Immune response of the long-tailed field mouse (Apodemus sylvaticus) to tick-borne encephalitis virus infection.

The immune response following infection with a virulent strain of Central European encephalitis (CEE) virus in a natural host, long-tailed field mouse (Apodemus sylvaticus L.) and white laboratory-bread ICR mouse, was compared. Viraemia was demonstrated in ICR mice after intraperitoneal infection with a dose of 10(5) LD50/0.5 ml. The virus titres were high in the spleen and, particularly, in the brain. In A. sylvaticus the virus was detected in the blood and spleen, but not in the brain. CEE virus multiplied in peritoneal macrophages from ICR mice, but not from A. sylvaticus. The infection induced a strong interferon response in both hosts. The natural killer (NK) cell activity increase was twice as high in A. sylvaticus compared to ICR mice. The neutralization antibodies appeared sooner in A. sylvaticus and reached higher titres in the early phases of infection.

Isolation of TBE virus from the tick Ixodes hexagonus.

Tick-borne encephalitis (TBE) virus was isolated from a sample of Ixodes hexagonus ticks collected from the hair of the western European hedgehog (Erinaceus europaeus) which had been captured on the edge of a new housing estate in Ceské Budejovice at the end of September 1986. This was the first isolation of TBE virus from this vector, supporting the previous experimental results. The virus was identified in immunofluorescent and plaquereduction neutralization tests.

Rapid serological diagnosis of tick-borne encephalitis by means of enzyme immunoassay.

A system of IgM-capture EIA made up from Czechoslovak immunopreparations (SEVAC) was developed for a rapid serological diagnosis of tick-borne encephalitis (TBE). The method was tested on clinical material. The total IgM antibody titres were detected using pig antiserum and the selection of specific IgM antibodies was made with TBE antigen with following indirect way of detection. The antibody analysis made by means of this method is sensitive and fully conforms to the clinical picture of the disease.

Interaction of tick/borne encephalitis virus with mouse peritoneal macrophages. The effect of antiviral antibody and lectin.

The interaction between tick-borne encephalitis (TBE) virus strain Hypr and mouse peritoneal macrophages was followed in vitro. Macrophages from juvenile mice (8-11 days old) were more permissive for virus infection than macrophages from adults (25-30 days old). Anti-TBE antibody in the subneutralizing dose increased the number of infected macrophages as well as virus release into the culture medium. Concanavalin A (con A), which bound to the virus as well as to the surface of macrophages, increased the uptake of the virus, but it neither enhanced the number of infected cells nor increased the virus release into medium. Antibody and lectin can modify the interaction between TBE virus and macrophages. Nevertheless, the Fc receptor-mediated endocytosis seems to be a necessary prerequisite for enhancing the effect of a ligand.

Monoclonal antibodies to tick-borne encephalitis (TBE) virus: their use for differentiation of the TBE complex viruses.

Monoclonal antibodies (MoAbs) to Central European tick-borne encephalitis virus (strain Hypr) were used for differentiation of eight viruses of the TBE complex by indirect immunofluorescence. MoAb 11/B3 (in Western blot recognizing 52 and 70 kD polypeptides) reacted with five out of the eight TBE complex viruses, MoAb 13/E5 (anti-52 kD protein) reacted with the western or eastern subtype of TBE virus only, while MoAb 12/G4 (anti-70 kD protein) distinguished the western subtype of TBE virus from the rest of the TBE complex. These three MoAbs were able to differentiate the virulent strain Hypr from attenuated strains Skalica and Hy-HK-18-"3". MoAb 2/10C (anti-56 and 70 kD proteins) which reacted with all viruses of the TBE complex, recognized both virulent and attenuated strains of TBE virus.

Aerobic resistance of Trichomonas vaginalis to metronidazole induced in vitro.

