Cytomegalovirus cell tropism.

The human cytomegalovirus (HCMV) can infect a remarkably broad cell range within its host, including parenchymal cells and connective tissue cells of virtually any organ and various hematopoietic cell types. Epithelial cells, endothelial cells, fibroblasts and smooth muscle cells are the predominant targets for virus replication. The pathogenesis of acute HCMV infections is greatly influenced by this broad target cell range. Infection of epithelial cells presumably contributes to inter-host transmission. Infection of endothelial cells and hematopoietic cells facilitates systemic spread within the host. Infection of ubiquitous cell types such as fibroblasts and smooth muscle cells provides the platform for efficient proliferation of the virus. The tropism for endothelial cells, macrophages and dendritic cells varies greatly among different HCMV strains, mostly dependent on alterations within the UL128-131 gene locus. In line with the classification of the respective proteins as structural components of the viral envelope, interstrain differences concerning the infectivity in endothelial cells and macrophages are regulated on the level of viral entry.

Evaluation of the COBAS Amplicor HCMV Monitor for early detection and monitoring of human cytomegalovirus infection after allogeneic stem cell transplantation.

Early diagnosis of human cytomegalovirus (HCMV) infection and the introduction of preemptive antiviral therapy have reduced HCMV-related mortality after allogeneic stem cell transplantation. A critical goal remains stratifying risk profiles and minimizing potential harm owing to antiviral overtreatment. We compared the commercially available standardized COBAS Amplicor CMV Monitor (CACM) to an in-house PCR assay, for the monitoring of HCMV infection. Seventy-two patients were surveyed by an in-house PCR of whole blood, quantitative viral load assessment by CACM and virus culture assays in a prospective and a retrospective study. A high concordance between CACM and PCR was documented. The viral load at onset correlated with the peak viral load (Spearman rank correlation R=0.634, P=0.0004). In patients developing HCMV disease, both viral loads were in trend higher (P=0.823, respectively P=0.053), and the viremic episodes longer (P=0.015), as compared to asymptomatically HCMV-infected patients. The serological pre-transplant status was the major risk factor for the development of HCMV disease, showing highest risk for seropositive patients receiving a seronegative graft, whereas donor type (related or unrelated) and graft type (bone marrow or peripheral blood mobilized stem cells) did not have an influence. HCMV infection proved to be a risk factor for the development of non-viral opportunistic infections (P=0.002).

Cytomegalovirus infection after orthotopic liver transplantation is restricted by a pre-existing antiviral immune response of the recipient.

Infection of the liver by the human cytomegalovirus (HCMV) frequently occurs after orthotopic liver transplantation (OLT). However, the role of viral replication and the inflammatory reaction in the development of HCMV-associated liver dysfunction is unclear. To address this question in vivo, 84 liver biopsy specimens from 74 patients who received an orthotopic liver transplant were investigated by immunohistochemical detection of viral antigens and cell type specific marker proteins. The extent of viral replication was correlated with the HCMV antibody status of donor and recipient. HCMV immediate early antigens were found in 25 of 84 liver tissue sections investigated, hepatocytes being the predominant target cells. Bile duct epithelial cells, endothelial cells, mesenchymal cells and sinusoidal lining cells were also found susceptible to HCMV infection. The detection of viral capsid antigens, nuclear inclusions in infected cells and foci of infected cells were suggestive of permissive infection in these cells. In 25 HCMV-positive liver biopsy specimens, the median extent of HCMV infection was 0.33 (0.02-5.67) infected cells/mm(2) liver tissue. Primary infection of liver transplant recipients (D+/R-) was associated with a significantly higher extent of organ involvement as compared to reinfection or reactivation (D+/R+). In contrast, the extent of inflammatory infiltrates in areas of infected liver cells was higher in tissues of patients with pre-existing immunoreactivity (R+) compared to patients without pre-existing immunoreactivity (R-). In conclusion, these results favour the assumption that the immune response to HCMV is effective in restricting viral spread in the liver.

