Anti-hepatitis E virus markers in hemodialysis patients.
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Antibodies, Viral↗
Biomedical subjects
G Jahn
Publications and source records attributed to G Jahn.
Monitoring of CMV infection: a comparison of PCR from whole blood, plasma-PCR, pp65-antigenemia and virus culture in patients after bone marrow transplantation.
Rapid, specific and sensitive methods are essential for early detection of CMV infection in patients after marrow transplantation. Thus, in a prospective study, PCR from whole blood, plasma-PCR, pp65-antigenemia and virus culture were used and compared in 20 consecutive marrow transplant recipients for early diagnosis of CMV infection and monitoring of antiviral therapy. Moreover, semi-quantification of the viral load in blood samples by PCR from whole blood or plasma and pp65-antigenemia was performed. Fifteen out of 20 patients were found to be CMV positive by PCR from whole blood, plasma-PCR and pp65-antigenemia, whereas only 9/20 developed culture-proven viremia and/or viruria. PCR from whole blood, plasma-PCR and pp65-antigenemia revealed identical results in 96 and discordant results in 13 of 109 blood samples (P < 0.01). The efficacy of antiviral therapy was monitored by semi-quantitative scoring of pp65-antigen-positive leukocytes and/or CMV-DNA levels in blood and plasma samples. Twelve of 13 patients were found to be CMV negative by all methods after 14 days of ganciclovir therapy. A good correlation of the semi-quantitative evaluation of the three assays was demonstrated. Thus, all three highly sensitive assays seem to be suitable for screening patients at risk for CMV infection and monitoring the efficacy of antiviral therapy.
Adolescent↗
Polymerase chain reaction monitoring reduces the incidence of cytomegalovirus disease and the duration and side effects of antiviral therapy after bone marrow transplantation.
Culture-based preemptive therapy with ganciclovir was shown to reduce the incidence of cytomegalovirus (CMV) disease after bone marrow transplantation (BMT). Culture techniques did not detect CMV in 12% to 13% of patients before the onset of CMV disease. In a prospective study, 71 patients either received preemptive therapy based on polymerase chain reaction (PCR) technique (37 patients) or on culture assays (34 patients). In both groups, therapy was continued until clinical signs disappeared and PCR negativity was documented. Twenty-two patients in the PCR group and 15 patients in the culture group received antiviral therapy. PCR allowed detection of the virus (median day, +32 v day +49; P = .006) and introduction of antiviral therapy (median day, +44 v day +54; P = .02) earlier than did culture assays. The incidences of CMV disease (2 of 37 v 8 of 34 in PCR group v culture group, respectively; P = .02) and CMV-associated mortality (0 of 37 v 5 of 34 in PCR group v culture group, respectively; P = .02) were decreased, and the duration of ganciclovir therapy (P < .001) was shorter in the PCR-monitored group. Incidence and median duration of severe neutropenia (less than 500/microL) were lower in the PCR group (two v eight episodes, P = .02; median duration, 1.5 v 5 days, P = .04), as was the incidence of nonviral infections during/after antiviral therapy (2 of 37 v 9 of 34; P = .012). Thus, preemptive therapy based on more sensitive detection methods such as the PCR assay reduces the incidence of CMV disease and CMV-related mortality. Additionally, stopping and withholding antiviral therapy in a PCR-negative patient is safe and allows reduction of the duration and side effects of antiviral therapy.
Adolescent↗
Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues.
High titre replication of human cytomegalovirus (HCMV) in cell culture is restricted to primary human fibroblasts. During acute infection in vivo, HCMV nucleic acids and antigens have been found in various organs. Using only morphological criteria, inconsistent data have been reported about the cell types that can be infected by HCMV. In particular, the role of fibroblasts in organ infections has remained unclear. To define accurately the target cells of HCMV in vivo, tissue sections from lung and gastrointestinal tract of patients suffering from acute HCMV infection were investigated using immunohistochemical double-labelling analyses. Monoclonal antibodies with defined specificity against immediate early (IE), early (E) and late (L) viral antigens and antibodies directed against cell marker proteins were employed to identify infected cells. The results demonstrated that a broad spectrum of cells was infected by HCMV in vivo. Consistent with their susceptibility in culture, fibroblasts formed a major population of HCMV-infected cells. In contrast, haemopoietic cells were only infrequently stained with virus-specific antibodies. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells and macrophages appeared to be permissive for HCMV replication. Contrary to this, polymorphonuclear cells showed only IE gene expression, indicating that these cells were abortively infected. The analysis of the distribution of infected cells in tissue supported the hypothesis that endothelial cells and monocytes/macrophages may play a crucial role in the haematogenous spread of HCMV; in contrast, fibroblasts, smooth muscle cells and epithelial cells may form the cell populations important for the multiplication and spread of the virus in infected tissues.
