Characterization of a mutant strain of murine cytomegalovirus which fails to grow in salivary glands

An understanding of the mechanisms of pathogenesis of cytomegaloviruses is essential for future work towards control and prevention of cytomegalovirus disease. Towards this end, a comparison of the Smith, Vancouver and K-181 strains of murine cytomegalovirus (MCMV), which differ in virulence, revealed differences in growth characteristics in vivo and in vitro. The Vancouver strain of MCMV grows to a limited extent in the spleen while it fails to grow in the salivary glands of inoculated mice (salivary gland growth mutant, Sgg⁻). This mutation probably arose during multiple in vitro passage of the parental Smith strain. In vitro, the Vancouver strain replicated more quickly and produced a greater yield of virus per cycle which resulted in a larger plaque size relative to the Smith or K-181 strains. The Vancouver strain has a 9.4 kb deletion, which spans the XbaI I/L junction of the parental Smith strain (0.960 to 0.995 map units), and a 0.9 kb insertion which maps to the EcoRI K fragment (0.37 to 0.47 map units). Recombinant MCMV was generated by co-transfection of cells with whole Vancouver strain DNA and cloned fragments of wild-type DNA. Three independent Sgg⁺ recombinant viruses were selected by passage of the co-transfected virus through mouse salivary glands. The three recombinants had recovered the DNA deletion, while they maintained the insertion associated with the Vancouver strain. Thus, the genotypic location of the defect responsible for the Sgg⁻ phenotype of the Vancouver strain of MCMV maps to 0.960 to 0.995 map units of the Smith virus genome. In addition, the restoration of this region of the genome correlated with the detection in the recombinants of a 42 kDa protein present during E and L times post infection in Smith- and K-181-infected cells, but not in Vancouver strain-infected cells. The K-181 strain was associated with higher salivary gland titres and a low LD₅₀ titre consistent with the increased virulence of this strain compared with the Smith strain. Differences in the K-181 restriction endonuclease pattern relative to the Smith strain were mapped, and were mainly confined to 0.0 to 0.06 map units of the genome without any large insertions or deletions detected. Minor differences in the proteins of the Smith and K-181 strains at early and late times post infection were also detected. The identification of a gene involved in the salivary gland tropism of MCMV and the characterization of differences between the Smith and K-181 strains of MCMV should increase our understanding of the virulence mechanisms of cytomegaloviruses.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat