Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

BACKGROUND: During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. RESULTS: Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. CONCLUSIONS: Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells.

During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release.

Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection.

Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected.

Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens.

Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections.

The host immune response contributes to Haemophilus influenzae virulence.

BACKGROUND: There is compelling evidence that infections with non-typeable Haemophilus influenzae (NTHi) are associated with exacerbations in COPD patients. However, NTHi has also been isolated frequently during clinically stable disease. In this study we tested the hypothesis that genetically distinct NTHi isolates obtained from COPD patients differ in virulence which could account for dissimilarities in the final outcome of an infection (stable vs. exacerbation). RESULTS: NTHi isolates (n = 32) were obtained from stable COPD patients, or during exacerbations. Genetically divergent NTHi isolates were selected and induction of inflammation was assessed as an indicator of virulence using different in vitro models. Despite marked genomic differences among NTHi isolates, in vitro studies could not distinguish between NTHi isolates based on their inflammatory capacities. Alternatively, when using a whole blood assay results demonstrated marked inter-, but not intra-individual differences in cytokine release between healthy volunteers irrespective of the origin of the NTHi isolate used. CONCLUSION: Results suggest that the individual immune reactivity might be an important predictor for the clinical outcome (exacerbation vs. no exacerbation) following NTHi infection.

Interferon-ß induces a long-lasting antiviral state in human respiratory epithelial cells.

OBJECTIVES: Interferon-ß (IFNß) induces strong antiviral effects and is therefore an attractive agent to prevent or reduce the incidence of virus-mediated exacerbations in asthmatic or chronic obstructive pulmonary disease (COPD) patients. We therefore investigated the effects of prophylactic IFNß on respiratory epithelial cells infected with rhinovirus (RV). METHODS: A549 cells and primary bronchial epithelial cells (PBECs) were exposed for 18 h to IFNß. Then, IFNß was either removed or maintained in the supernatant for the rest of the experiment and cells were infected with RV-1B at t = 0 or 72 h after the initial exposure to IFNß. RESULTS: Viral RNA levels were decreased in both cell types. Furthermore, both viral RNA and infectious virus levels in the supernatant of infected A549 cells were still significantly reduced at 72 h after removal of IFNß. This pronounced antiviral pre-treatment effect was associated with increased expression of the antiviral genes IFN-stimulated protein of MR15000 (ISG15) and Myxovirus resistance 1 (Mx1) and the effect was maintained even when IFNß levels in the supernatant of A549 cells were undetectable. CONCLUSIONS: These data show that IFNß has not only a strong, but also a long-lasting protective effect against RV infection of respiratory epithelium.

CpG and poly(I:C) stimulation of dendritic cells and fibroblasts limits herpes simplex virus type 1 infection in an IFNß-dependent and -independent way.

Viral activation of toll-like receptors (TLRs) on dendritic cells (DCs) leads to production of various cytokines, including antiviral type I interferons (IFNs). Synthetic ligands specific for TLRs are also able to induce the production of type I IFNs (IFNα/ß) by DCs, suggesting that these ligands have potential as antiviral drugs. In this in vitro study we extensively investigated the antiviral activity of various TLR ligands. Mouse bone marrow (BM) cells were differentiated into plasmacytoid and conventional DCs (pDCs and cDCs), stimulated with various TLR ligands and tested the antiviral abilities of collected supernatants in an in vitro herpes simplex virus type 1 (HSV-1) infection model. We observed a significant IFNß-, (but not IFNα-) dependent reduction in HSV-1 infection when a mixed pDC/cDC population was stimulated with the TLR9 ligand CpG. In the absence of pDCs, TLR stimulation resulted in less pronounced antiviral effects. The most pronounced antiviral effect was observed when both DC subsets were stimulated with poly(I:C). A similar noticeable antiviral effect was observed when fibroblasts (L929 cells) were stimulated directly with poly(I:C). These poly(I:C)-mediated antiviral effects were only partially IFNß-mediated and probably TLR independent. These data demonstrate that TLR ligands are not only able to produce type I IFN but can indeed act as antiviral drugs. In particular poly(I:C), which exerts its antiviral effects even in the absence of DCs, may become a promising drug e.g. to prevent respiratory infections by topical intranasal application.

Lack of evidence for the role of human adenovirus-36 in obesity in a European cohort.