Animals↗
The DNA-binding protein pUL57 of human cytomegalovirus is a major target antigen for the immunoglobulin M antibody response during acute infection.
A small polypeptide from pUL57 of human cytomegalovirus was identified as a major target for the immunoglobulin M antibody response. This antigen seems to be superior to antigenic fragments from pp150 and p52 in the identification of sera from acutely infected patients. It may therefore represent an essential antigen for recombinant immunoglobulin M antibody tests for human cytomegalovirus.
Acute Disease↗
The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture.
The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts. As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus. This efficient transport is mediated by unique nuclear localization signals. To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter. The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation. The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses. The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies. Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus. Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts. Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed. However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the late gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses. In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion-associated kinases in RVAd65 compared with wild-type virus. We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts.
Base Sequence↗
Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport.
Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.
Amino Acid Sequence↗
Comparative immunoblot analysis with ten different, partially overlapping recombinant fusion proteins derived from five different cytomegalovirus proteins.
Ten fusion proteins derived from five various CMV encoded proteins were used for the detection of specific antibody response by immunoblot technique in sera from renal transplant recipients. The fusion proteins were derived from the following CMV specific proteins: the assembly protein ppUL80a with an apparent molecular weight of 38 kD, ppUL99 (MW of 28 kD), the basic phosphoprotein ppUL32 (MW of 150 kD), the lower matrix protein ppUL83 (65 kD), and the DNA binding protein ppUL44 (MW of 52 kD). Some fragments overlapped and responses to these recombinant proteins were compared. Antibody responses appeared to be predominately directed at fusion proteins of ppUL32, the basic phosphoprotein and at ppUL44, the major DNA binding protein. Some differences were found in the responses to these partly overlapping fusion proteins. In addition, an IgM response mainly present in primary infections to fusion proteins XP1 and G2, again representing ppUL32 and ppUL44, was found.
Antibodies, Viral↗
Reduced levels of IE2 gene expression and shutdown of early and late viral genes during latent infection of the glioblastoma cell line U138-MG with selectable recombinants of human cytomegalovirus.
To establish stable culture conditions which support persistence of the human cytomegalovirus (HCMV) genome in a latent state, the expression of the bacterial neomycin phosphotransferase (neo) from HCMV recombinants was used for selection. Different cell lines were infected with HCMV recombinants. The human glioblastoma line U138-MG was rendered resistant to G418 and retained the viral genome. More than 90% of the cells expressed the viral IE1 protein of 72 kDa for a culture period of 18 months. Many fewer cells expressed IE2-encoded proteins. No late gene expression or infectious virus was detectable. IE2 gene expression in latently infected cells appeared to be restricted at the level of RNA accumulation. Treatment with TPA or retinoic acid led to enhanced expression of the IE2 gene and the early genes encoding pp65 (UL83) and p52 (UL44). Superinfection with wild-type HCMV led to replication of neo-recombinant virus, indicating that replication-competent virus had been retained in latently infected U138-MG and that the cells had kept their permissive phenotype. Latent HCMV infection in U138-MG cells provides a useful model system for studying the role of particular viral and cellular genes in latent and permissive infections.
Cytomegalovirus↗
Comparative analysis of intrathecal antibody synthesis and DNA amplification for the diagnosis of cytomegalovirus infection of the central nervous system in AIDS patients.
We evaluated 49 paired cerebrospinal fluid (CSF) and serum samples of 35 patients infected with the human immunodeficiency virus type 1 (HIV-1) for laboratory evidence of cytomegalovirus (CMV) infection. The patients were grouped according to clinical criteria as probable CMV encephalitis/polyradiculomyelitis, CMV retinitis, cerebral toxoplasmosis, progressive multifocal leukoencephalopathy, HIV-1-related cognitive/motor complex, HIV-1-associated myelopathy, and other neurological diseases. Paired CSF and serum samples were analysed for CMV deoxyribonucleic acid (DNA) by polymerase chain reaction (PCR), quantitative intrathecal synthesis of immunoglobulin G (IgG) antibodies specific for recombinant phosphoprotein 150 (pp150) of CMV and CMV-specific serum IgM. Intrathecal synthesis of pp150-specific IgG was detected in 26% of patients (9/35), serum IgM was found in 23% of patients (8/35), and PCR of CSF was positive in 11% of patients (4/35). Detection of CMV-specific DNA in CSF preceded the intrathecal antibody synthesis in three patients for whom serial samples were available. PCR results of the CSF became negative in one patient with CMV polyradiculomyelitis after successful therapy with 9-[2-hydroxy-1-(hydroxymethyl) ethoxymethyl] guanine (DHPG). PCR has a higher diagnostic specificity in the acute phase of CMV infection than intrathecal antibody synthesis. The serum IgM response to CMV cannot be used to monitor a compartmentalized immune response in the central nervous system while an intrathecal immune response seems to be associated with recovery either spontaneously or as a result of treatment.