Human cytomegalovirus as a direct pathogen: correlation of multiorgan involvement and cell distribution with clinical and pathological findings in a case of congenital inclusion disease.

The human cytomegalovirus (HCMV), a member of the Herpesviridae, is the most frequent cause of congenital virus infections and a major cause of morbidity and mortality in immunocompromised patients. Due to the lack of an appropriate animal model, insight into the pathogenesis of HCMV infections originates primarily from in situ examination of HCMV-infected tissues. Although in immunocompromised adults such tests are complicated frequently by the presence of additional misleading pathogens, the absence of additional pathogens renders congenital inclusion disease the most suitable access for investigation of pathogenetic aspects of HCMV infections. Immunohistochemical examination of tissue sections from a boy with fatal congenital inclusion disease was undertaken to detect the extent of multiorgan and cell involvement. Adrenal gland, bone marrow, diencephalon, heart, kidney, liver, lung, pancreas, placenta, small bowel and spleen were included in this study. Detection of virus antigens from different phases of viral replication revealed that all investigated organs were infected by HCMV. Simultaneous detection of cell type specific marker molecules showed that a variety of cell types stained positive for HCMV antigens including endothelial cells, epithelial cells, smooth muscle cells, mesenchymal cells, hepatocytes, monocytes/macrophages and granulocytes. The lung, the pancreas, the kidneys and the liver were the major target organs with a high number of HCMV infected cells. This correlated with multiorgan failure as the cause of death and strongly indicates direct pathogenetic effects of HCMV.

Induction of CMV-specific T-cell lines using Ag-presenting cells pulsed with CMV protein or peptide.

BACKGROUND: CMV disease is still associated with a high morbidity and mortality in recipients of a solid organ or stem cell graft, especially in patients undergoing allogenic stem cell transplantation. Reconstitution of CMV-specific CD4(+) and CD8(+) cytotoxic T cell responses are essential to control CMV infection following allogenic stem cell transplantation. The transfer of unselected populations of lymphocytes from the peripheral blood of a CMV-scropositive donor to a transplant recipient can be used to control CMV infection. However, such transfer of unselected donor lymphocytes is limited by potentially fatal complications that arise from alloreactive T cells, also present in the unselected donor lymphocytes. Thus to make infusion of donor T cells safe and also more effective in controlling CMV infection in the recipient of the T cell infusion, T cells are manipulated in vitro to deplete alloreactive T cells and to enrich for CMV-specific T cells. METHODS: Using various antigen-presenting cells (monocytes/PBMNCs/dendritic cells) and different modes of antigen presentation (infected APCs, pulsing of protein or peptide antigen) different CMV-specific T cell populations can be generated and expanded. RESULTS: Using protein-/or peptide-pulsed DCs CMV-specific CD8(+) cytoxic T cell lines (can be generated and expanded) in addition CMV-specific CD4(+) T cell lines can be generated when CMV-protein-pulsed DCs are used as antigen-presenting cells. When peripheral blood mononuclear cells were stimulated with CMV lysates predominantly CMV-specific CD4(+) T cells are generated and expanded ex vivo. DISCUSSION: Depending on the APC used (monocytes versus DC) and the mode of antigen presentation (protein versus peptide pulsing) different CMV-specific T cell populations of varying purity can be generated which show preserved function when tested for specific proliferation, cytokine production and cytotoxicity.

Evaluation of the Murex CMV DNA Hybrid Capture assay (version 2.0) for early diagnosis of cytomegalovirus infection in recipients of an allogeneic stem cell transplant.

