Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 29
Filter
1.
Clin Microbiol Infect ; 23(8): 516-523, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28712666

ABSTRACT

BACKGROUND: The WHO European Region (EUR) has adopted the goal of eliminating measles and rubella but individual countries perform differently in achieving this goal. Measles virus spread across the EUR by mobile groups has recently led to large outbreaks in the insufficiently vaccinated resident population. As an instrument for monitoring the elimination process and verifying the interruption of endemic virus transmission, molecular surveillance has to provide valid and representative data. Irrespective of the country's specific situation, it is required to ensure the functionality of the laboratory surveillance that is supported by the WHO Global Measles and Rubella Laboratory Network. AIMS: To investigate whether the molecular surveillance in the EUR is adequate for the challenges in the elimination phase, we addressed the quality assurance of molecular data, the continuity and intensity of molecular monitoring, and the analysis of transmission chains. SOURCES: Published articles, the molecular External Quality Assessment Programme of the WHO, the Centralized Information System for Infectious Diseases of the WHO EUR and the WHO Measles and Rubella Nucleotide Surveillance databases served as information sources. CONTENT: Molecular proficiency testing conducted by the WHO in 2016 has shown that the expertise for measles and rubella virus genotyping exists in all parts of the EUR. The analysis of surveillance data reported nationally to the WHO in 2013-2016 has revealed some countries with outbreaks but not sufficiently representative molecular data. Long-lasting supranational MV transmission chains were identified. IMPLICATIONS: A more systematic molecular monitoring and recording of the transmission pattern for the whole EUR could help to create a meaningful picture of the elimination process.


Subject(s)
Epidemiological Monitoring , Measles virus/isolation & purification , Measles/epidemiology , Rubella virus/isolation & purification , Rubella/epidemiology , Disease Outbreaks , Disease Transmission, Infectious , Europe/epidemiology , Genotyping Techniques/methods , Genotyping Techniques/standards , Humans , Laboratory Proficiency Testing , Measles/transmission , Measles virus/classification , Measles virus/genetics , Molecular Epidemiology/methods , Molecular Epidemiology/standards , Rubella/transmission , Rubella virus/classification , Rubella virus/genetics , World Health Organization
2.
Vox Sang ; 98(3 Pt 2): 403-14, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20412171

ABSTRACT

BACKGROUND AND OBJECTIVES: This study was conducted by the International Consortium for Blood Safety (ICBS) to identify high-quality test kits for detection of hepatitis B virus (HBV) surface antigen (HBsAg) for the benefit of developing countries. MATERIALS AND METHODS: The 70 HBsAg test kits from around the world were evaluated comparatively for their clinical sensitivity, analytical sensitivity, sensitivity to HBV genotypes and HBsAg subtypes, and specificity using 394 (146 clinical, 48 analytical and 200 negative) ICBS Master Panel members of diverse geographical origin comprising the major HBV genotypes A-F and the HBsAg subtypes adw2,4, adr and ayw1-4. RESULTS: Seventeen HBsAg enzyme immunoassay (EIA) kits had high analytical sensitivity <0.13 IU/ml, showed 100% diagnostic sensitivity, and were even sensitive for the various HBV variants tested. An additional six test kits had high sensitivity (<0.13 IU/ml) but missed HBsAg mutants and/or showed reduced sensitivity to certain HBV genotypes. Twenty HBsAg EIA kits were in the sensitivity range of 0.13-1 IU/ml. The other eight EIAs and the 19 rapid assays had analytical sensitivities of 1 to >4 IU/ml. These assays were falsely negative for 1-4 clinical samples and 17 of these test kits showed genotype dependent sensitivity reduction. Analytical sensitivities for HBsAg of >1 IU/ml significantly reduce the length of the HBsAg positive period which renders them less reliable for detecting HBsAg in asymptomatic HBV infections. Reduced sensitivity for HBsAg with genetic diversity of HBV occurred with genotypes/subtypes D/ayw3, E/ayw4, F/adw4 and by S gene mutants. Specificity of the HBsAg assays was >or=99.5% in 57 test kits and 96.4-99.0% in the remaining test kits. CONCLUSION: Diagnostic efficacy of the evaluated HBsAg test kits differed substantially. Laboratories should therefore be aware of the analytical sensitivity for HBsAg and check for the relevant HBV variants circulating in the relevant population.


