Studies with 6 ts mutants of mouse cytomegalovirus indicated that mutants tsm1, tsm2, tsm3, and tsm6, like wild-type (wt) virus, produced acute infection in mice, became latent, and were reactivated as infectious virus immunosuppression. Using PCR, all five viruses expressed immediate-early (IE)-1, early (E)-1, and late (L, gB) genes during acute infection in all tissues examined (salivary glands, lung, spleen, liver, kidney, and heart). DNA was present in most tissues during latent infection with all five viruses, but transcription was restricted to the IE-1 gene in the salivary glands of wt infected mice only, suggesting true molecular latency rather than low level virus persistence. Similarly, mutant tsm5 expressed all three genes following primary inoculation. Although no detectable virus was produced, tsm5 subsequently entered the latent state as evidenced by DNA detection without RNA transcription indicating that productive infection is not required to initiate latency. This mutant also failed to reactivate from latency, although all three marker genes were expressed in most tissues. In contrast, tsm4 expressed all three marker genes and produced infectious virus during acute infection, then became latent. However, upon immunosuppression to reactivate tsm4, IE-1 and E-1 transcription occurred but neither gB transcription nor infectious virus was detectable in salivary glands, lung, spleen, liver, kidney, heart, or blood. The significance of this with regard to reactivation from latency is discussed.

SUMMARYTo study the pathogenicity of murine cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterized a set of temperature-sensitive mutants. One mutant, PP269/38, was avirulent for 1-week-old BALB/c mice and restricted in its plaque formation and replication at 39 °C. Mutants PP242/68 and PP268/38 were 100-fold less virulent than salivary gland-grown virus (SGV), even after two passages in the salivary glands of l-week-old mice. The former mutant was unable to replicate or form plaques at 39 °C whereas the latter replicated poorly at 39 °C but not at all at 40 °C although it was able to form plaques at 40 °C with a reduced plaque size. PP31/15 exhibited a 40-fold reduction in virulence compared to SGV after two passages in vivo and was unable to form plaques or to replicate at 40 °C; at 39 °C it was able to, with reduced efficiency. The remaining two mutants, PP99/3 and PP392/31, were 10-fold less virulent than SGV and were restricted at 40 °C. The six mutants have been classified into at least four complementation groups. These mutants may be useful for studying various aspects of MCMV pathogenicity.

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