Adenovirus infection has been shown to increase adiposity in chickens, mice, and nonhuman primates. Adenovirus type 36 (Ad-36) DNA was detected in adipose tissues in these animal trials. In the United States, Ad-36 significantly correlates with obesity as illustrated by an Ad-36 seroprevalence of 30% in obese individuals and 11% in nonobese individuals. We investigated the possibility of a similar correlation of Ad-36 in Dutch and Belgian persons. In total, 509 serum samples were analyzed for Ad-36 antibodies using a serum neutralization assay. In addition, PCR was used to detect adenoviral DNA in visceral adipose tissue of 31 severely obese surgical patients. Our results indicated an overall Ad-36 seroprevalence of 5.5% increasing with age. BMI of Ad-36 seropositive humans was not significantly different from seronegative humans. No adenoviral DNA could be found using PCR on visceral adipose tissue. In conclusion, this first Ad-36 study in the Netherlands and in Belgium indicates that Ad-36 does not play a role as a direct cause of BMI increase and obesity in humans in Western Europe.

FK778 attenuates cytomegalovirus-enhanced vein graft intimal hyperplasia in a rat model.

BACKGROUND: Venous grafts are commonly used to treat drug-resistant coronary artery disease, although long-term functionality is limited because of proliferation and migration of smooth muscle cells (SMC). As proliferating SMC are particularly susceptible for the stimulating effects of cytomegalovirus (CMV), we hypothesized that CMV infection may enhance cell proliferation and graft failure. Furthermore, we evaluated the potential of FK778 to prevent intimal hyperplasia. Apart from its antiviral properties, FK778 is a new immunosuppressive agent which may also affect SMC proliferation, making it an interesting drug to prevent (CMV-enhanced) venous graft intimal hyperplasia. METHODS: Epigastric vein-to-common femoral artery interposition grafts were placed in four groups of 10 rats each. Rats received either FK778 (oral treatment, 15 mg/kg), were infected with CMV (1.25 x 10(6) plaque-forming units) or were both treated and infected. RESULTS: CMV infection resulted in a significant increase in intimal and medial cross-sectional area and medial wall thickness of the vein grafts. This effect was diminished by administration of FK778. Moreover, FK778 treatment alone resulted in a significant decrease in neointimal area and percentage of stenosis versus the control group. CONCLUSIONS: These data suggest a role of CMV in venous graft failure. Also, our results suggest a prospective role for the new immunosuppressive drug FK778 in the prevention of (CMV-mediated) vein graft intimal hyperplasia.

Adenoviral activin A expression prevents vein graft intimal hyperplasia in a rat model.

Autologus vein grafts are used for coronary artery and infra-inguinal bypass procedures. Although initially successful, long-term patency rates are limited by lumen occlusion due to neointima formation by smooth muscle cell hyperplasia. Gene therapy to prevent this smooth muscle cell proliferation has been studied extensively with limited success. Activin A, a member of the transforming growth factor-beta super family, promotes the contractile phenotype of smooth muscle cells. Maintaining the contractile phenotype could be a novel strategy to prevent intimal hyperplasia. In an epigastric vein-to-common femoral artery interposition grafts rat model, activin A over-expression resulted in a significant decrease in intimal cross-sectional area and percentage stenosis as compared to the control group. BrdU staining identified lower proliferation rates of the smooth muscle cells in the group treated with activin A. We report for the first time evidence that activin A can diminish vein graft failure in a rat model supporting a novel strategy to prevent intimal hyperplasia.

Chlamydia pneumoniae aggravates vein graft intimal hyperplasia in a rat model.

BACKGROUND: Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n = 10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*108 IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis. RESULTS: Cpn administration immediately after vein grafting resulted in a significant increase in medial cross-sectional area, wall thickness and total wall area. There were no significant differences in T-cell or macrophage influx. Likewise, although positive immunostaining for both HSP60 and CRP could be detected, no differences were found between groups. Based on the observation that the number of cells/microm2 was also not altered, we conclude that Cpn infection stimulates smooth muscle cell proliferation by hereunto unknown molecular mechanisms, resulting in a significant increase in intimal hyperplasia. CONCLUSION: In conclusion, in a well defined animal model we present here for the first time evidence for a role of Chlamydia pneumoniae in the process of venous graft failure.

Chlamydia pneumoniae infection of brain cells: an in vitro study.