Subject(s)
Hepatitis B Surface Antigens/blood , Hepatitis B/epidemiology , Immunoenzyme Techniques/methods , Africa/epidemiology , Amino Acid Sequence , Asia/epidemiology , Developing Countries , False Negative Reactions , Genes, Viral , Genotype , Global Health , Hepatitis B/blood , Hepatitis B/diagnosis , Hepatitis B/virology , Hepatitis B Surface Antigens/classification , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/classification , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Humans , Molecular Sequence Data , Reagent Kits, Diagnostic , Sensitivity and Specificity , Sequence Alignment , Sequence Homology, Amino Acid , South America/epidemiology , United States/epidemiology
3.
Gene Ther ; 14(12): 960-71, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17377597

ABSTRACT

As coxsackievirus B3 (CoxB3) and adenoviruses may cause acute myocarditis and inflammatory cardiomyopathy, isolation of the common coxsackievirus-adenovirus-receptor (CAR) has provided an interesting new target for molecular antiviral therapy. Whereas many viruses show high mutation rates enabling them to develop escape mutants, mutations of their cellular virus receptors are far less likely. We report on antiviral efficacies of CAR gene silencing by short hairpin (sh)RNAs in the cardiac-derived HL-1 cell line and in primary neonatal rat cardiomyocytes (PNCMs). Treatment with shRNA vectors mediating RNA interference against the CAR resulted in almost complete silencing of receptor expression both in HL-1 cells and PNCMs. Whereas CAR was silenced in HL-1 cells as early as 24 h after vector treatment, its downregulation in PNCMs did not become significant before day 6. CAR knockout resulted in inhibition of CoxB3 infections by up to 97% in HL-1 cells and up to 90% in PNCMs. Adenovirus was inhibited by only 75% in HL-1 cells, but up to 92% in PNCMs. We conclude that CAR knockout by shRNA vectors is efficient against CoxB3 and adenovirus in primary cardiac cells, but the efficacy of this approach in vivo may be influenced by cell type-specific silencing kinetics in different tissues.


Subject(s)
Adenoviridae Infections/therapy , Coxsackievirus Infections/therapy , Genetic Therapy/methods , Myocarditis/therapy , RNA Interference , Receptors, Virus/genetics , Adenoviridae , Animals , Cell Line , Cells, Cultured , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Enterovirus B, Human , Gene Silencing , Genetic Engineering , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Myocarditis/virology , Myocytes, Cardiac/virology , RNA, Small Interfering/administration & dosage , Rats , Virus Replication/genetics
4.
J Mol Med (Berl) ; 84(10): 842-51, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16924471

ABSTRACT

Coxsackie adenovirus receptor (CAR) is involved in immunological processes, and its soluble isoforms have antiviral effects on coxsackievirus B3 (CVB3) infection in vitro. We explored in this study the impact of CAR4/7, a soluble CAR isoform, on CVB3-induced myocarditis in BALB/c mice. BALB/c mice were treated daily with recombinant CAR4/7, beta-galactosidase (beta-Gal; as control protein) or buffer for 9 days. Half of each group was infected with CVB3 on day 3, and all mice were killed on day 9. Myocardial CVB3 titer, histology, and serology were analyzed. Treatment with CAR4/7 led to a significant reduction of myocardial CVB3 titer, whereas the application of beta-Gal had no detectable effect on the myocardial virus load. CAR4/7 application, however, resulted in increased myocardial inflammation and tissue damage in CVB3-infected hearts, whereas beta-Gal caused a degree of cardiac inflammation and injury similar to that in buffer-treated CVB3-infected control animals. CAR4/7 and beta-Gal treatment induced the production of antibodies against the respective antigens. CAR4/7-, but not beta-Gal-specific, virus-negative sera reacted against myocardial tissue and cellular membranous CAR, and significantly inhibited CVB3 infection in vitro. Thus, CAR4/7 suppressed CVB3 infection in vivo, supporting the concept of receptor analog in antiviral therapy. However, CAR4/7 treatment also leads to an aggravation of myocardial inflammation and injury most likely secondary to an autoimmune process.


