Human Cytomegalovirus Immediate-early Antigen Research Articles

The multi-targeted kinase inhibitor sorafenib inhibits human cytomegalovirus replication.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised individuals. Here, non-toxic concentrations of the anti-cancer kinase inhibitor sorafenib were shown to inhibit replication of different HCMV strains (including a ganciclovir-resistant strain) in different cell types. In contrast to established anti-HCMV drugs, sorafenib inhibited HCMV major immediate early promoter activity and HCMV immediate early antigen (IEA) expression. Sorafenib is known to inhibit Raf. Comparison of sorafenib with the MEK inhibitor U0126 suggested that sorafenib inhibits HCMV IEA expression through inhibition of Raf but independently of signaling through the Raf downstream kinase MEK 1/2. In concordance, siRNA-mediated depletion of Raf but not of MEK-reduced IEA expression. In conclusion, sorafenib diminished HCMV replication in clinically relevant concentrations and inhibited HCMV IEA expression, a pathophysiologically relevant event that is not affected by established anti-HCMV drugs. Moreover, we demonstrated for the first time that Raf activation is involved in HCMV IEA expression.

Detection of Human Cytomegalovirus in Atherosclerotic Carotid Arteries in Humans

Atherosclerosis plays an important role in stroke. A microbiological infection has been suggested to be involved in the pathogenesis of atherosclerosis, in particular the human cytomegalovirus (HCMV). The objective of this study was to determine the association between HCMV infection and atherosclerosis of the internal carotid arteries in patients of Chinese Han ethnicity with ischemic stroke. HCMV DNA and antigen were detected in atherosclerotic internal carotid arteries from 35 patients with ischemic stroke and 20 controls from a Chinese Han ethnic population. Immunohistochemistry, in situ hybridization and polymerase chain reaction were used to detect the HCMV immediate early (IE) and late (L) antigen as well as DNA in the vascular walls. We observed that the proportion of cases that tested positive for HCMV IE but not L antigen and DNA was statistically greater in stroke patients compared with the control population. HCMV IE antigen and DNA were associated with the pathological process of atherosclerosis. The vessel wall might be the infection site of the dormant virus.

An Animal Model of Human Cytomegalovirus Infection

To develop a rat model that allowed in vivo progressive human cytomegalovirus (HCMV) infection, allogeneic liver transplantation was performed across a rat combination of Dark Agouti (DA) to Brown Norway (BN). AD169, a well-characterized laboratory strain of HCMV, was used to establish a rat model of HCMV infection by injection of 0.4 mL (30.0 logTCID50) supernate into the rat peritoneum. Histological and blood specimens were obtained from animals sacrificed at predetermined timepoints. We performed immunohistochemical staining in liver, heart, kidney, spleen, and lung for HCMV immediate-early antigen (IE), lower matrix protein (pp65) detection in peripheral blood leukocytes, and HCMV early antigen (EA) and late antigen (LA). We compared survival rates. Our results showed positive HCMV IE and pp65 antigenemia detected in peripheral blood leukocytes in transplanted recipients from day 1 to day 30. Positive HCMV EA and LA staining cells were only detected in sections 10 days after liver transplantation, namely, in hepatocytes, mononuclear cells, bile duct epithelial cells, and endothelial cells. Successful HCMV replication was due to the combination of liver transplantation and cyclosporine (CsA) immunosuppression. Survival analysis showed no significant differences between the HCMV-infected group and HCMV-uninfected group. This new rat model of HCMV infection may be helpful to understand immune system modulation of HCMV infection.

Macrophage cultures are susceptible to lytic productive infection by endothelial-cell-propagated human cytomegalovirus strains and present viral IE1 protein to CD4+ T cells despite late downregulation of MHC class II molecules

The contribution of CD4(+) T cells to control of human cytomegalovirus (HCMV) has been shown and infected tissue macrophages might contribute to this response by antigen presentation. As shown previously, CD4(+) T cells recognize HCMV immediate-early antigen IE1 on glioblastoma cells manipulated to express MHC class II molecules. Here, the possible interference of virus-induced MHC class II downmodulation with the presentation of IE1 by natural target cells was analysed. The capacity of IE1-specific CD4(+) T-cell clones to recognize HCMV-infected monocyte-derived macrophages was tested. Various HCMV strains were used to achieve efficient infection of macrophages. Activation of CD4(+) T cells by infected macrophages was evaluated at different time points after infection. Endothelial-cell-adapted HCMV strains efficiently infected cultured human macrophages. However, the immediate-early and early phases of replication were prolonged. Infected cells entered the late replication phase only after 3 days of infection, which was associated with downmodulation of MHC class II molecules at the surface of infected cells. Strong stimulation of IE1-specific CD4(+) T cells resulted from endogenous de novo antigen production and presentation by infected macrophages during the first 3 days of virus replication, despite MHC class II downmodulation in the late replication phase. Therefore, infected macrophages are assumed to contribute to the antiviral immune response in infected organs.

