="_blank">Materials and Methods</a>). Data shown for intraperitoneal inoculations is representative of three independent experiments, 4–5 mice per group, with IL-10 levels measured in individual mice and splenocytes pooled for viral latency assays. (A and B) Total serum IL-10 levels were significantly lower in mice infected with MHV68/M2.Stop compared to MHV68/M2.MR virus, independent of the route of infection. Intranasal infection data is from 10 individual mice from two independent infections; intraperitoneal data is from 15 individual mice from three independent infections. Two naïve animals were analyzed per infection * p = 0.0117, ** p = 0.0005. Significance of IL-10 data was determined by two-tailed, unpaired Student's T test with a confidence level of 95%. (C and D) Frequencies of splenocytes from MHV68/M2.MR and MHV68/M2.Stop infected mice harboring latent viral genomes and reactivating from latency upon explant. The frequency of latency was determined by nested, limiting-dilution PCR (LD-PCR) as previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000039#ppat.1000039-Weck1" target="_blank">[3]</a>. The frequency of cells reactivating from latency upon in vitro culture was determined by plating serial dilutions of live, intact splenocytes on mouse embryonic fibroblast monolayers and scoring cytopathic effect 14–21 days post-explant, as previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000039#ppat.1000039-Weck1" target="_blank">[3]</a>. Data from both assays was subjected to nonlinear regression analysis with a sigmoidal dose-response algorithm for best fit. Data points represent individual experiments with splenocytes pooled from 4–5 mice per condition.