AIDS-Related Opportunistic Infections↗
Triple retinal infection with human immunodeficiency virus type 1, cytomegalovirus, and herpes simplex virus type 1. Light and electron microscopy, immunohistochemistry, and in situ hybridization.
PURPOSE: This report describes the histopathologic and virologic findings of the retina from a 55-year-old bisexual patient with the acquired immune deficiency syndrome (AIDS), who had concurrent human immunodeficiency virus type 1 (HIV-1), cytomegalovirus (CMV), and herpes simplex virus type 1 (HSV-1) retinitis, and was treated with ganciclovir. METHODS: The eyes were obtained at autopsy and processed for light microscopy and transmission electron microscopy. Immunohistochemical stains for HSV-1, CMV, HIV-1, varicella zoster virus, and glial fibrillary acidic protein were carried out using the peroxidase-antiperoxidase and streptavidin-biotin-alkaline phosphatase techniques. For in situ hybridization, a radiolabeled CMV DNA probe (Eco-RI-Y fragment of strain AD 169) was used. RESULTS: Results of histopathologic examination showed a full-thickness necrotizing retinitis with cytomegalic and herpes viral intranuclear inclusions in cells of the neurosensory retina, retinal vascular endothelium, and the retinal pigment epithelium. Some areas of the retina were replaced by glial tissue. The choroid contained only a few chronic inflammatory cells. Immunoperoxidase studies disclosed CMV antigens diffusely distributed throughout all layers of the retina and the retinal pigment epithelium. Herpes simplex virus type 1 antigens were present in retinal cells and the retinal vascular endothelium. Human immunodeficiency virus type 1 antigens were found in mononuclear cells in all layers of the sensory retina. Dual infections with HIV-1 and CMV of individual multinucleated giant cells of glial origin were demonstrated immunohistochemically. Transmission electron microscopy showed herpes viral particles in the vascular endothelium of the retinal vessels and the choriocapillaris. Human immunodeficiency virus particles were identified in the endothelium of the choriocapillaris. CONCLUSIONS: The possibility of multiple viral infections of the retina, mimicking classic CMV retinitis, should be considered in the clinical and histologic differential diagnosis of necrotizing retinitis in patients with AIDS.
Antibodies, Viral↗
[Injury to the surgeon in treatment of an HIV infected patient].
The number of HIV-infected female patients undergoing surgery is increasing. Injuries of the surgeon can never be completely excluded. The risk of HIV infection via a needle puncture lesion is estimated to be approximately 0.4%. Besides the well-known protective measures, careful wound disinfection followed by HIV tests for a period of up to six month after the lesion are indicated. It is indeed high time to consider the patient HIV test as a matter of course, not least because it is our duty to be concerned also about the health of our co-workers and staff directly engaged in patient care.
Adult↗
Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens.
DNA fragments from eight different reading frames of human cytomegalovirus (HCMV) were generated by PCR and subsequently cloned and expressed in Escherichia coli in fusion with glutathione S-transferase. The recombinant viral antigens were evaluated in immunoblot analyses. The most reactive antigens were purified and further evaluated in ELISAs. For this, sera from healthy blood donors and immunocompetent individuals with acute HCMV infection, and follow-up sera from transplant recipients with acute primary HCMV infection were used. The results of our experiments indicate that only three particular recombinant polypeptides from two viral proteins are necessary for serodiagnosis. While a fragment covering amino acids (aa) 495 to 691 of pp150 (150/1) was the most suitable antigen for the identification of infected individuals in general, immunoglobulin M antibodies against the C-terminal parts of pp150 (aa 862 to 1048; 150/7) and p52 (aa 297 to 433; 52/3) proved to be excellent serological markers to monitor acute HCMV infection. The selected recombinant antigens enable the improvement of serodiagnosis of HCMV-related diseases, especially during the early stages of infection.
Antibodies, Viral↗
[Cytomegaloviruses--clinical aspects and therapy].
Human cytomegalovirus (HCMV) is an important pathogen for the fetus, recipients of solid organ transplants, bone marrow allograft patients, individuals infected with human immunodeficiency virus and other immunosuppressed patients. The clinical features of congenital cytomegalovirus infection as well as HCMV infection and HCMV disease in immunosuppressed transplant recipients are described. Diagnostic methods for HCMV monitoring are discussed from a clinical perspective. Antivirals as Ganciclovir and Foscarnet are used for induction and maintenance regimes for the treatment of HCMV-associated retinitis, pneumonitis, hepatitis, gastrointestinal involvement and neurological disorders. Drug resistance both to Ganciclovir and Foscarnet of HCMV strains isolated from immunosuppressed patients has already been reported. The development of rapid diagnostic tools for the detection of HCMV drug resistance is urgently required.
