In situ PCR for the detection of human cytomegalovirus in suspension cells during the latent phase of infection.
Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of latent viral DNA and transcripts in these cells was investigated using methods based on polymerase chain reaction (PCR)-driven in situ hybridization (ISH) and reverse transcription (RT)-PCR-driven ISH. Using a conventional thermal cycler, latent viral DNA or transcripts were amplified within suspension cells. Amplified products were then detected by nonisotopic ISH on cells cytospun on glass microscope slides. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in more than 90% of cells. During natural infection, viral genomes were detected in 0.004 to 0.01% of mono-nuclear cells from granulocyte colony-stimulating factor mobilized peripheral blood or bone marrow from healthy seropositive donors. When evaluated by RT-PCR-ISH, only a small proportion of experimentally infected cells (approx 2%) had detectable latent transcripts. The application of PCR-ISH and RT-PCR-ISH has enabled the identification of the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode latent transcripts.
Slobedman
Barry
B
Centre for Virus Research, Westmead Millennium Institute, Westmead, New South Wales, Australia.
D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D023461:Myeloid Progenitor Cells / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D017735:Virus Latency / Q000235:genetics*
D004279:DNA, Viral; D012367:RNA, Viral