Renate Mentel

Screening of Nepalese medicinal plants for antiviral activity

In an ethnopharmacological screening, plants used in Nepalese traditional medicine were evaluated for antiviral activity. Methanolic and methanolic-aqueous extracts derived of 23 species were assayed in two in vitro viral systems, influenza virus/MDCK cells and herpes simplex virus/Vero cells. Two species, Bergenia ligulata and Nerium indicum showed the highest antiinfluenzaviral activity with 50% inhibitory dose of 10 microg/ml. Holoptelia integrifolia and N. indicum exhibited considerable antiviral activity against herpes simplex virus. None of these extracts showed cytotoxic effects. Additionally for B. ligulata and H. integrifolia partial protease inhibitory activity was estimated.

Mapping of linear epitopes on fibre knob of human adenovirus serotype 5.

Linear antigenic epitopes on the Ad5 fibre knob (FK5) were characterised with fibre- and virion-specific antisera, using 15-mer overlapping peptides covering the knob of the fibre. They were compared with epitopes on the Ad2 fibre knob (FK2) domain. The stronger reactive FK5 epitopes were represented by peptides P3 (amino acids (aa A419-L433)), P6 (aa S449-E463), P7 (aa I459-L473), P12 (aa G509-N523), P14 (aa P529-G543) and P16 (aa A549-Y563). P3 spans the B beta-strand and the left portion of the C beta-strand, P6 and P7 the D beta-strand and the adjacent parts of the CD and DE loops, P12, P14 and P16 the G, H and I beta strands and the adjacent parts of the loops, respectively. The stronger reactive epitopes on FK2 were located in P2 (aa P409-L423), P6 (aa T449-Q463), P8 (aa E469-G483), P13 (aa Q519-T533) and P16 (aa S549-K563). The positions of FK5 and FK2 derived peptides, representing epitopes, are either identical or overlapping or adjacent, as determined by amino acid sequence alignment. Antisera obtained against several longer peptides showed virus neutralising capacity, indicating neutralising epitopes in these peptides.

Adenovirus-receptor interaction with human lymphocytes

Lymphocytes play a key role in cell‐mediated immunity and are host cells for several viral and bacterial pathogens. Their importance in adenovirus (Ad) infections is not yet fully understood. The initial event, the attachment of Ad to lymphocytes and their subsets, was examined using flow cytometry. The study included analysis of stimulated T cells in binding assays with FITC‐labeled Ad fiber. The results confirm that native peripheral lymphocytes express very small amounts of Ad receptors. Stimulation with PHA and interleukin 2 induced the expression. The presence of Ad DNA as a sign of internalization in stimulated cells was demonstrated using the polymerase chain reaction. The findings suggest that lymphocytes after stimulation can turn into target cells for Ad. This is particularly important if there are indications for persistence of Ad, and in the case of immunocompromised patients severe, life‐threatening diseases can develop. J. Med. Virol. 51:252–257, 1997.

Balticols A–F, New Naphthalenone Derivatives with Antiviral Activity, from an Ascomycetous Fungus

Six new naphthalenone derivatives, balticols A-F and the known metabolite altechromone A were isolated from the AcOEt extract of the culture broth of fungal strain 222 belonging to the Ascomycota, which was found on driftwood collected at the coast of the Greifswalder Bodden, Baltic Sea, Germany. All structures were elucidated on the basis of NMR spectroscopic data and mass spectrometric analyses. The balticols were found to exhibit inhibitory activity against influenza virus A and herpes simplex virus. The most potent antiviral activity was observed for balticol E with an IC(50) value of 0.01 microg/ml against Herpes simplex virus type I.

No evidence for simian virus 40 DNA sequences in malignant non-Hodgkin lymphomas

DNA sequences coding for simian virus 40 (SV40) large T antigen have been detected at different frequencies in human non‐Hodgkins lymphomas (NHL) by PCR techniques as well as immunohistochemistry. A highly sensitive quantitative real‐time PCR specific for a sequence of SV40 large T antigen was established to test whether SV40 DNA is present in malignant lymphomas of German patients. Thirty‐three lymph node samples obtained from 27 patients with NHL and 6 patients with Hodgkins disease (HD) were tested in addition to 48 samples of peripheral blood mononuclear cells (PBMNC) from patients with NHL containing between 0.1% and >90% circulating lymphoma cells determined by PCR. Fourteen lymph nodes obtained from patients with other diseases than malignant lymphomas and 47 PBMNC samples from healthy volunteers served as controls. All samples from patients with malignant lymphomas and all controls were negative for SV40 DNA by quantitative real‐time. In contrast, EBV‐DNA could be detected in 29 of 46 DNA preparations isolated from lymph nodes (63%) and in 20 of 47 DNA preparations from PBMNC. EBV‐positive samples contained between 5 and 80,000 EBV copies per 100,000 cells. Our results do not support the hypothesis that SV40 plays a major role in the etiology of malignant lymphomas and, in addition, they exclude a clonal SV 40 infection of malignant lymphoma cells in all samples investigated.