Inspired by the suggested associations between neurological diseases and infections, we determined the susceptibility of brain cells to Chlamydia pneumoniae (Cpn). Murine astrocyte (C8D1A), neuronal (NB41A3) and microglial (BV-2) cell lines were inoculated with Cpn. Infection was established by immunofluorescence and real-time PCR at various time points. Productive infection was assessed by transferring medium of infected cells to a detection layer. Finally, apoptosis and necrosis post-infection was determined. Our data demonstrate that the neuronal cell line is highly sensitive to Cpn, produces viable progeny and is prone to die after infection by necrosis. Cpn tropism was similar in an astrocyte cell line, apart from the higher production of extracellular Cpn and less pronounced necrosis. In contrast, the microglial cell line is highly resistant to Cpn as the immunohistochemical signs almost completely disappeared after 24 h. Nevertheless, significant Cpn DNA amounts could be detected, suggesting Cpn persistence. Low viable progeny and hardly any necrotic microglial cells were observed. Further research is warranted to determine whether these cell types show the same sensitivity to Cpn in an in vivo setting.

Mouse cytomegalovirus antigenic immune stimulation is sufficient to aggravate atherosclerosis in hypercholesterolemic mice.

We have previously demonstrated that mouse cytomegalovirus (MCMV) infection aggravates atherosclerosis by stimulating the ongoing inflammatory process in the vascular wall. Here we investigated whether MCMV antigenic immune stimulation by UV-MCMV injection is sufficient to aggravate atherosclerosis. In addition we analyzed whether low viral doses are sufficient to stimulate atherosclerosis. Therefore, apoE(-/-) mice received a low dose injection with infectious virus (MCMV) or replication-deficient virus (UV-inactivated MCMV, UV-MCMV). Atherosclerosis progression, influx of inflammatory cells in atherosclerotic lesions and internal organs and the number of MCMV DNA copies in various organs were determined at 2 weeks after injection. After injection with infectious virus, MCMV DNA was present in internal organs, while no MCMV DNA could be detected after UV-MCMV injection. Interestingly, both MCMV and UV-MCMV significantly increased mean atherosclerotic lesion area and T cell number in the atherosclerotic lesions, while only MCMV infection increased T cell numbers in the internal organs. These data indicate that in apoE(-/-) mice both low dose infectious MCMV as well as MCMV antigenic injections are sufficient for atherosclerosis aggravation.

Cytomegalovirus-enhanced development of transplant arteriosclerosis in the rat; effect of timing of infection and recipient responsiveness.

Cytomegalovirus (CMV) is put forward as a risk factor for transplant arteriosclerosis (TA). In this article, we studied CMV-enhanced development of TA in rats in different donor/recipient combinations in relation to the timing of infection. Recipient rats transplanted with an aortic allograft (BN to Lew) were infected with rat CMV (RCMV) at different time-points relative to transplantation. The virus-induced effects on TA development were also determined in other strain combinations (PVG to AO and DA to WF). Finally, transmission of RCMV from aortic grafts and its effect on TA was studied. RCMV infection enhanced TA development only in Lew recipients and only after infection early post-transplantation (days 1-5). Virus transmission to the recipient only occurred from 5 and 10 days infected aortic donor-grafts, however without affecting TA development. These data indicate that the acute alloresponse and acute CMV infection need to occur simultaneously to enhance TA. This effect, however, appears to be strain combination dependent and therefore cannot be generalized.

Cytomegalovirus aggravates intimal hyperplasia in rats by stimulating smooth muscle cell proliferation.

Epidemiological and animal studies suggest a role for cytomegalovirus (CMV) in restenosis. Previously, we demonstrated that proliferating smooth muscle cells (SMCs) in the injured arterial wall are particularly susceptible to CMV-induced effects. Therefore, we hypothesised that, depending on the time point of infection after vascular injury, CMV infection may affect cell proliferation either in the media or in the neointima, thereby aggravating the process of restenosis. In the present study, we focused on the individual layers of the arterial wall by evaluating, besides the neointima-to-media ratio, the medial and neointimal area and cellularity in the rat femoral artery. Vascular injury was photochemically induced in rat femoral arteries. Immediately or 14 days thereafter, rats were infected with rat CMV (RCMV) or mock infected. The presence of RCMV in the vascular wall was determined at 3, 5, 14 and 35 days after infection by quantitative real-time PCR. When rats were infected immediately after injury, a significant increase was seen only in the medial but not in the neointimal cross-sectional area. On the other hand, when rats were infected 14 days after the initial injury, a significant increase was only seen in the neointimal area, thereby confirming our hypothesis that RCMV infection primary affects proliferating SMCs. As the mean number of SMCs per microm2 in both cell layers was unchanged despite an increase in cross-sectional area, this implies that RCMV stimulated SMC proliferation. Furthermore, these vascular effects were observed without the virus being abundantly present in the vascular wall, suggesting that inflammatory and immune-mediated responses to RCMV infection are more important in aggravating the response to vascular injury than the virus itself.

