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B Rombaut

Publications and source records attributed to B Rombaut.

At least 19 recordsLinked to original sources
PubMed2004

Mode of action of 2-furylmercury chloride, an anti-rhinovirus compound.

2-Furylmercury chloride (2-FMC), an organic mercury derivative, has been found to inhibit the replication of all tested human rhinovirus (HRV) serotypes belonging to the antiviral group B and a limited number of HRV serotypes belonging to the antiviral group A. The mechanism of action of 2-FMC was tested against HRV-2 (antiviral group B, minor receptor group), and compared with an antiviral compound for which the viral target was already determined (enviroxime). 2-FMC was found to bind reversibly to virus particles. However, time-dependent plaque reduction assays revealed that 2-FMC did not interfere with early events of HRV-2 replication. Using a quantitative RT-PCR ELISA assay, we were able to prove that 2-FMC inhibits the synthesis of viral RNA. However, the mode of action of 2-FMC is not identical to that of enviroxime, another inhibitor of viral RNA synthesis. Time-of-addition and time-of-withdrawal experiments demonstrated that 2-FMC acted during a broader time interval than enviroxime.

Antiviral Agents↗
PubMed2003

Quantitative RT-PCR ELISA to determine the amount and ratio of positive- and negative strand viral RNA synthesis and the effect of guanidine in poliovirus infected cells.

Quantitative reverse transcription-polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR ELISA) is the method of choice to study positive- and negative strand viral RNA synthesis during poliovirus replication. In comparison with other methods used for this purpose, it fulfils all necessary requirements to accurately determine RNA of different polarity. It combines high specificity, high sensitivity, safety, speed, and the ability to perform quantitative analysis. The enterovirus specific RT-PCR ELISA method described in this work, was used to determine quantitatively the amount of de novo poliovirus positive- and negative strand RNA synthesis at different time-points in the viral replication cycle, both in presence and absence of the viral RNA synthesis inhibitor guanidine hydrochloride.

Biotin↗
PubMed2002

Screening and optimisation of an ELISA method for the quantitative detection of enterovirus specific RT-PCR products by means of a two-level experimental design.

In a previous paper, optimal reaction conditions were determined for the RT-PCR part of a quantitative enterovirus specific RT-PCR ELISA method (J. Pharm. Biomed. Anal., 25 (2001) 131-142). In order to obtain a detection limit as low as possible, the ELISA part of the procedure was optimised as well. This was done by investigating the influence of seven factors at three levels in a multivariate approach. A reflected two-level screening design, derived from a Plackett-Burman design, was used. Optimal reaction conditions were established by calculation and by evaluation of the effects of the factors on the measured absorbance of the ELISA detection. Under these conditions, the linear range and detection limit of the test were determined and compared with the ELISA conditions before optimisation. The optimised RT-PCR ELISA will be used to study a possible longitudinal relationship between enteroviruses and the development of multiple sclerosis and juvenile diabetes.

Enterovirus↗
PubMed2001

Capsid and RNA stabilisation of the oral polio vaccine.

The oral polio vaccine is the least stable vaccine of the common childhood vaccines. Two different inactivation mechanisms are responsible for the thermolability of the vaccine, i.e. denaturation of the viral capsid and degradation of the viral RNA within the capsid. Pirodavir, a capsid-binding compound, inhibits the viral capsid thermodenaturation. In this paper we show that deuterium oxide is able to stabilise the viral RNA against thermodegradation and that a combination of pirodavir and deuterium oxide leads to an additive effect indicating that both stabilisers work indeed on different inactivation mechanisms. Furthermore, it is shown that the variation in thermostability of the different vaccine strains is due to the different thermostability of their capsids.

Capsid↗
PubMed2001

Screening of an enterovirus specific RT-PCR ELISA method for the quantification of enterovirus genomes in human body fluids by means of a three-level experimental design.

In order to obtain a detection limit as low as possible for a quantitative enterovirus specific RT-PCR ELISA assay, optimal reaction conditions, which give rise to the highest response, need to be determined. This was done by investigating the influence of 13 factors, selected from RT and PCR, in a multivariate approach by means of a well-balanced three-level screening design, derived from a three-level Plackett--Burman design. Optimal reaction conditions could be determined by calculation and evaluation of the effects of the different factors on the response, i.e. the measured absorbance of the ELISA detection. The method will be used to study a possible longitudinal relationship between enteroviruses and the development of multiple sclerosis and juvenile diabetes.

Body Fluids↗
PubMed2000

Antibody-dependent induction of type I interferons by poliovirus in human mononuclear blood cells requires the type II fcgamma receptor (CD32).

