FIG. 8.

Effect of RV7 vaccination on reactivation of MCMV from spleen explants. BALB/c mice were vaccinated s.c. with 2 × 10⁵ PFU of RV7, or were mock vaccinated, on days 0 and 14 of the experiment and then challenged i.p. with the indicated doses of sgMCMV on day 28. On day 88 of the experiment, one-fourth of the spleen was sonicated and cocultured with MEFs to detect persistent infection. No cultures scored positive for persistent infection in these experiments, demonstrating that spleens were latently infected. (A) One half of the spleen was homogenized and explanted into two tissue culture wells. Every 3 to 4 days, samples of the supernatant from spleen explant wells were harvested and cultured with MEFs to detect infectious virus. Data were pooled from three experiments and are presented as the percentage of wells reactivating versus the days of explant culture. The number of wells per condition (two wells per mouse evaluated) are noted. (B) To identify the virus reactivating in the spleen explants shown in panel A, MCMV was grown from individual reactivated explant wells. Viral DNA was prepared, digested with HindIII and BglII, and analyzed by Southern blotting with ³²P-labeled HindIII J fragment of the MCMV genome. The patterns expected from wild-type MCMV and mutant RV7 are demonstrated by the controls on the left side; sizes in kilobases are indicated. No band at 6.0 kb (specific to RV7) was detected in MCMV from reactivating wells even on prolonged exposures. We show here data from eight isolates. An additional seven isolates were analyzed, with comparable results.