Diagnosis and monitoring of acute cytomegalovirus infection in peripheral blood of transplant recipients by nested reverse transcriptase polymerase chain reaction (RT-PCR).

The aim of this novel diagnostic approach is to monitor cytomegalovirus (CMV) infection in immunocompromised transplant recipients using early, sensitive, and specific predictors before and during antiviral therapy. The peripheral blood cells of 20 patients after transplantation (9 liver, 7 kidney, 4 simultaneous kidney-pancreas) were studied for an early diagnosis of acute infection. The mRNA and DNA of human CMV immediate-early antigen (IEA) were detected by nested-polymerase chain reaction (PCR) assay. Results of nested PCR were compared with the immunological detection of antigen pp65 and serological diagnosis of CMV infection. All data were correlated with clinical symptoms like leukopenia, thrombopenia, pneumonia, and allograft-rejection reaction. Of 20 transplant recipients, 12 were infected by CMV, and 9 suffered from a CMV-related disease. CMV mRNA were detected simultaneously with antigen pp65 and CMV DNA in all patients with symptomatic infection. Additionally, CMV mRNA was found over a longer period after ganciclovir treatment of infected recipients. Nested reverse transcriptase (RT)-PCR for CMV-IEA mRNA allows a sensitive and specific diagnosis of an acute CMV infection. CMV mRNA was found to be a good marker of acute viremia and could be a useful tool for CMV monitoring over the whole period of disease management, even during antiviral therapy.

Effect of human cytomegalovirus infection on apoptosis and expression of bcl-2 and Fas mRNA

To develop a rat model that allowed in vivo progressive human cytomegalovirus (HCMV) infection, allogeneic liver transplantation was performed across a rat combination of Dark Agouti (DA) to Brown Norway (BN). AD169, a well-characterized laboratory strain of HCMV, was used to establish a rat model of HCMV infection by injection of 0.4 mL (30.0 logTCID50) supernate into the rat peritoneum. Histological and blood specimens were obtained from animals sacrificed at predetermined timepoints. We performed immunohistochemical staining in liver, heart, kidney, spleen, and lung for HCMV immediate-early antigen (IE), lower matrix protein (pp65) detection in peripheral blood leukocytes, and HCMV early antigen (EA) and late antigen (LA). We compared survival rates. Our results showed positive HCMV IE and pp65 antigenemia detected in peripheral blood leukocytes in transplanted recipients from day 1 to day 30. Positive HCMV EA and LA staining cells were only detected in sections 10 days after liver transplantation, namely, in hepatocytes, mononuclear cells, bile duct epithelial cells, and endothelial cells. Successful HCMV replication was due to the combination of liver transplantation and cyclosporine (CsA) immunosuppression. Survival analysis showed no significant differences between the HCMV-infected group and HCMV-uninfected group. This new rat model of HCMV infection may be helpful to understand immune system modulation of HCMV infection.

Rapid ganciclovir susceptibility assay using flow cytometry for human cytomegalovirus clinical isolates.

Rapid, quantitative, and objective determination of the susceptibilities of human cytomegalovirus (HCMV) clinical isolates to ganciclovir has been assessed by an assay that uses a fluorochrome-labeled monoclonal antibody to an HCMV immediate-early antigen and flow cytometry. Analysis of the ganciclovir susceptibilities of 25 phenotypically characterized clinical isolates by flow cytometry demonstrated that the 50% inhibitory concentrations (IC50s) of ganciclovir for 19 of the isolates were between 1.14 and 6.66 microM, with a mean of 4.32 microM (+/-1.93) (sensitive; IC50 less than 7 microM), the IC50s for 2 isolates were 8.48 and 9.79 microM (partially resistant), and the IC50s for 4 isolates were greater than 96 microM (resistant). Comparative analysis of the drug susceptibilities of these clinical isolates by the plaque reduction assay gave IC50s of less than 6 microM, with a mean of 2.88 microM (+/-1.40) for the 19 drug-sensitive isolates, IC50s of 6 to 8 microM for the partially resistant isolates, and IC50s of greater than 12 microM for the four resistant clinical isolates. Comparison of the IC50s for the drug-susceptible and partially resistant clinical isolates obtained by the flow cytometry assay with the IC50s obtained by the plaque reduction assay showed an acceptable correlation (r2 = 0.473; P = 0.001), suggesting that the flow cytometry assay could substitute for the more labor-intensive, subjective, and time-consuming plaque reduction assay.

Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins

Human cytomegalovirus (HCMV) viremia in peripheral blood polymorphonuclear leukocytes (PMNLs) from 187 immunosuppressed patients (79 heart transplant recipients and 108 patients with acquired immunodeficiency syndrome [AIDS]) was investigated. Five mouse monoclonal antibodies (MAbs), one specifically reactive to HCMV immediate-early antigen (IEA) in PMNLs, two specifically reactive to IEA in infected cell cultures, and two specifically reactive to late antigens, were used in immunofluorescence and/or immunoperoxidase test systems for (i) detection of HCMV IEA in human embryonic lung fibroblast (HELF) cell cultures inoculated with PMNL samples, (ii) direct detection of HCMV IEA in PMNL nuclei of cytospin preparations, and (iii) identification of HCMV isolates from PMNL samples in HELF cells. Quantification of viremia was achieved by counting the number of infected PMNLs per 2 x 10(5) cells examined directly on cytospin preparations as well as by counting the number of IEA-positive HELF cells inoculated with 2 x 10(5) PMNLs. A significant correlation was found between the number of infected PMNLs and the number of infected HELF cells. When the number of infected PMNLs per 2 x 10(5) cells was greater than 10, both methods (PMNL IEA and HELF IEA) gave concordant results; when it was greater than 80/2 x 10(5) cells, clinical symptoms were consistently associated with HCMV viremia. Ten patients with heart transplants and three patients with AIDS who had high or increasing levels of HCMV viremia underwent antiviral treatment with ganciclovir. Treatment was discontinued only after disappearance of IEA-positive PMNLs from blood (the last marker of infection to become negative). On the other hand, in the presence of low levels of viremia (less than 10 infected PMNLs per 2 x 10(5) cells), different methods often provided discordant results and overt clinical symptoms were never observed. IEA-negative results with PMNL samples or HELF cells in the presence of positive virus isolation could never be attributed to the inability of IEA MAbs to recognize individual HCMV strains, since all of the relevant viral isolates were recognized by IEA MAbs. In addition, all five MAbs used in the study were capable of identifying all 135 conventional HCMV isolates obtained from the study population. It was concluded that (i) maximal sensitivity in diagnosing HCMV viremia may be achieved by combining different techniques; (ii) direct detection of HCMV IEA in PMNLs, yielding results on the day of blood collection, is a very rapid and sensitive technique, and thus one can rely on it for clinical management of HCMV infections; and (iii) low levels of viremia are clinically irrelevant, whereas high levels are associated with clinical symptoms.

Human cytomegalovirus

Exposure of human lung fibroblasts to human cytomegalovirus (HCMV) stimulated a rapid increase in the release of [3H] from cells prelabelled with radiolabelled arachidonic acid ([3H]AA). Maximum stimulation of [3H] release was observed at 20 min postinfection and was quantitatively similar to that induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA: 10 nM) or fetal calf serum (5%). The level of [3H] release was dependent on the multiplicity of infection, and appeared to be mediated by a component(s) of the virion, since the findings from three series of experiments suggested that neither infectious virus, nor HCMV-specific macromolecular synthesis was required for stimulation of [3H] release. (1) Inactivation of HCMV infectivity with ultra-violet (UV) light (approximately 254 nm, 4.80 x 10(4) ergs/mm2) did not diminish the stimulation of [3H] release. (2) Significant reduction in the level of [3H] release was not observed when infected cells were maintained in the presence of a protein synthesis inhibitor, cycloheximide (50 micrograms/ml), or an inhibitor of mRNA synthesis, 3'-deoxyadenosine (cordycepin, 50 micrograms/ml). (3) No correlation was established between the expression of HCMV immediate early (IE) antigens and the induction of [3H] release, since there was little, if any, synthesis of HCMV IE antigen detectable by anticomplement immunofluorescence through the first 30 min postinfection. These findings suggesting that the HCMV particle rapidly stimulates AA metabolism are consistent with the view that the interaction of a HCMV virion component(s) with the cell surface may initiate membrane-associated events similar to those induced by growth factors.

Detection of human cytomegalovirus immediate early antigen in leukocytes as a marker of viremia in immunocompromised patients

Peripheral blood polymorphonuclear (PMN) cells from 35 immunocompromised patients (22 heart transplant recipients and 13 AIDS patients) and four normal subjects were tested for the presence of human cytomegalovirus (HCMV) immediate early antigen (IEA) (antigenemia) by indirect immunofluorescence (IFA) and IEA-specific monoclonal antibodies (MAb). PMN samples were tested in parallel for HCMV isolation (viremia) by using MAb to viral early antigens (EA) and the IFA technique 24-48 hr after inoculation onto human fibroblast monolayers. HCMV was isolated from 26 of 83 PMN samples examined: of these, 25 were also positive for HCMV IEA (96% sensitivity). Seven additional PMN samples negative for viral isolation resulted IEA-positive (87.7% specificity). Six of the seven discordant samples were taken from four patients during ganciclovir treatment. The transitory dissociation between positive HCMV antigenemia and negative viremia during antiviral treatment was followed, at the end of the therapy, either by virus clearance and disappearance of IEA-positive PMNs (one patient) or by reappearance of viremia (three patients). Among concordant positive samples, a significant correlation was observed between the number of IEA-positive PMN leukocytes and EA-positive nuclei of infected fibroblasts, when the same number of PMNs were used for both tests.