Quantitative measurement of infectious murine cytomegalovirus genomes in real-time PCR.
Acute virus replication in murine (M) CMV infected C57BL/6 (Cmv1(r)) mice is severely limited by Ly49H+ NK cells, but not in MCMV infected BALB/c or BXD8 (Cmv1(s)) mice that lack Ly49H+ NK cells. Interestingly, other NK cell receptors may also play a role in MCMV immunity, as CMV encoded gp40 protein can diminish expression of protein ligands recognized by the NK cell receptor NKG2D. To determine whether other additional gene products might influence MCMV immunity, we designed an efficient, sensitive and reliable method for screening resistance or susceptibility phenotypes in mice. Although multiple methods are frequently used to detect and quantify infectious MCMV in mouse tissue samples collected during acute viral infection, these are not readily adaptable to high-throughput screening strategies. Hence, we utilized real-time PCR for detection and quantitative measurement of infectious MCMV genomes present in various tissues of infected mice. MCMV genomic sequence was accurately and reproducibly detected over the range 10(2)-10(8) molecules in mouse genomic DNA samples using this methodology. Importantly, it was found that quantitative real-time PCR and viral plaque assay measurements of MCMV in tissues collected from infected mice, including resistant and susceptible strains, were directly correlated. Moreover, quantitative real-time PCR results obtained during a 3-week time-course study of virus replication in spleens, livers and salivary glands of infected mice demonstrated sensitive, accurate and reproducible detection and measurement of infectious MCMV.
Wheat
Roy L
RL
Department of Internal Medicine, Division of Rheumatology and Immunology, University of Virginia School of Medicine, Box 800412, Charlottesville, VA 22908, USA. mgb4n@virginia.edu
Clark
Patricia Y
PY
Brown
Michael G
MG
D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000821:virology; D004279:DNA, Viral / Q000032:analysis*; D016679:Genome, Viral; D008099:Liver; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D016133:Polymerase Chain Reaction / Q000379:methods*; D012469:Salivary Glands / Q000821:virology; D012680:Sensitivity and Specificity; D013154:Spleen
D004279:DNA, Viral