Klaus Ritter

High Prevalence of Human Bocavirus Detected in Young Children with Severe Acute Lower Respiratory Tract Disease by Use of a Standard PCR Protocol and a Novel Real-Time PCR Protocol

ABSTRACT The human bocavirus (HBoV) was recently isolated from respiratory tract samples. Within a study collective of children with severe lower respiratory tract disease, the patients testing positive for HBoV (12.8%) had a higher rate of underlying cardiopulmonary disease. Viral loads in respiratory tract specimens varied from 102 to 1010 genome equivalents/ml.

Affinity purification of antibodies from sera using polyvinylidenedifluoride (PVDF) membranes as coupling matrices for antigens presented by autoantibodies to triosephosphate isomerase

Polyvinylidenedifluoride (PVDF) membranes covered with antigen can be used for affinity purification of antibodies from sera. These antibodies serve as an appropriate substitute for monoclonal antibodies when performing cytological and analytical experiments with the respective antigen. The usefulness of this method is demonstrated with the purification of IgM autoantibodies produced against human triosephosphate isomerase in acute Epstein-Barr virus infection. Compared with the original serum, the specific activity of the purified antibodies was up to 1400-fold higher, and the recovery of the anti-triosephosphate isomerase activity between 25 and 33%.

Generation of macaque B lymphoblastoid cell lines with simian Epstein-Barr-like viruses: transformation procedure, characterization of the cell lines and occurrence of simian foamy virus

Two simian Epstein-Barr-like viruses, a rhesus Epstein-Barr virus and Herpesvirus papio, were used to transform B cells from rhesus or cynomolgus macaques. The resulting cell lines exhibited predominantly a B lymphocyte phenotype and expressed Epstein-Barr virus antigens. The majority of B lymphoblastoid cell lines from macaques, which were seropositive for simian foamy virus, developed giant cells in culture. The cytopathic agent was identified as a foamy virus and was transmissible to human embryonal fibroblasts. Treatment of cell cultures with AZT abolished giant cell formation.

Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR

ABSTRACT The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available.

Detection of Cytomegalovirus DNA in Human Specimens by LightCycler PCR: Melting Point Analysis Is Mandatory To Detect Virus Strains with Point Mutations in the Target Sequence of the Hybridization Probes

In our recently published paper we described the application of the LightCycler technology for the detection of cytomegalovirus (CMV) DNA in human plasma and urine (1). We are now able to give further information gained since September 2000, when this PCR procedure was introduced as the standard assay for the detection of CMV DNA in our diagnostic laboratory. As of June 2001, we had tested 912 specimens using this assay. According to the described test protocol, 185 specimens (20.3%) were positive for CMV DNA. A melting point (Tm )o f 59.2°C was observed for the hybridization probes with PCR amplicons of all positive specimens. Another 12 specimens (1.3%) were negative in quantitative analysis but generated a significant peak, with a Tm of 53.1°C, in the melting point analysis. Gel electrophoresis revealed a distinct PCR product of about 250 bp with these specimens that corresponds to the targeted 254-bp amplicon. DNA sequencing of the PCR products with the decreased melting point confirmed the specific amplification of CMV DNA using these specimens as a template. The decline of the melting point is caused by a point mutation in position 630 of the CMV glycoprotein B gene (GenBank accession no. A13758), resulting in a shift from cytosine to thymidine. This strain variant is not included in the databases and causes a mismatch with position 5 of the LCRed 640-labeled acceptor fluorophore probe. However, the existence of further strain variations in the amplified region cannot be excluded. The melting point analysis allows the reliable detection of CMV strain variants harboring this point mutation. Specificity of the signal should be further confirmed by gel electrophoresis. Melting point analysis is mandatory to detect virus strain variants in the target sequence of the hybridization probes with the described LightCycler CMV PCR assay.

Cytostatic effect of gangliosides present in the membrane of macrophages

Stimulated macrophages are known to inhibit the growth of certain tumor cells. Using mouse peritoneal exudates as a source of macrophages and the mastocytoma cell line P815 as the target, the inhibition was found to depend on direct contact between the macrophages and the growing cells. Cytostatic activities were detected in extracts of macrophages as well as in membranes of macrophages bound to substances of low molecular weight. Physical and biochemical characteristics of the cytostatic activity hint toward N-acetylneuraminic acid containing glycosphingolipids (gangliosides). The different macrophage gangliosides were separated by thin-layer chromatography. All types showed cytostatic activity, but the most effective gangliosides were identified as monosialoganglioside GM1 and disialoganglioside GD3.

Low to Medium WU-Virus Titers in Young Children with Lower Respiratory Tract Infections

The WU-virus (WUV), a novel polyomavirus, has recently been recovered from respiratory tract samples. Within a study collective of children with severe lower respiratory tract disease, 3% of the patients tested WUV positive. Viral loads ranged from 5 × 102 copies/ml to 1 × 104 copies/ml. The WUV genome-positive patients did not display specific clinical or radiological characteristics to be distinguished from other respiratory tract infections.

Antibodies to adult T-cell leukemia virus (ATLV/HTLV-I) in AIDS patients and people at risk of AIDS in Germany.

A total of 2048 serum samples from Germany were examined for antibodies to adult T-cell leukemia virus (ATLV) structural polypeptides with an enzyme-linked immuno sorbent assay (ELISA) and confirmative immuno precipitation. The origin of the sera samples was: 850 samples taken for virological or protozoal diagnosis; 626 samples from male homosexuals, about 20% of whom had lymphadenopathy syndrome; 164 from hemophiliacs; 184 were from multiple transfused, mostly dialysis patients; 9 from intravenous drug abusers; 182 from suspected cases of acquired immuno deficiency syndrome (AIDS) and 33 from AIDS-patients. In none of these sera did we detect antibodies to ATLV, except in the serum of one patient who had been on hemodialysis for over 11 years. Obviously infection with ATLV or a serologically related agent is very rare in our country and an association with AIDS could not be observed.

Molecular diagnosis of Epstein-Barr virus

Molecular techniques have become an important tool in Epstein-Barr virus diagnostics. In recent years novel real-time PCR formats and in situ techniques have been developed that offer increased time efficiency, reduced cross-contamination, high reproducibility, high sensitivity and allow determination of viral loads. In the near future, widespread clinical application of these diagnostic modalities may provide increased knowledge of the pathophysiology of Epstein-Barr virus infection and may optimize treatment or even explore novel Epstein-Barr virus-related diseases. The monitoring of Epstein-Barr virus viral loads in different tissue compartments is currently being effectively used to assess the treatment response or prognosis in patients with oncological diseases or immunosuppression. This may also gain increasing importance in the nononcological environment. However, the general acceptance of molecular techniques will largely depend on improved standardization.

Haemolysis in hepatitis A virus infections coinciding with the occurrence of autoantibodies against triosephosphate isomerase and the reactivation of latent persistent Epstein-Barr virus infection.

Haemolysis has been observed frequently as a complication of acute hepatitis A virus (HAV) infection. However, the pathogenic mechanism has not been elucidated completely. In individual cases the detection of anti‐erythrocyte antibodies of unknown specificity was described. The raised serum IgM fraction was shown to consist partially of autoantibodies. Previously, we detected autoantibodies of immunoglobulin class M directed against triosephosphate isomerase (IgM anti‐TPI) in patients with infectious mononucleosis. These autoantibodies are able to induce haemolysis.