Subject(s)
Coxsackievirus Infections/drug therapy , Enterovirus B, Human/drug effects , Receptors, Virus/therapeutic use , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Cell Survival/drug effects , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Coxsackievirus Infections/pathology , Coxsackievirus Infections/virology , Creatine Kinase/blood , Enterovirus B, Human/growth & development , Enzyme-Linked Immunosorbent Assay , HeLa Cells , Humans , Immune Sera/pharmacology , Immunohistochemistry , Injections, Intraperitoneal , Male , Mice , Mice, Inbred BALB C , Myocarditis/chemically induced , Myocarditis/pathology , Myocarditis/virology , Random Allocation , Receptors, Virus/genetics , Receptors, Virus/immunology , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Recombinant Proteins/therapeutic use , Serum Amyloid A Protein/analysis , Solubility
5.
Biochem Pharmacol ; 58(11): 1781-90, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10571253

ABSTRACT

Human immunodeficiency virus (HIV)-specific peptide antibody-brefeldin A conjugates and antibody-glaucarubolone conjugates directed to cell surface viral glycoprotein epitopes were prepared and tested for antiviral activity. A selective response was observed both on survival of cell lines permanently infected with lentiviruses and on HIV infectivity. With human peripheral blood mononuclear cells (PBMCs), the conjugate also was effective in reducing virus titers. The effectiveness of an HIV-specific peptide antibody-brefeldin A conjugate was enhanced by combination with 3'-azido-3'-deoxythymidine (AZT) and was effective against AZT-resistant isolates in combination with AZT. The conjugates reduced virus production in MOLT-4 cells and in HIV-1-infected PBMCs without affecting the viability of uninfected cells.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Antibodies/pharmacology , HIV/drug effects , Immunoconjugates/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Zidovudine/pharmacology , Amino Acid Sequence , Anti-HIV Agents/chemistry , Glycoproteins/immunology , HIV/immunology , HIV/physiology , HIV Envelope Protein gp120/immunology , Humans , Immunodeficiency Virus, Feline/immunology , Immunodeficiency Virus, Feline/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/virology , Microbial Sensitivity Tests , Molecular Sequence Data , Tumor Cells, Cultured , Virus Replication/drug effects
6.
Life Sci ; 62(3): 213-9, 1998.
Article in English | MEDLINE | ID: mdl-9488099

ABSTRACT

Growth of Crandall feline kidney cells permanently infected with feline immunodeficiency virus was inhibited by the anti-cancer quassinoid glaucarubolone whereas growth of uninfected cells was not inhibited. Similar results were obtained for human MOLT-4 cells infected with HIV-1. The results suggest that cell lines permanently infected with either the feline or the human lentivirus exhibit growth response characteristics to the quassinoids in common with other cell lines malignantly transformed. In addition the quassinoids may delay viral infection suggesting some commonality between the mechanism responsible for inhibition of the growth of the transformed phenotype and viral infection.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cell Division/drug effects , Cell Transformation, Viral/drug effects , Glaucarubin/analogs & derivatives , HIV-1/drug effects , Immunodeficiency Virus, Feline/drug effects , Quassins , Animals , Cats , Glaucarubin/pharmacology , Humans
7.
J Virol Methods ; 67(1): 45-55, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9274817

