Quantity of cytomegalovirus DNA in different leukocyte populations during active infection in vivo and the presence of gB and UL18 transcripts.
The quantity of human cytomegalovirus (HCMV) DNA in the blood of immunocompromised individuals correlates with the development of HCMV disease. We wished to determine which leukocytes harboured DNA and whether this represented active viral replication. Magnetic bead separation techniques were used to obtain pure polymorphonuclear leukocyte (PMNL), monocyte, B and T cell fractions, and RT-PCR and quantitative-competitive PCR (QC-PCR) to detect HCMV glycoprotein B (gB; UL55) transcripts and quantify HCMV DNA levels, respectively, in each cell fraction. QC-PCR revealed that PMNLs contribute the greatest to the overall viral load in blood (median viral load: PMNLs, 10(5.37) genomes/ml of blood; monocytes, 10(4.40) genomes/ml; B cells, 10(3.70) genomes/ml; and T cells, 10(4.08) genomes/ml). However, monocytes have a viral burden of 0.65 genomes/monocyte which is greater than that within the other leukocyte populations (0.11 genomes/PMNL, 0.23 genomes/B cell, and 0.20 genomes/T cell). Glycoprotein B transcripts were detected in all four cell populations: 3/10 PMNL fractions, 6/13 monocyte fractions, 5/13 B cell fractions, and 4/13 T cell fractions. The data show that productive infection of these leukocyte subpopulations, including PMNLs, can occur in vivo. Furthermore, transcripts of gpUL18, the putative natural killer (NK) cell decoy, were detected in 2/6 monocyte fractions with active replication, and 1/4 T cell fractions but not in the other leukocyte fractions. The transient nature of UL18 gene expression, and the low abundance of the transcript relative to gB were confirmed.
Hassan-Walker
A F
AF
Department of Virology, Royal Free and University College Medical School, University College London, Royal Free Campus, Hampstead, London, United Kingdom. aycan@rfhsm.ac.uk
Mattes
F M
FM
Griffiths
P D
PD
Emery
V C
VC
D001402:B-Lymphocytes / Q000276:immunology / Q000821:virology; D002452:Cell Count; D002469:Cell Separation; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology; D003586:Cytomegalovirus Infections / Q000097:blood / Q000235:genetics / Q000821:virology; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics; D005434:Flow Cytometry; D005814:Genes, Viral; D006801:Humans; D016867:Immunocompromised Host; D007159:Immunologic Tests; D007694:Killer Cells, Natural / Q000276:immunology; D007962:Leukocytes / Q000821:virology*; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000821:virology; D009504:Neutrophils / Q000821:virology; D016377:Organ Transplantation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology / Q000821:virology; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000097:blood / Q000235:genetics; D019562:Viral Load; D014766:Viremia / Q000821:virology; D014779:Virus Replication
D004279:DNA, Viral; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus