| Latent human cytomegalovirus (HCMV) is carried life-long. This thesis investigated: cell types harboring latent HCMV, evidence of viral transcription in cells carrying latent HCMV, and novel putative latency transcripts in productive infection.; Latent HCMV genome has been detected in peripheral blood (PB) CD33 +CD14+ cells. However, this population is heterogeneous. It consists of the major monocyte population (CD14bright) and small monocytes/dendritic cells (CD14dim). DNA, extracted from sorted cells, was amplified by polymerase chain reaction (PCR). Results demonstrated that the CD14dim population harbored the predominant cell type carrying HCMV DNA in naturally infected healthy subjects.; Novel sense cytomegalovirus latency-associated transcripts (CLTs) have been identified in latent HCMV infection. Using reverse transcription-PCR (RT-PCR) and 5′ rapid amplification of cDNA ends (RACE), we investigated whether PB CD33+CD14+ cells obtained from naturally infected hosts express the sense CLTs. Sense CLTs were not detected in any samples. Although CD14dim cells harbor latent HCMV DNA, sense CLTs are not expressed or are expressed at levels below the limits of detection for our assay.; Sense CLT expression was evaluated in HCMV productive infection. Using RT-PCR, 5′ RACE, and sequence analysis, transcripts consistent with sense CLTs were detected. RNase protection assay demonstrated that the transcripts were expressed over a wide range of times post-infection. Sense CLTs were detected in HCMV-infected fibroblasts, dTHP-1 monocytes, monocyte-derived macrophages, and endothelial cells as well as in clinical isolate-infected fibroblasts. Although the sense CLTs were not detected in latent infection, they were clearly expressed during productive infection.; Our findings indicate that PB CD14dim cells are enriched for latent HCMV genome in the naturally infected host. Novel CLTs, previously detected in an experimental system and in bone marrow, are undetectable in circulating PB CD14+ cells. However, these latency-associated transcripts were detected during productive infection. This might suggest that detection of CLTs was a reflection of low-level productive or abortive infection and not latent infection. Future studies are warranted to further define the PB CD14+ cell type that harbors HCMV. Following identification, cells may be enriched from naturally infected hosts and investigated for gene expression using techniques to screen expression from the entire viral genome. |