ABSTRACT

Eleven German laboratories and one Swiss laboratory initiated a quality assessment study to evaluate the specificity and sensitivity of their polymerase chain reaction (PCR) for detection of HIV-1 DNA. Following its own PCR protocols, each laboratory tested a panel of ten coded samples consisting of cell pellets containing 0, 0.1, 1, 10, 10(2), 10(3) and 10(4) ACH-2 cells per 1.5 x 10(5) uninfected peripheral blood mononuclear cells. Of the twelve participating laboratories, three reported correct results for the dilution series as well as for uninfected specimens. One or more classification errors were recorded for 12% of the samples for which the diagnosis was expected to be positive or negative. Samples containing 10 copies of the target template were correctly reported by eleven of the twelve participants. The average sensitivity was 97%. The results of the study revealed no significant differences between the Amplicor kit and in-house procedures. Most of the classification errors occurred in specimens from HIV-negative samples. Out of 36 negative samples, 5 were reported false positive, showing that contamination remains a problem for some laboratories, regardless of the PCR test performed. Careful laboratory techniques and internal as well as external quality control procedures will help avoiding carryover contamination.


Subject(s)
HIV Infections/diagnosis , HIV-1/isolation & purification , Polymerase Chain Reaction/standards , DNA, Viral/analysis , False Negative Reactions , False Positive Reactions , HIV-1/genetics , Humans , Lymphocytes , Quality Control , Reagent Kits, Diagnostic , Sensitivity and Specificity
8.
Med Microbiol Immunol ; 186(1): 1-9, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9255760

ABSTRACT

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.


Subject(s)
Intercellular Adhesion Molecule-1/physiology , Rhinovirus/physiology , Animals , Cricetinae , HeLa Cells/virology , Humans , Intercellular Adhesion Molecule-1/genetics , Kidney/virology , Transfection , Viral Proteins/biosynthesis
9.
J Mol Med (Berl) ; 75(2): 145-52, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9083932

ABSTRACT

Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi's sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8.


Subject(s)
Capsid/immunology , Herpesvirus 8, Human/immunology , Sarcoma, Kaposi/immunology , Amino Acid Sequence , Antibodies, Viral/analysis , Humans , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino Acid
10.
Virus Res ; 41(1): 101-8, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8725106

ABSTRACT

We have previously shown that the pseudorabies virus (PrV) glycoproteins gB and gC (former PrV-gII and PrV-gIII) exhibit heparin-binding properties. While PrV-gC functions as the major adsorption protein, the biological role of the heparin-binding properties of PrV-gB are not understood. We used a gC-deleted PrV-mutant, PrV (dlg92/dltk), to analyse the heparin-binding properties of PrV-gB and the biological role of the PrV-gB-protein in adsorption. PrV-gB was the only glycoprotein of this vaccine strain binding to immobilised heparin in in vitro assays. Presence of the gC-protein was not necessary for the interaction of gB with heparin. Soluble heparin also interfered with adsorption of this mutant virus to a similar extent as it blocked adsorption of wild-type PrV (Ka), but it had only a minor inhibitory effect on infectivity of the mutant strain. These results show that PrV-gB interacts specifically with immobilized heparin and heparin-like structures on the cell surface, but this interaction is not required for a productive infection.


Subject(s)
Glycosaminoglycans/metabolism , Heparin/metabolism , Herpesvirus 1, Suid/metabolism , Viral Envelope Proteins/metabolism , Adsorption , Cell Line , Herpesvirus 1, Suid/pathogenicity , Protein Binding , Solubility
11.
Med Microbiol Immunol ; 184(2): 97-103, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7500917

ABSTRACT

Pseudorabies virus (PrV), an alphaherpesvirus of swine, uses cellular heparan sulfate residues as a receptor for attachment. Interaction of the virus with its receptor is mediated by the envelope glycoprotein C (PrV-gC), a protein with heparin-binding properties. We have previously shown that a region of this protein shows structural similarities to the high-affinity heparin-binding site of the serum protease-inhibitor antithrombin III (ATII). In this publication, we describe the effect of ATIII on interaction of PrV with its cellular receptor. ATIII bound specifically to heparan sulfate residues on the surface of herpesvirus-permissive RK13 cells. Binding of ATIII to RK13 cells interfered with adsorption of radioactively labelled PrV to these cells. Enzymatic treatment using heparinase I (E.C. 4.2.2.7) removed the receptor for PrV as well as the receptor for ATIII. Since amino acids 130-137 of the high affinity heparin-binding site of ATIII show structural similarities to amino acids 134-141 of PrV-gC, both sequences were synthesized as synthetic peptides. Although interaction of the peptide derived from ATIII with heparin was significantly stronger, both peptides interacted specifically with heparin in assays in vitro. These results suggest that PrV and ATIII interact with the same structure on the cellular surface.


