Quantitation of the spliced late gene of human cytomegalovirus and its kinetics during experimental infection

Abstract.

Human cytomegalovirus (hCMV) infection is still a cause of morbidity and mortality after solid organ and bone marrow transplantation and in other immunocompromised states. 'Preemptive therapy' strategies necessitate sensitive and specific methods for rapid diagnosis of symptomatic hCMV infection and monitoring of antiviral treatment. For analysis of the lytic stage of viral replication, the molecular determination of late transcripts is a useful approach for diagnosis of hCMV disease. In the present study we established an absolute quantitation of hCMV spliced late gene (SLG) RNA transcripts by real-time reverse transcription-polymerase chain reaction. Intron spanning primers were used for amplification to discriminate between viral DNA and cDNA. For standardization of the varying amounts of cDNA analyzed, cytoplasmic β₂-microglobulin (β₂-MG) cDNA was quantitated in parallel. cDNA copy numbers of both target sequences could be quantitated in a wide linear range from 10 to 10⁷ copies. To investigate the applicability of the developed assay, diploid lung fibroblasts were infected with the virus strain AD169. SLG expression was measured during a 48-h period after inoculation. After an only low expression during the first 10 h (approximately 5×10² copies/sample or SLG/β₂-MG ratio <0.001), SLG transcription increased dramatically after 24 h, peaking at 5×10⁴ copies/sample or SLG/β₂-MG ratio of 0.035 after 48 h. Intra- and interassay variability was less than 5% for calibrators and less than 10% samples. We conclude that quantitation of SLG transcripts by the presented method might be a powerful tool for differentiating between hCMV latency and active replication in vitro and in vivo, and thus may be a promising tool for diagnosis of symptomatic hCMV infection and monitoring of antiviral treatment.