Early diagnosis of CMV infection based on sensitive diagnostic assays has helped to reduce CMV-related mortality after allogeneic stem cell transplantation (SCT). In this study, the commercialized Murex CMV DNA Hybrid Capture assay (version 2.0) (HCS) was prospectively compared to an in-house CMV-DNA PCR assay from whole blood in patients after allogeneic stem cell transplantation. Overall, a high concordance between HCS and PCR was documented (kappa = 0.686; n = 385). The HCS assay was found to be as sensitive as the PCR indicating active CMV infection at a median of 35 and 34 days after transplantation, respectively. None of the HCS-negative patients developed CMV-related symptoms (negative predictive value 100%). Declining CMV DNA load in the blood was found to be an indicator for effective antiviral therapy, whereas persistence of a high viral load was associated with fatal CMV disease. In conclusion, the Hybrid Capture CMV DNA assay (v 2.0) allows early diagnosis of CMV infection after allogeneic SCT and assessment of the efficacy of antiviral therapy.

Ex vivo generation of human cytomegalovirus-specific cytotoxic T cells by peptide-pulsed dendritic cells.

Adoptive transfer of donor-derived human cytomegalovirus (HCMV)-specific T-cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV-specific T cells using HCMV-infected fibroblasts as stimulator cells confines this approach to HCMV-seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV-specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV-specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide-pulsed dendritic cells as antigen-presenting cells. HCMV-specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV-seropositive and 2 out of 11 HCMV-seronegative donors. The CTLs recognized HCMV-infected autologous fibroblasts. No lysis was observed with either non-infected autologous or HLA-mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide-specific T cells of up to 28% and > 90% of CD8(+) T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 x 10(9) HCMV-specific T cells was possible after 6--7 weeks when cultures were initiated with 1--5 x 10(6) responder cells. Thus, the approach with peptide-pulsed DCs to generate HCMV-specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.

Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus.

Marked interstrain differences in the endothelial cell (EC) tropism of human cytomegalovirus (HCMV) isolates have been described. This study aimed to define the step during the replicative cycle of HCMV that determines this phenotype. The infection efficiency of various HCMV strains in EC versus fibroblasts was quantified by immunodetection of immediate early (IE), early and late viral antigens. Adsorption and penetration were analysed by radiolabelled virus binding assays and competitive HCMV-DNA-PCR. The translocation of penetrated viral DNA to the nucleus of infected cells was quantified by competitive HCMV-DNA-PCR in pure nuclear fractions. The intracytoplasmic translocation of capsids that had penetrated was followed by immunostaining of virus particles on a single particle level; this was correlated with the initiation of viral gene expression by simultaneous immunostaining of viral IE antigens. The infectivity of nonendotheliotropic HCMV strains in EC was found to be 100-1000-fold lower when compared to endotheliotropic strains. The manifestation of this phenotype at the level of IE gene expression indicated the importance of initial replication events. Surprisingly, no interstrain differences were detected during virus entry. However, dramatic interstrain differences were found regarding the nuclear translocation of penetrated viral DNA. With nonendotheliotropic strains, the content of viral DNA in the cell nucleus was 100-1000-fold lower in EC when compared to endotheliotropic strains, thereby reflecting the strain differences in IE gene expression. Simultaneous staining of viral particles and viral IE antigen revealed that interstrain differences in the transport of penetrated capsids towards the nucleus of endothelial cells determine the EC tropism of HCMV.

Efficient lytic infection of human arterial endothelial cells by human cytomegalovirus strains.

Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) in vivo during acute disease. However, studies of HCMV-EC interactions in vitro have generated discordant results. While lytic infection of cultured venous EC has been well established, Fish et al. (K. N. Fish, C. Soderberg Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson, J. Virol. 72:5661-5668) have reported noncytopathic persistence of the virus in cultured aortic EC. We propose that interstrain differences in viral host cell tropism rather than the vascular bed of origin of infected EC might account for these discrepancies. In the present investigation we compared the responses of EC derived from human adult iliac artery, placental microvasculature, and umbilical vein to infection with various HCMV strains. Regardless of the vascular bed of origin, infection with EC-propagated HCMV strains induced 100% efficient cytopathic change progressing to complete lysis of inoculated monolayers. While fibroblast-propagated strains persisted at low titer in infected arterial EC cultures, they were also cytolytic for individual infected cells. The finding of cytopathic lytic infection of arterial EC by HCMV implicates a mechanism of vascular injury in the pathogenesis of HCMV infection.

Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection.

PCR-based preemptive therapy with ganciclovir has been shown to reduce the incidence of CMV disease after BMT. Failures of this treatment strategy are CMV disease and secondary non-viral infections. Eighty-six consecutive patients at high risk for CMV disease who received PCR-based preemptive therapy with ganciclovir were assessed for treatment failures and possible risk factors. Ganciclovir was initiated in 57 of 86 patients (66%). Only 28 of 86 (32%) patients received 4 or more weeks of ganciclovir. Recurrence of CMV infection after successful treatment was more frequent among recipients of a BMT from an unrelated compared to a sibling donor (P = 0.004). Three (3.5%) patients developed non-fatal early onset CMV disease and seven of 68 (10.3 %) late onset CMV disease (>100 days post transplant). Risk factors for late onset CMV disease were cGVHD (P = 0.0017) and duration of prior antiviral therapy >4 weeks (P = 0. 0073). The incidence of secondary non-viral infections was 28% with the duration of antiviral treatment being a significant risk factor for secondary bacterial (P = 0.0045) and invasive fungal infections (P = 0.006). Thus, PCR-based preemptive treatment with ganciclovir reduces early onset CMV disease, but the duration of antiviral therapy prior to day +100 is a significant risk factor for late onset CMV disease as well as secondary non-viral infections.

Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus.

The susceptibility of monocyte-derived immature dendritic cells (DC) to infection by various strains of human cytomegalovirus (HCMV) was analysed. Immature DC were generated by incubation of peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor for 7 days and were characterized by a CD1a+/CD40+/CD80+/CD86+/HLA-DR+/CD14- phenotype. Viral antigen expression and production of infectious progeny virus were analysed in infected immature DC cultures. Immature DC were 80-90 % susceptible to HCMV strains that had been propagated in endothelial cell culture, whereas the infection rate was negligible with fibroblast-adapted HCMV strains. Immature DC infection resulted in expression of viral immediate early, early and late genes. Productive infection was proven by the detection of infectious virus in single-step growth curves and in infectious centre assays. It is concluded that HCMV might interfere with the host immune reaction by permissive, lytic infection of immature DC.

Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome.

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.

Hepatocytes are permissive for human cytomegalovirus infection in human liver cell culture and In vivo.

The cytopathic potential of human cytomegalovirus (HCMV) in human liver cells was analyzed in cell culture and in tissue sections from patients with HCMV hepatitis. Liver cell cultures, consisting of hepatocytes, bile duct epithelial cells, and stromal cells were infected by various HCMV strains. Cytopathic effects, viral gene expression, and virus production were detected. Infected cell types were identified by immunocytochemical double labeling. Hepatocytes were the predominant target cells of HCMV infection in liver tissues and in cell culture. Late-stage infected cultured hepatocytes produced infectious progeny virus, and infectious virus was propagated from liver tissue specimens. HCMV infection in cultured liver cells closely resembled in vivo infection of the liver with regard to the target cell spectrum and the permissive course of infection. It is concluded that HCMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes.

A simple and rapid method for preparation of viral DNA from cell associated cytomegalovirus.

