Angelika Vallbracht

Polymerase chain reaction to evaluate antiviral therapy for cytomegalovirus disease

A sensitive and specific method to monitor suppression of cytomegalovirus (CMV) replication is essential in patients treated with ganciclovir after allogeneic bone-marrow transplantation. In this study, antiviral therapy of eighteen episodes of symptomatic CMV infection in 15 such patients were followed up clinically and by virus culture and polymerase chain reaction (PCR). Clinical improvement, culture, and PCR were assessed for their ability to predict the efficacy of ganciclovir therapy in each patient. In eleven successfully treated episodes of CMV disease (disappearance of symptoms and improvement in biochemical variables) clinical improvement was associated with an effective suppression of virus replication as shown by negative culture and PCR assays of blood and urine specimens obtained after antiviral therapy. 1 patient who did not improve clinically when receiving antiviral therapy remained both culture positive and PCR positive for CMV. 6 patients with early relapse of CMV disease or who died after an initial clinical improvement were PCR positive but culture negative after termination of therapy. Demonstration of CMV in blood and urine by PCR after stopping antiviral therapy (even when culture is negative) points to incomplete suppression of virus replication. The findings show that PCR is a better predictor of the efficacy of antiviral therapy than are culture or clinical assessment.

Correlation of pathogenicity and gene constellation of influenza A viruses. II. Highly neurovirulent recombinants derived from non-neurovirulent or weakly neurovirulent parent virus strains.

Abstract Mixed infections with various nonneurovirulent or weakly neurovirulent influenza A strains yielded recombinants that were highly neurovirulent for mice. A correlation was detected between gene constellation and neurovirulence of these recombinants. For the recombination pair FPV/A2-England the Pol 1 and Ptra genes had to be derived from the human strain; while the HA and/or M gene has to be from FPV in order to obtain highly neurovirulent recombinants. For the FPV/PR8 pair only the Pol 1 gene needs to be derived from PR8 while the HA gene had to be from FPV in order to get highly neurovirulent recombinants. The derivation of the other genes does not appear to be important in this respect. Recombinants between FPV and the A2-Singapore influenza strain do not exhibit significant neurovirulence suggesting that at least one parent strain should be adapted to grow in mice in order to obtain neurovirulent recombinants. In addition, there is a correlation between pneumovirulence and growth in mouse kidney cells, but neurovirulence for mice and pathogenicity for chickens was not correlated. The data presented here demonstrate in principle the possiblity of an increase in pathogenicity in recombinants derived from less or nonpathogenic parent viruses.

Hepatitis A-virus in cell culture. V. Neutralizing antibodies against hepatitis A-virus

A test system for the detection of neutralizing antibodies against hepatitis A-virus (anti-HAV-Nt) is presented. The anti-HAV-Nt assay is performed with Frhk-4/R cells and the hepatitis A virus (HAV) strain GBM/Frhk-4/R which has been adapted to these cells. Non-neutralized HAV is demonstrated 14 days after infection of Frhk-4/R cells using a radio-immunoassay for detecting newly grown HAV.The influence of differing amounts of HAV on the anti-HAV-Nt titre and the effect of variations in incubation time of virus-serum mixtures are described. The time course of anti-HAV-Nt is shown in sera from a hepatitis-A patient which were taken at different stages of the disease. Anti-HAV-Nt is compared with anti-HAV and anti-HAV-IgM. It is shown that anti-HAV-Nt correlates closely with anti-HAV and separated anti-HAV-IgG, but only slightly with anti-HAV-IgM.The test system presented makes possible the demonstration of neutralizing antibodies against HAV in gamma-globulin preparations and during vaccination studies.

Recombination of influenza a strains with fowl plague virus can change pneumotropism for mice to a generalized infection with involvement of the central nervous system

Abstract Recombination between fowl plague virus FPV/Rostock (Hav1N1), an influenza A strain which produces a generalized fatal infection with involvement of the central nervous system in chickens, and mouse lung-adapted human strains England/1/61 (H2N2) or PR/8/34 (H0N1 results in recombinants which produce a generalized infection in suckling mice with involvement of the central nervous system. A combination of the hemagglutinin and M protein from FPV/Rostock (Hav1N1) with certain polymerase proteins from the mouse lung-adapted strains produces generalized infections in mice. Infectious virus was present in lung, brain, and blood even after intranasal infection. A correlation between the ability to induce a generalized infection of suckling mice in vivo and the replication in mouse embryo fibroblasts in vitro could be demonstrated.

