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. 2014 Apr;14(4):820-30.
doi: 10.1111/ajt.12642. Epub 2014 Mar 4.

Perivascular Stromal Cells as a Potential Reservoir of Human Cytomegalovirus

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Free PMC article

Perivascular Stromal Cells as a Potential Reservoir of Human Cytomegalovirus

M A Soland et al. Am J Transplant. .
Free PMC article

Abstract

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality among both solid organ and hematopoietic stem cell transplant recipients. Identification of cells throughout the body that can potentially serve as a viral reservoir is essential to dissect mechanisms of cell tropism and latency and to develop novel therapies. Here, we tested and compared the permissivity of liver-, brain-, lung (LNG)- and bone marrow (BM)-derived perivascular mesenchymal stromal cells (MSC) to HCMV infection and their ability to propagate and produce infectious virus. Perivascular MSC isolated from the different organs have in common the expression of CD146 and Stro-1. While all these cells were permissive to HCMV infection, the highest rate of HCMV infection was seen with LNG-MSC, as determined by viral copy number and production of viral particles by these cells. In addition, we showed that, although the supernatants from each of the HCMV-infected cultures contained infectious virus, the viral copy number and the quantity and timing of virus production varied among the various organ-specific MSC. Furthermore, using quantitative polymerase chain reaction, we were able to detect HCMV DNA in BM-MSC isolated from 7 out of 19 healthy, HCMV-seropositive adults, suggesting that BM-derived perivascular stromal cells may constitute an unrecognized natural HCMV reservoir.

Keywords: Cytomegalovirus; HCMV; hematopoietic niche; pericytes; transplantation.

Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Figures

Figure 1
Figure 1
(A) Perivascular stromal cells are permissive to in vitro infection with HCMV FIX-BAC and are able to propagate the virus. Human foreskin fibroblasts (HF); Stro-1-selected bone marrow (BM)-, liver (LVR)-, lung (LNG)- and brain (BRN)-mesenchymal stromal cells (MSC) were infected at MOI of 3 with HCMV FIX-BAC strain and analyzed for 10 days for infection and viral growth/spread through the expression of GFP using an Olympus IX-71 microscope with a 10× objective. (B) Supernatants from HCMV FIX-BAC-infected cells were used to serially infect, de novo, respective stromal layers. BAC, bacterial artificial chromosome; GFP, green fluorescence protein; HCMV, human cytomegalovirus.
Figure 2
Figure 2. HCMV Davis strain infects perivascular stromal cells
Cytopathic effects were observed in human fibroblasts (HF), Stro-1-selected bone marrow (BM)-, liver (LVR)-, lung (LNG) and brain (BRN)-mesenchymal stromal cells (MSC) after infection with HCMV Davis strain at MOI of 3. These representative images of the cytopathic effects were taken at 10 dpi with Olympus IX-71 microscope with a 10× objective. HCMV, human cytomegalovirus; MOI, multiplicity of infection.
Figure 3
Figure 3. Perivascular stromal cells produce and release infectious viral particles
In order to determine the number of infectious units (IU) produced by infected BM-, LNG-, LVR- and BRN-MSC, these cells were infected with HCMV Davis strain at MOI of 3, and supernatants collected at 3 h postinfection, 1, 3, and 5 dpi and used to infect HF. The number of IE-positive HF present at the different time points were calculated, and the values were log 10–transformed and plotted graphically, to facilitate comparison between the supernatants from the different MSC types. The results represent the mean ± SD from three independent experiments. BM, bone marrow; BRN, brain; dpi, day postinfection; HCMV, human cytomegalovirus; HF, human foreskin fibroblasts; IE, immediate early; LNG, lung; LVR, liver; MOI, multiplicity of infection; MSC, mesenchymal stromal cells.
Figure 4
Figure 4. Perivascular stromal cells are permissive to HCMV infection in vitro
(A) Stro-1-selected bone marrow (BM)-, liver (LVR)-, lung (LNG)- and brain (BRN)-derived mesenchymal stromal cells (MSC) were infected at MOI of 3 with HCMV Davis strain, and the presence of viral UL123 (immediate early gene) DNA was analyzed at 7 dpi by PCR. Negative control (NC) consisted of PCR in absence of DNA. Positive control (PC): PCR in presence of viral HCMV Davis strain DNA; Mock: PCR performed with DNA from respective cell populations prior to infection, to establish the specificity of the assay and to ensure that amplification was not due to latent HCMV in stromal layers. Amplification of β-actin was used to verify DNA integrity. Marker: 1 kb plus Ladder. (B) Quantitative PCR determined the viral copy number present in the different cultured cells at 7 dpi. To prepare a calibration/standard curve, known number of viral copies per microliter and serial 10-fold dilutions (from 1 × 108 to 1 × 102 viral copies per reaction) were amplified simultaneously and under the same conditions as the various MSC samples. The Ct values obtained from each standard dilution were then plotted against the viral copy number to construct the standard curve. Each Ct value, obtained from viral DNA amplification for each sample, was used to extrapolate from the standard curve the absolute viral copy number for that sample. Finally, the viral copy numbers for each sample were statistically compared to the numbers obtained for the HCMV negative samples. The amplification results were validated based on the quality of dissociation curves and high amplification efficiency of the primers. Each sample was analyzed at least in triplicate, along with nontemplate controls, serial dilutions of the solution containing known viral copies per microliter, and mock infected samples. HCMV, human cytomegalovirus; MOI, multiplicity of infection; PCR, polymerase chain reaction; UL, unique long.
Figure 5
Figure 5. Transcription of UL123 and UL83 genes confirms HCMV infection of Stro-1+ bone marrow (BM)-, liver (LVR)-, lung (LNG)- and brain (BRN)-derived MSC
HF and Stro-1-selected human LVR-, LNG-, BRN- and BM-MSC were infected at MOI of 3 with HCMV Davis strain. RNA from each cell population, prior to and after infection, was purified, DNase-treated, reverse-transcribed and analyzed for the presence of UL123 and UL83 transcripts up to 5 dpi. Amplification of β-actin was used as a positive control to verify RNA integrity and ensure that the RT-PCR reactions were successfully performed. Negative control (NC): PCR in absence of cDNA. Positive control (PC): PCR in presence of viral HCMV DNA. Mock: PCR performed with cDNA from uninfected cells to guarantee that amplification was not due to reactivation of latent HCMV in stromal layers. In addition, for each RNA sample, RT-PCR was performed in the absence of reverse transcriptase enzyme (NO RT). dpi, day postinfection; HCMV, human cytomegalovirus; HF, human foreskin fibroblasts; MOI, multiplicity of infection; MSC, mesenchymal stromal cells; PCR, polymerase chain reaction; UL, unique long.
Figure 6
Figure 6. Bone marrow (BM)-, liver (LVR)-, brain (BRN)- and lung (LNG)-derived perivascular Stro-1+ mesenchymal stromal cells (MSC) are naturally infected with HCMV
DNA isolated from BM-MSC obtained from healthy CMV-seropositive adult donors, and DNA isolated from LNG-, BRN- and LVR-MSC from different donors, for which serologic status/presence of active disease was unknown were analyzed by nested qPCR. Negative controls included BM-MSC from CMV serologically negative adult donors, as well as stromal cells obtained from cord tissue from serologically negative mothers. CMV, cytomegalovirus; HCMV, human cytomegalovirus; qPCR, quantitative polymerase chain reaction.

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