Aerobic resistance of Trichomonas vaginalis to metronidazole was induced in vitro by anaerobic cultivation of drug-susceptible trichomonads with low concentrations of the drug (2-3 micrograms/ml) for 50 days. Minimal lethal concentrations (MLC) for metronidazole of the resistant derivatives were high in aerobic susceptibility assays (MLC = 216-261.5 micrograms/ml) but low in anaerobic assays (MLC = 4.2-6.3 micrograms/ml), surpassing MLC values of their parent strain approximately 50-fold and 3-fold under aerobiosis and anaerobiosis, respectively. Sensitivity to metronidazole under anaerobic conditions and activity of the hydrogenosomal enzyme pyruvate: ferredoxin oxidoreductase indicated that the resistance was of the aerobic type. Dependence of the resistance manifestation on O2 was further confirmed by susceptibility assays in vitro performed in defined gas mixtures of different oxygen content (1-20%). Five percent concentration of O2 proved to be the threshold required for resistance demonstration and the MLC values further increased with increasing O2 concentrations. The in vitro-induced resistance was also demonstrated in vivo by subcutaneous mouse assay. The dose of metronidazole needed to cure 50% of infected mice (DC50) was 223 mg/kg x 3 for resistant derivative MR-3a but 6.6 mg/kg x 3 only for its drug-susceptible parent strain. The metronidazole-resistant strains developed in this study correspond by their properties to drug-resistant T. vaginalis strains isolated from patients refractory to treatment, and promise to be a useful tool in the study of 5-nitroimidazole aerobic resistance.

Analysis of tick-borne encephalitis virus antigens by monoclonal antibodies.

Monoclonal antibodies (MAbs) against Central European tick-borne encephalitic virus, strain Hypr, were used for determination and characterization of viral antigens of infected PS cells. The MAbs reacted in immunoblotting with flavivirus glycoprotein E (56 kD), and nonstructural protein NS3 (70 kD). According to enzyme immunoassay with infected cells, NS3 antigen is expressed in the plasmalemma.

[Antigens for the diagnosis of tick-borne encephalitis using the ELISA technique]. / Antigen pro diagnostiku klístové encefalitidy technikou ELISA.

From the brains of suckling mice infected with the virus of tickborne encephalitis, strain Hypr, the authors concentrated by precipitation with polyethylene glycol - 6000 and purified by differential ultracentrifuging on a linear saccharose gradient the virion antigen KEV-II for assessment of specific human antibodies, using the indirect ELISA technique. The antigen was characterized as to haemagglutination activity, electrophoretic properties, immunoblotting with a monoclonal antibody against external layer gpE and enzyme immune analysis to assess the relative ratio of gpE in the diagnostic antigen. Using the technique of indirect ELISA with antigen KEV-II, the authors examined in 1988 and 1989 sera of 631 patients with a specificity of estimations of 99.4% (0.6% falsely positive results) and sensitivity expressed by the liminal serum dilution rate of 1:1000.

Metronidazole-resistant strains of Trichomonas vaginalis display increased susceptibility to oxygen.

Susceptibility to oxygen and properties relative to oxygen metabolism were compared in metronidazole-resistant and susceptible strains of Trichomonas vaginalis. The study involved clinical isolates displaying the aerobic type of resistance, as well as resistant strains developed in vitro, both with aerobic (MR-3) and anaerobic (MR-5, MR-100) resistance. Elevated sensitivity to oxygen of the resistant clinical isolates was observed. Progressive increase of susceptibility to oxygen also accompanied in vitro development of resistance. No correlation was found between the activity of NADH oxidase and aerobic resistance, while the in vitro derivative with fully developed anaerobic resistance (MR-100) showed about 50% decrease of NADH oxidase activity. The superoxide dismutase (SOD) activity was elevated in both resistant clinical isolates and in in vitro-developed resistant strains. The changes in levels of ferredoxin were insufficient to support ferredoxin deficiency as a cause of aerobic metronidazole resistance. Western blot analysis and electron paramagnetic resonance spectroscopy of purified hydrogenosomes showed that ferredoxin is expressed in aerobically resistant strains and has intact iron-sulfur clusters. Down-regulation of ferredoxin was demonstrated only in the late phase of development of the anaerobic resistance (MR-100). The results support a link between aerobic resistance and defective oxygen scavenging. The increased levels of intracellular oxygen, beneficial to resistant parasites when they interact with the drug, may have adverse effects on their fitness as shown by their increased sensitivity to oxidative stress.

Humoral immunity against tick-borne encephalitis virus following manifest disease and active immunization.

Humoral immunity against tick-borne encephalitis virus (TBEV) in patients with a well-documented history of naturally acquired tick-borne encephalitis (TBE) was compared with immunity resulting from vaccination in a carefully controlled immunization programme. The vaccination study was performed with a highly purified, inactivated virus particle vaccine and the immune response was followed by tracing the course of IgG antibody formation in an enzyme-linked immunosorbent assay and a neutralization assay. It was shown that this TBE vaccine induced a strong immune response. TBE IgG antibody titres measured after three vaccinations were of the same order of magnitude as those determined in patients recovered from manifest TBE.