Influenza Virus: Appearance of High Mouse-Neurovirulent Recombinants

Recombinants from two influenza A strains that lacked mouse neurovirulence were tested, along with their parent strains, for mouse neurovirulence and for the ability to propagate in dissociated mouse embryo brain cells. The parents used were (i) strain A/Rostock/34 (FPV) (Hav1N1), with a high chicken neurovirulence, and (ii) the mouse-lung-adapted human strain Engl/1/61 (H2N2), lacking neurovirulence. In some of the recombinants high mouse neurovirulence could be detected after intracerebral inoculation of low virus doses. There was neither a correlation between surface antigen and neurovirulence nor between neurovirulence and mouse lung virulence in our system, although neurovirulence was only found in strains with Hav1 hemagglutinin. There was an association between replication in mouse embryo brain cells in culture and high mouse neurovirulence.

Interferon-γ secretion by in vivo activated cytotoxic T lymphocytes from the blood and cerebrospinal fluid during mumps meningitis ☆

Functional studies of cerebrospinal fluid T lymphocytes during acute viral infections of the nervous system are rare. Recently, we had the opportunity to investigate the requirements for interferon-gamma (IFN-gamma) production of human in vivo activated (primary) cytotoxic T lymphocytes (CTL) generated during acute viral meningitis. Two HLA-B7-restricted, CD4-, CD8+ CTL clones from cerebrospinal fluid of one patient with mumps meningitis were studied. Although lytic activity was restricted by HLA-B7, the clones produced similar amounts of IFN-gamma when stimulated with HLA-matched and mismatched mumps virus-infected target cells. In addition, peripheral blood mononuclear cells of infected patients secreted significant amounts of IFN-gamma when incubated with autologous or allogeneic (HLA-A/B-mismatched) mumps virus-infected target cells. T cells capable of lytic activity and IFN-gamma secretion could only be isolated from venous blood during the initial phase of the infection. We suggest that the ability of human in vivo activated CTL to secrete INF-gamma early during the course of inflammation and in a HLA-unrestricted fashion is important for the elimination of viruses invading the central nervous system.

Hybridization techniques provide improved sensitivity for HCMV detection and allow quantitation of the virus in clinical samples

Hybridization techniques (slot-blot and in-situ hybridization assays) and immunostaining using murine monoclonal antibodies directed against different proteins of the human cytomegalovirus (HCMV) were compared for their sensitivity and specificity for detection of HCMV. A model system with HCMV infected human embryonic lung fibroblasts and lung biopsy specimens obtained from patients with culture positive HCMV interstitial pneumonia were used for evaluation of these techniques. The hybridization techniques were found to provide an improved sensitivity compared to immunostaining. Additionally a good correlation was found between the virus dose determined by TCID50 and the amount of viral DNA detected by slot-blot hybridization and by the number of autoradiographic silver grains per 100 cells per 2 weeks exposure time detected in the infected fibroblasts by in-situ hybridization. Thus, at least in the model system quantification of the virus was achieved by hybridization assays.

Influenza Virus: Association of Mouse-Lung Virulence with Plaque Formation in Mouse Kidney Cells

In genetic recombination experiments with the mouse-lung-adapted human influenza A/Engl/1/61 (H2N2) and an avian influenza strain A/Rostock/34 (FPV) (Hav1N1) which is avirulent for the mouse lung, recombinants in which hemagglutinin and neuraminidase were either segregated (Hav1N2; H2N1) or not segregated (Hav1N1) were selected. The recombinants were studied for mouse-lung virulence and their ability to propagate in mouse kidney cells, mouse embryo fibroblasts, chick embryo kidney cells and chick embryo fibroblasts. An association between plaque formation in mouse kidney cells and mouse-lung virulence was found.

Frequent GvHR-Like Syndromes Following Syngeneic Bone Marrow Transplantation Suggest Inappropriately Controlled Autoreactivity

Development of graft versus host disease is the rule in major histocompatibility complex mismatched bone marrow transplants, but with variable incidence it is also observed in recipients of HLA-identical grafts (1) and even reported to occur in rare cases after syngeneic or autologous bone marrow transplant (BMT) (2), suggesting that differences in histocompatibility antigens are not an absolute requirement for the induction of GvHD.