In vivo activation of toll-like receptor-9 induces an age-dependent abortive lytic cycle reactivation of murine gammaherpesvirus-68.

Infection of mice with murine gammaherpesvirus-68 (γHV-68) serves as a model to understand the pathogenesis of persistent viral infections, including the potential for co-infections to modulate viral latency. We have previously found that infection of neonates (8-day-old mice) with γHV-68 resulted in a high level of persistence of the virus in the lungs as well as the spleen, in contrast to infection of adult mice, for which long-term latency was only readily detected in the spleen. In this study we investigated whether stimulation of toll-like receptor (TLR)9 would modulate viral latency in mice infected with γHV-68 in an age-dependent manner. Pups and adult mice were injected with the synthetic TLR9 ligand CpG ODN at 30 dpi, at which time long-term latency has been established. Three days after CpG injection, the lungs and spleens were removed, and a limiting dilution assay was done to determine the frequency of latently infected cells. RNA was extracted to measure viral transcripts using a ribonuclease protection assay. We observed that CpG injection resulted in an increase in the frequency of latently-infected cells in both the lungs and spleens of infected pups, but only in the spleens of infected adult mice. No preformed virus was detected, suggesting that TLR9 stimulation did not trigger complete viral reactivation. When we examined viral gene expression in these same tissues, we observed expression only of the immediate early lytic genes, rta and K3, but not the early DNA polymerase gene or late gB transcript indicative of an abortive reactivation in the spleen. Additionally, mice infected as pups had greater numbers of germinal center B cells in the spleen following CpG injection, whereas CpG stimulated the expansion of follicular zone B cells in adult mice. These data suggest that stimulation of TLR9 differentially modulates gammaherpesvirus latency via an age-dependent mechanism.