The induction of type I interferons (IFNs) in peripheral blood mononuclear cells (PBMCs) can be triggered by viral infection or exposure to viral glycoproteins. Here we show that the IFN-alpha-inducing capacity of attenuated poliovirus vaccine strains is dramatically enhanced in the presence of human polyvalent immunoglobulin G (IgG). The transcription of both IFN-alpha and IFN-beta genes was detected by RT-PCR in stimulated cells. This antibody-dependent activation of type I IFNs genes was also observed with Formalin-inactivated or UV-inactivated poliovirus, but not with empty poliovirus capsids. The ability of poliovirus-antibody complexes to induce IFN-alpha was specifically inhibited when PBMCs were preincubated with an excess of the Fc fragment of IgG. Monoclonal antibodies directed to FcgammaRII (CD32) were also inhibitory, whereas antibodies to the two other classes of Fcgamma receptors, CD16 and CD64, were not. Also, aggregation of FcgammaRII by anti-CD32 antibodies alone failed to induce IFN-alpha production. Our results suggest that induction of type I interferons by poliovirus-antibody complexes depends on CD32-mediated phagocytosis of RNA-containing viral particles. As suggested by the results of an ELISPOT analysis, only a fraction of the IFN-alpha-producing cells are able to synthesize IFN-alpha in response to poliovirus-IgG complexes.

Antibodies, Viral↗
PubMed2000

The antiviral compound 5-(3,4-dichlorophenyl) methylhydantoin inhibits the post-synthetic cleavages and the assembly of poliovirus in a cell-free system.

The mode of action of the antiviral drug 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin) was studied in a cell-free system allowing de novo synthesis of poliovirus. This cell-free system, which is programmed with viral RNA, is suitable for the study of the late stages of poliovirus replication and, thus, for a study of antiviral compounds acting on these late stages. It was shown that, apart from the known inhibition of the assembly of poliovirus, hydantoin also blocks post-synthetic cleavages of poliovirus proteins. Our data demonstrate that the cell-free system is a sensitive tool to study the mode of action of antiviral compounds.

Antiviral Agents↗
PubMed1998

Uptake of poliovirus into the endosomal system of HeLa cells.

To understand the topology and mechanism of poliovirus uncoating, the question of whether intact virions can be endocytosed by the host cell was studied by a combination of various techniques. In order to prevent alteration of the virus to subviral particles, Hela cells were infected at 26 degrees C. At this temperature the majority of cell-associated virions remained at the plasma membrane, whereas a smaller amount accumulated in vesicles having the same mobility (upon free-flow electrophoresis) and migration behaviour on Nycodenz density gradients as early and late endosomes. Co-localization of native poliovirions with endosomal markers was verified by peroxidase-induced diaminobenzidine density-shift of endosomal vesicles. Internalization of poliovirions into endosomes makes it likely, but does not prove that viral RNA can be released into the cytoplasm from the vesicular compartment.

Endocytosis↗
PubMed1998

Development of an enterovirus specific PCR method for the quantification of enterovirus genomes in blood of diabetes patients.

BACKGROUND: Insulin-dependent diabetes mellitus or type 1 diabetes is a disease with a diverse aetiology. Epidemiological studies examining newly diagnosed, recent onset IDDM patients have suggested a role for viruses in the aetiology of IDDM (Yoon, 1995, Diabetes/Metabolism Reviews 11, 83-107). Important candidates are the enteroviruses, in particular coxsackieviruses B3 and B4. The latter can cause diabetes in animals (Clements et al., 1995, Lancet 346, 221-223). OBJECTIVES: We have developed a quantitative PCR method for the detection of enterovirus genomes in biological samples. The quantitative PCR will be used to screen for enteroviruses in blood of diabetes patients and their relatives by testing a Blood Diabetes Register. STUDY DESIGN: A substantial amount of data has been collected on enterovirus induced IDDM, our study is original in so far as it will be: (1) a quantitative study, not only the presence of viral genome sequences in blood will be determined, but also their concentrations (viral load); and (2) a longitudinal study, samples are and will be collected as a function of time. Positive PCR samples will be quantified using the standard addition method. RESULTS: The test is specific for enteroviruses, since all enteroviruses were detected with equal sensitivity. Viruses belonging to other picornavirus genera scored negative (even up to 3 x 10(6) genome copies). An equal detection limit of 10 genome copies was found for all enteroviruses. CONCLUSIONS: The developed method will permit us to generate quantitative and longitudinal data of enterovirus genomes in blood of diabetes patients and their relatives, which might help in the elucidation of the relationship between enteroviruses and IDDM.

Diabetes Mellitus, Type 1↗
PubMed1997

Immunogenic, non-infectious polio subviral particles synthesized in Saccharomyces cerevisiae.

Polioviral genes coding for P1, the precursor for the structural proteins, and 3CD, the viral protease, were cloned in a Saccharomyces cerevisiae inducible expression system. N-antigenic empty capsids could be isolated from the yeast cell extract provided that pirodavir, a capsid-binding compound and capsid stabilizer, was added during the induction period and during purification. Purification was by immunoaffinity chromatography. The purified empty capsids had the same immunogenicity as poliovirus virions. The techniques described might be useful for the production of new non-infectious vaccines.

