Improvement of a quantitative cell tropism assay for rapid and reliable characterization of human cytomegalovirus mutants.
Endothelial cell tropism is an inherent property of isolates of human cytomegalovirus (HCMV), and has been proposed as a pathogenicity factor of HCMV. Mutational approaches can be applied for analysis of the molecular determinants of endothelial cell tropism, but the evaluation of mutants is limited by the low reliability of the widely employed cell tropism assay. The aim of this study was to improve the assay conditions in order to allow for accurate discrimination of phenotypic differences between HCMV mutants. Target cell density and input virus titres were identified to account for most of the variation in the apparent endothelial cell tropism of a given cytomegalovirus strain. Coating of culture dishes, cell attachment at ambient temperature, and standardization of virus input titres together with automated counting of immunofluorescence signals enabled the discrimination of differences of as little as 25% in endothelial cell tropism of HCMV strains. This improvement will facilitate rapid and reliable quantitation of cell tropism of HCMV, and is particularly suitable for the analysis of large numbers of mutants with only minor changes in tropism.
Schuessler
Andrea
A
Institute of Medical Virology and Epidemiology of Virus Diseases, University of Tuebingen, Elfriede-Aulhorn-Str. 6, D-72076 Tuebingen, Germany.
Sampaio
Kerstin Laib
KL
Sinzger
Christian
C
D001331:Automation / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology*; D042783:Endothelial Cells / Q000821:virology*; D006801:Humans; D009154:Mutation; D012680:Sensitivity and Specificity; D013696:Temperature; D019562:Viral Load; D056189:Viral Tropism; D014773:Virology / Q000379:methods*