Inhibition of cell adhesion to the virus by synthetic peptides of fiber knob of human adenovirus serotypes 2 and 3 and virus neutralisation by anti-peptide antibodies.

Abstract The fiber knob of adenovirus (Ad) causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s) several synthetic peptides derived from Ad2 and Ad3 fiber head sequences and their antisera were tested for interference with virus attachment to HeLa and FL cells and cell adhesion to viruses. The anti-peptide sera were also evaluated in ELISA and virus neutralisation test. Ad2 (of subgroup C) and Ad3 (of subgroup B) attachment was not significantly inhibited by peptides corresponding to the amino acid residues 535–554, 555–573, 562–582 of Ad2 fiber or 210–225, 267–283, 291–306 and 300–319 of Ad3 fiber. However, microplate pre-adsorbed Ad3 fiber residues 210–225 and 267–283 could bind FL and HeLa cells, and 1 mg/ml of Ad3 fiber residues 267–283 inhibited the cell adhesion to Ad3 virus to approximately 90%. This peptide may participate in the receptor binding site of Ad3 fiber. ELISA reactive anti-peptide antibodies against the homologous peptide and virus did not significantly reduce the cell adhesion to the immobilised virus or the virus attachment to cells, but in the neutralisation assay antibodies raised to Ad2 fiber residues 555–573 and 562–582 and Ad3 fiber residues 210–225 caused neutralisation of the homologous virus at serum dilutions of 1:500 and 1:32, respectively. The corresponding peptides and one further peptide of Ad2 fiber and two of Ad3 fiber seem to contain neutralisation epitopes.

Inhibition of adenovirus DNA polymerase by modified nucleoside triphosphate analogs correlate with their antiviral effects on cellular level

Abstract Adenovirus (Ad) infection results in significant morbidity and mortality in both immunocompetent and immunosuppressed hosts. There is currently no licensed chemotherapy effective in dealing with this virus infection. In this study the anti-adenoviral activity of a group of modified nucleoside analogs was investigated. The most efficient 3-fluorosubstituted nucleoside triphosphate inhibitors of Ad DNA polymerase were 3′-fluorothymidine triphosphate (IC50 0.63 μM), 2′,3′-dideoxy-3′-fluoroguanosine triphosphate (IC50 0.71 μM) and 2′,3′-dideoxy-3′-fluorouridine triphosphate (IC50 2.96 μM). The most efficient 2′,3′-dideoxynucleoside triphosphates were 2′,3′-dideoxycytidine triphosphate (ddCTP; IC50 1.0 μM), 2′,3′-dideoxyadenosine triphosphate (IC50 1.6 μM) and 2′,3′-dideoxythymidine triphosphate (IC50 1.82 μM). Kinetic studies indicate competitive inhibition of adenovirus DNA polymerase by ddCTP. These data confirm results previously obtained at the cellular level using a focus reduction assay involving Ad2-infected FL cells. Whereas the D-enantiomers 3′-fluorothymidine and 2′,3′-dideoxycytidine are potent inhibitors of adenoviral replication, the corresponding L-enantiomers exhibited no inhibitory activity.

Quantification of adenovirus particles

A physical method for quantification of the adenovirus was tested. It implies a centrifugation of the clarified or chloroform- (or freon-) treated crude virus suspension (0.5 ml) in a gentle sucrose gradient, following the analysis in a sensitive UV flow photometer and calculation of virus mass. The result (micrograms/ml) is obtained after about 1 h. The sensitivity of detection at wavelength 254 nm, 278 nm and 226 nm was compared. Virus yield of several serotypes from monolayer cultures of FL-cells was determined in a range of < 0.1 to 7 micrograms/ml. The ratio of infectious to physically complete virus (about the 770S component), the influence of freezing and thawing, storing at 4 degrees C and the effectiveness of concentration steps were also determined. There was no significant difference between the sucrose density gradient method (sedimentation rate) and the density equilibrium ultracentrifugation in a CsCl gradient.

Fluorescent focus reduction assay for the screening of antiadenoviral agents

A method for screening of antiviral compounds against adenoviruses was established. Test compounds were diluted and plated in chamber slides for tissue culture. Drug-treated, virus-infected cultures were stained with fluorescein isothiocyanate conjugated rabbit antibodies against adenovirus hexon type 2 and fluorescent cells were counted by microscopy. This assay is more sensitive than the colorimetric method and requires smaller volumes of compounds when compared with the standard method using plaque assay.

Molecular and clinical characteristics of respiratory syncytial virus infections in hospitalized children

The objective of this study was to determine the importance of respiratory syncytial virus (RSV) for hospitalization in the north east of Germany and to obtain molecular epidemiological data of the circulating strains. Using a rapid and sensitive reverse transcriptase-PCR, it was found that a quarter of pediatric respiratory disease admissions were due to RSV. Infections caused by RSV in hospitalized patients were determined over the whole year. Both RSV groups A and B were identified with a predominance of RSV A (86%) over the entire period. The analysis of the deduced amino acid sequences by direct sequencing showed that very similar RSV strains are circulating in the community.