Inflammatory markers in depressed post-myocardial infarction patients.

BACKGROUND: Depressive disorder in the post-myocardial infarction (MI) period has been associated with increased cardiac morbidity and mortality. Possible pathophysiological mechanisms behind this association are not clear. Major depression in physically healthy subjects has been related to immune abnormalities including increased plasma levels of interleukin-6 (IL-6), tumor necrosis factor alfa (TNF-alpha) and C-reactive protein (CRP). In patients with MI, increased inflammatory markers, such as CRP and TNF-alpha, have been associated with increased cardiovascular events. It was the aim of this study to test the hypothesis that depression in post-MI patients is associated with increased inflammation as compared to non-depressed post-MI patients. METHODS: The cytokines IL-6 and TNF-alpha ; the soluble cytokine receptors sIL-6R, sTNF-RI and sTNF-RII; neopterin; and the inflammation-sensitive plasma proteins (ISPs) CRP and haptoglobin were assessed in a group of 57 patients with a diagnosis of depression post-MI and in a control group of 46 non-depressed post-MI patients, matched for age, gender and time elapsed since MI. RESULTS: Cytokine, neopterin and ISP levels were not statistically different in the depressed post-MI group as compared to the non-depressed post-MI group. Several inflammatory markers were however elevated in both cohorts when compared with levels reported in healthy subjects, indicating persistent inflammation several months after MI. CONCLUSIONS: There was no indication of increased inflammation in depressed post-MI patients as compared to non-depressed post-MI patients.

The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication.

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1.1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVDeltar127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

Cytomegalovirus infection aggravates atherogenesis in apoE knockout mice by both local and systemic immune activation.

Since the 1970s, cytomegalovirus (CMV) infection has been associated with atherosclerotic disease. However, the exact contribution of the virus remains uncertain. In this article we describe both a direct and indirect immune-mediated effect of the virus on the disease process. Eight-week-old apolipoprotein E (apoE) knockout mice were infected with mouse CMV (MCMV) or mock injected, and they were sacrificed at 2 and 20 weeks post-injection (p.i.) to study atherosclerosis, vascular wall IFNgamma and TNFalpha expression and MCMV spread. To study plasma IFNgamma and TNFalpha levels, blood was collected at 1, 2, 4 and 6 days p.i. in addition to days of sacrifice. Plasma cytokine levels were increased after MCMV infection at early time points and decreased to mock levels at 2 and 20 weeks p.i. At 2 weeks p.i., more aortic arch samples showed local cytokine expression after MCMV infection. The number of early atherosclerotic lesions and the percentage of mice containing early lesions were increased at 2 weeks p.i., while at 20 weeks p.i., the MCMV-induced effect on atherogenesis was seen on the late lesions. In conclusion, MCMV infection induces a systemic immune response reflecting an indirect effect of MCMV infection on atherosclerosis in addition to a local aortic immune response reflecting a direct effect of the virus on the atherosclerotic process.

Improved detection and quantification of mouse cytomegalovirus by real-time PCR.

During latent cytomegalovirus (CMV) infection, viral presence cannot be detected by plaque assay. Therefore, we assessed the applicability of real-time PCR for temporal determination of virus dissemination in two different mouse strains. Eight-week-old BALB/c and C57BL/6J mice were infected with mouse CMV (MCMV) and sacrificed at 1, 2, 4, 6, 14 and 28 days post infection. Real-time PCR was used to determine MCMV copy number in the heart, bone marrow cells, aorta and blood. In lung, liver, salivary gland and spleen the presence of MCMV was determined both by plaque assay and real-time PCR. In analogy with the plaque assay, the real-time PCR technique revealed higher numbers of MCMV genomic copies in all organs obtained from BALB/c mice when compared with C57BL/6J mice, demonstrating the applicability of the technique. A significant correlation was observed between both assays when a positive test result was seen with both assays. Nonetheless, lower viral infectivity titers were found compared to real-time PCR data. Thus, the real-time PCR technique is more sensitive in detecting the presence of MCMV and is therefore well suited for (dose-response) intervention studies aimed at studying virus eradication.