Quantitative competitive NASBA for measuring mRNA expression levels of theimmediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus

Different cell types were infected with human cytomegalovirus (HCMV) and RN A expression dynamics were analyzed by quantitative NASBA assays for IE1 (U L123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The qu antitative NASBA assays gave reproducible quantification in the range of 10 (3)- 10(6) RNA copies for IE1 and pp67 RNA, from 3 x 10(3) to 1 x 10(6) RNA copies for US6 and US11 RNA, and from 1 x 10(4) to 1 x 10(6) RNA copies fo r US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells , propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within th e first 4 h post-infection. The first gene for which expression could be de tected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected w ith HCMV strain AD169 showed a different RNA expression pattern for US3. Tr anslation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence. (C) 2001 Elsevier Science B.V. All rights reserved.