Isolation and Preliminary Characterization of Temperature-sensitive Mutants of Mouse Cytomegalovirus of Differing Virulence for 1-week-old Mice

Abstract

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.

Mutations in the temperature‐sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: A study of genes involved in immune evasion, DNA packaging and processing, and DNA replication

“…93061524) was propagated in Growth Media (GM) comprising of Dulbecco's modified Eagle's medium (DMEM) (Sigma, Poole, UK) supplemented with 10% (v/v) new-born calf serum (Biowhittaker; Walkersville, Maryland), 2 mM L-glutamine (GibcoBRL, Paisley, UK), 200 U/ml penicillin (GibcoBRL), and 200 mg/ml streptomycin (GibcoBRL). Primary murine embryo fibroblast (MEF) cells were prepared and maintained as described previously [Sammons and Sweet, 1989] from outbred CD-1 or inbred BALB/c mice purchased from Charles River, UK. The origin of the K181 (Birmingham) strain of MCMV and the generation of the temperature-sensitive mutants tsm5 and tsm30 have been described previously as has their plaque assay in MEF cells [Sammons and Sweet, 1989;Bevan et al, 1996].…”

“…A panel of 31 ts mutants of the K181 (Birmingham) strain of murine cytomegalovirus (MCMV) were created by mutagenesis with N-methyl-N 0 -nitro-N-nitrosoguanidine and selected for a restricted growth phenotype at temperatures of 398C or above [Sammons and Sweet, 1989;. One such mutant, tsm5, did not replicate to detectable levels in any tissue of 1-week-old mice for up to 21 days following i.p.…”

Antiviral Therapy Can Reverse the Development of Immune Senescence in Elderly Mice with Latent Cytomegalovirus Infection

“…The mice were inoculated by intraperitoneal injection (i.p.) with 1 ϫ 10 6 PFU of MCMV strain Smith (19). The influenza A virus (IAV) A/Puerto Rico/8/34/England/939/69 clone 7a (H3N2) was cultured and titrated in fertilized hen's eggs (20).…”

Recent advances in cloning herpesviral genomes as infectious bacterial artificial chromosomes

“…Following the isolation of temperature-sensitive mutants of herpesvirus simplex virus-1 (HSV-1), 4 mutants of various herpesviruses were generated using the same or similar methods. [5][6][7][8][9][10][11][12][13][14][15] While this is a useful method for obtaining herpesviral mutants, the mutation frequency is usually low, and the isolation and purification steps are time-consuming and labor-intensive. Furthermore, the mutation is often scattered through the entire viral genome and mutants with multiple mutations are common, making it difficult to precisely map the mutation site(s) and elucidate the function of a viral gene.…”