In the field of human cytomegalovirus pathogenesis there is growing interest in analyzing recent clinical isolates rather than cell culture adapted laboratory strains. However, true low passage isolates are strictly cell associated prior to cell culture adaptation and only a minor fraction of cells are infected at low passage number. Both conditions hinder the preparation of pure viral DNA. To date, genetic analyses had been carried out mostly with supernatant associated cytomegalovirus. A rapid and simple method is described for preparation of viral DNA from low passage cell associated isolates with little cytopathogenic effect. The protocol is based on a combination of Triton X-100 lysis, nuclease treatment, and subsequent phenol chloroform extraction. Cellular background was reduced significantly to enable clear detection of all viral DNA fragments in restriction fragment length analysis. The method yielded DNA which was suitable for downstream applications like cloning of viral DNA fragments or transfection of genomic viral DNA. This method may facilitate genomic analyses of pathogenic cell associated recent cytomegalovirus isolates.

Human cytomegalovirus infection of immature dendritic cells and macrophages.

A central aspect of human cytomegalovirus (HCMV) pathogenesis is the interaction of the virus with different antigen-presenting cell (APC) types of the host. In principle, a number of various cell types have the potential of antigen presentation when MHC II expression is induced by appropriate stimuli. The most potent antigen presenters are monocytes/macrophages and dendritic cells (DCs), therefore called professional APCs. Interestingly, these cells seem to be targets of productive HCMV infection. The susceptibility of the monocyte/macrophage system has been analyzed intensively during the past decade. Investigation of the role of DCs during HCMV infection, however, has begun only recently.

Human trophoblast cells are permissive to the complete replicative cycle of human cytomegalovirus.

Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.

Evaluation of Murex CMV DNA hybrid capture assay for detection and quantitation of cytomegalovirus infection in patients following allogeneic stem cell transplantation.

Murex hybrid capture DNA assay (HCS) is a solution hybridization antibody capture assay for detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. To determine whether CMV HCS is sensitive enough to initiate and monitor antiviral therapy after allogeneic stem cell transplantation (SCT), 51 consecutive SCT recipients were prospectively screened for the appearance of CMV infection by HCS, PCR, and culture assays from blood samples. Preemptive antiviral therapy was initiated after the second positive PCR result in all patients, as previously reported, and HCS was not considered for clinical decision making. A total of 417 samples were analyzed. Of these, 21 samples were found to be positive by PCR and HCS, 88 samples were PCR positive but HCS negative, and 308 were negative by both assays. Concordance of results between PCR and HCS and between HCS and blood culture was observed in 78.9 and 95.9% of the samples assayed, respectively. PCR was found to be more sensitive than HCS, and HCS was more sensitive than the blood culture assay (P < 0.0001). Four patients with symptomatic CMV infection were PCR positive prior to the onset of CMV-related symptoms, whereas HCS detected CMV DNA in three patients prior to and one at onset of CMV disease. The numbers of genomes per milliliter of blood were higher in patients with symptomatic CMV infection than in those with asymptomatic CMV infection (P = 0.06). None of the HCS-negative patients developed CMV disease. Thus, all patients with CMV disease were correctly identified by HCS; however, the lower sensitivity limit of the HCS assay may still be insufficient to allow diagnosis of CMV infection early enough to prevent CMV disease in patients following allogeneic SCT.

Quantification of replication of clinical cytomegalovirus isolates in cultured endothelial cells and fibroblasts by a focus expansion assay.

A method for quantitative analysis of the growth properties of human cytomegalovirus (HCMV) in various cell culture systems was developed. Recent HCMV isolates are, in most cases cell associated, causing only limited cytopathic effect. This renders comparative analysis of the biological properties of such isolates difficult. The focus expansion assay described in this study is based on cocultivation of infected fibroblasts with a cell type of choice, relying on cell mediated infectivity. The extent of replication of a given isolate in cell culture is quantified by determining the size of resulting infectious foci. Analysis of various clinical isolates and laboratory strains indicated that this assay is a reliable and valid method to define growth properties of HCMV in cell culture. Remarkable differences in the cytopathogenicity of these isolates in fibroblasts as well as in endothelial cells were found. The assay will be useful in studies regarding cell tropism and virulence of recent HCMV isolates and for the quick and easy phenotypic characterization of HCMV deletion mutants.