3C Viral Proteases↗
PubMed1996

Stabilisation of poliovirus with pirodavir.

Two hundred and forty pyridazinamine derivates were assayed for their ability to stabilize the Oral Polio Vaccine (OPV). Of these, pirodavir (R 77975) was selected for vaccine potency tests. Although pirodavir turned out to be an effective stabilizer of OPV, the protection induced by pirodavir was not better, nor additive to the effect by MgCl2 1 M, the usual stabilizer. Both stabilizers were effective in preventing damage to the viral proteins, but could not prevent damage of viral RNA.

Antibodies, Viral↗
PubMed1994

Formation of poliomyelitis subviral particles in the yeast Saccharomyces cerevisiae.

The sequence of the poliovirus genome encoding 3CD (a protease) was transferred to the yeast Saccharomyces cerevisiae on expression vectors with either a constitutive or an inducible promoter. Transformants could only be obtained with vectors carrying the inducible transcription unit. Extracts of induced cells were able to cleave cell-free synthesized P1, the precursor of the poliovirus capsid proteins, into VP0, VP3 and VP1. In yeast cells constitutively expressing P1, induction of 3CD expression resulted in only trace amounts of processed products. Processing could be improved considerably by simultaneous induction of both P1 and 3CD expression. Analysis of extracts of such induced cells revealed the presence of particles that resembled authentic subviral particles.

3C Viral Proteases↗
PubMed1994

A competition immunoprecipitation assay of unlabeled poliovirus antigens.

An assay method is described for unlabeled, unpurified N(ative) and H(eated) antigens of poliovirus. The method is based on competition between the unlabeled antigen and a standard quantity of radiolabeled antigen, in the presence of a limiting amount of a N- or H-specific, monoclonal antibody. The immune complexes are removed by protein A-bearing, fixed staphylococci. The method is free from cross-reaction between N and H antigen, and has a detection limit of approximately 2 nM. It was applied successfully to the quantitation of poliovirus antigen synthesized by recombinant yeast expressing the viral proteins P1 and 3CD.

Antibodies, Monoclonal↗
PubMed1994

Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation.

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.

Antiviral Agents↗
PubMed1994

Discrepancy between infectivity and antigenicity stabilization of oral poliovirus vaccine by a capsid-binding compound.

Two hundred forty pyridazinamine derivatives were tested for the ability to stabilize the antigenicity and infectivity of oral poliovirus vaccine subjected to 45 degrees C for 2 h. Seven compounds stabilized the antigenicity of all three vaccine strains and neutralized the viral particles in a way that is reversible by dilution. Of these, R 77975 (pirodavir) was selected for vaccine potency tests. Sabin type 2 and type 3 strains were subjected to 4, 25, 42, and 45 degrees C for 1 week in the presence and absence of R 77975. Although R 77975 particularly stabilized the infectivity of the most thermolabile vaccine strain (Sabin type 3), the protection did not exceed that of 1 M MgCl2. When virus was inactivated in the absence of R 77975, the native or N antigenicity changed in H antigenicity. However, in the presence of the capsid-binding compound, N antigenicity was preserved in particles that had lost infectivity.

Antiviral Agents↗
PubMed1991

In vitro assembly of poliovirus 14 S subunits: identification of the assembly promoting activity of infected cell extracts.

Purified poliovirus 14 S subunits are assembled into empty capsids in vitro only if their concentration exceeds a 1.6 nM threshold. This also holds true for the 14 S subunits in unpurified extracts of infected cells. Such an extract may promote the assembly of extraneous 14 S subunits, but only if it contributes enough 14 S subunits to raise their total concentration over the threshold. As a result of assembly, the concentration of 14 S subunits in an infected cell extract decreases exponentially, with a half life of 15 min at 37 degrees. When purified 14 S subunits of serotype 1 are mixed with extracts of cells infected with type 2 or 3, chimeric empty capsids are formed, thus showing the pooling of endogenous and extraneous 14 S subunits. In conclusion, the assembly promoting activity of infected cell extracts amounts to nothing more than the supply of endogenous 14 S subunits.

Antibodies, Monoclonal↗
PubMed1991

In vitro assembly of poliovirus 14 S subunits: disoxaril stabilization as a model for the antigenicity conferring activity of infected cell extracts.

14 S subunits of poliovirus at 37 degrees undergo a rapid thermodenaturation, whereby all neutralizing epitopes are lost. This denaturation is prevented by 1 microgram/ml disoxaril, up to 42 degrees. In the absence of disoxaril, self-assembly of 14 S subunits at 37 degrees yields H-antigenic empty capsids. However, in the presence of disoxaril, and provided the molar ratio of disoxaril per 14 S subunit exceeds 7, the empty capsids generated by self-assembly are N-antigenic, and indistinguishable from native procapsids extracted from infected cells.

Antibodies, Monoclonal↗