AIDS-Related Opportunistic Infections↗
[Acute cytomegalovirus gastritis after kidney transplantation. Its diagnosis by the polymerase chain reaction, antigenemia assay and immunohistochemistry].
To diagnose possible cytomegalovirus (CMV) infection in a 64-year-old man after renal transplantation, polymerase chain reaction (PCR), pp65 antigenaemia assay (pAA) and virus isolation in cell culture were routinely performed on a weekly basis. The PCR obtained virus DNA in peripheral blood lymphocytes for the first time in the fifth week. Two weeks later the patient complained of feeling unwell with abdominal pain and vomiting on eating. Two days later he developed a fever up to 38 degrees C and nocturnal sweats. Gastroscopy revealed marked antral gastritis which histologically showed typical cytomegalic "owl-eye" cells. The pAA was clearly positive and the cell culture started in the fifth week now showed a cytopathogenic effect. CMV gastritic having been diagnosed treatment consisted of 175 mg ganciclovir intravenously twice daily for 10 days. He became symptom-free after two days. The only side effects were thrombocytopenia down to 67,000/microliters and a rise in transaminase activities, changes which regressed later. To ensure early diagnosis and treatment of any CMV infection, specific virus diagnostic tests should be routinely undertaken after transplantation of organs from CMV-positive persons into CMV-negative patients.
Acute Disease↗
Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene.
Studies on the biology and function of human cytomegalovirus (HCMV) genes have been hampered by the limited number of viral mutants available for genetic analyses. We have developed a simple procedure to generate and enrich for HCMV recombinants. By inserting the bacterial neo gene, encoding neomycin/kanamycin phosphotransferase, into the large HCMV DNA genome using homologous recombination, selectable mutants of this complex herpesvirus were isolated for the first time. The synthesis of Neo from the viral genome was used to effectively enrich for recombinant viruses (re-viruses) in permissive culture cells grown in the presence of Geneticin (G418). A quick assay for Neo activity in infected cells, based on phosphorylation of kanamycin (Km), was used to easily identify viral recombinants in the process of screening and isolation. This procedure, not used previously to identify re-viruses, proved to be very useful for screening of large numbers of HCMV recombinants. Analysis of re-virus by Southern blotting revealed that the insertion of the marker gene had resulted in the expected deletion of the open reading frames, TRL 13/14 and UL 1-5, of HCMV. Re-virus was stable and showed no differences in growth kinetics as compared to wild-type (wt) virus. The insertion of a selectable marker gene into the HCMV genome and identification of viral recombinants by the Km phosphorylation assay, as presented here, provides the rationale for effective generation, enrichment and stable propagation of HCMV mutants.
Blotting, Southern↗
Inhibition of HIV-1 replication by a high-copy-number vector expressing antisense RNA for reverse transcriptase.
We report the construction of a high-copy-number (hcn) expression vector for human cells. Amplification of this vector occurs due to the presence of an element derived from the murine DNA encoding ribosomal RNA (rDNA). HIV-1 replication in Jurkat T lymphocytes is nearly abolished when antisense RNA directed against the gene encoding reverse transcriptase is expressed from this hcn vector. The replication of the virus is only slightly reduced by the plasmid control version lacking the murine amplification-promoting element. This kind of hcn vector may represent an important improvement for the genetic engineering of eukaryotic cells and may also provide some ideas for the future gene therapy of some human diseases.
Culture Media, Conditioned↗
Analysis of proteins encoded by IE regions 1 and 2 of human cytomegalovirus using monoclonal antibodies generated against recombinant antigens.
The genomic region of human cytomegalovirus (HCMV) encoding the major immediate-early (IE) proteins was cloned as overlapping fragments into a prokaryotic expression vector. Three recombinant polypeptides were used as antigens to generate monoclonal antibodies specifically reactive with the proteins encoded by IE region 1 and IE region 2. At least 10 different antigenic regions were identified on the IE proteins of HCMV. One monoclonal generated against an IE-2 polypeptide of 156 amino acids (termed SMX) was found to react with a viral pentapeptide, which was also a constituent of the beta-chain of human HLA-DR. This peptide was contained in the 40-kDa (p40) protein encoded by IE region 2. This protein appeared to be an abundant product of the major IE region in infected cells at late times after infection. The putative translation initiation site for p40 was mapped to position 3348 of the IE2 sequence using monoclonal antibody SMX. It is therefore proposed that p40 consists of amino acids 242-580 encoded by the IE2 gene of HCMV (strain AD169). Neither this late protein nor any other of the IE1/IE2-encoded proteins was detectable in extracellular virions.
Amino Acid Sequence↗