Subject(s)
Antithrombin III/pharmacology , Herpesvirus 1, Suid/physiology , Receptors, Virus/metabolism , Amino Acid Sequence , Antithrombin III/metabolism , Cells, Cultured , Heparin/metabolism , Molecular Sequence Data
12.
13.
Virology ; 186(2): 533-42, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1310182

ABSTRACT

Glycoproteins in the range 50 and 23/25 kDa were identified as poliovirus specific binding sites on HeLa cells with the monoclonal antibody mAb 122. mAb 122 is characterized by its partial inhibiting effect on poliovirus reproduction and adsorption when prebound to HeLa cells. The binding sites are endocytosed in native cells and specific for poliovirus as mAb 122 did not interfere with the adsorption of human rhinovirus type 14 (HRV 14). The poliovirus binding sites are present also on nonprimate so called nonsusceptible cells, e.g., mouse L-cells, as could be shown with sensitive ELISA based binding assays and performance of binding studies with fixed cells at 37 degrees.


Subject(s)
Membrane Glycoproteins/metabolism , Poliovirus/metabolism , Receptors, Virus/metabolism , Animals , Antibodies, Monoclonal/immunology , HeLa Cells , Humans , L Cells , Mice , Poliovirus/immunology , Poliovirus/physiology , Rhinovirus/metabolism , Virus Replication
14.
J Virol Methods ; 35(1): 1-14, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1666110

ABSTRACT

A combined assay consisting of a pre-cpe-protection assay and a double-antibody sandwich ELISA for detecting poliovirus was developed on a microtiter scale in order to quantify inhibition of virus replication caused by cell protective antibodies. The system was of high sensitivity and allowed the measurement of the protecting effect caused by a broad range of antibody concentrations before appearance of cytopathic effects. It was applied as a screening test for a large number of hybridomas secreting antibodies specific to the surface of HeLa cells and allowed the identification of four monoclonal antibodies (mAbs) with partial protection activity against poliovirus infection. One of the antibodies, mAb 122, detected SDS-PAGE-separated HeLa cell membrane proteins of 23-25 kDa and 50 kDa by immunoblot, indicating that these proteins are involved in poliovirus adsorption.


Subject(s)
Antibodies, Monoclonal/immunology , Immunologic Techniques , Membrane Proteins , Poliomyelitis/immunology , Poliovirus/growth & development , Receptors, Virus/immunology , Animals , Blotting, Western , Enzyme-Linked Immunosorbent Assay/methods , HeLa Cells/microbiology , Humans , Hybridomas , Immunotherapy, Adoptive , Mice , Poliovirus/immunology
15.
Virology ; 168(2): 417-20, 1989 Feb.
Article in English | MEDLINE | ID: mdl-2536990

ABSTRACT

Poliovirus type 1 enters HeLa cells by receptor-mediated endocytosis as an intact virus. Up to 30 min after adsorption complete virus particles still containing VP4 and sedimenting with 156 S could be recovered from the cells. These virus particles were N-antigenic and infectious. Thirty minutes after adsorption the recovery of intact and infectious virus decreased. This decrease presumably reflects viral uncoating in the acidic endosomes and/or lysosomes because virus particles could be localized in endosomes at this time. The direct involvement of clathrin-coated structures in the endocytosis of poliovirus has been deduced from the enclosure of poliovirus in coated vesicles at 10 min after adsorption. At this time intact and infectious virus could be recovered only after the coated vesicles were disrupted by treatment with 0.5 M Tris at pH 7.0.


Subject(s)
Endocytosis , Poliovirus/metabolism , Receptors, Virus/metabolism , Adsorption , Coated Pits, Cell-Membrane/microbiology , HeLa Cells , Humans , Hydrogen-Ion Concentration , Lysosomes/microbiology , Organelles/microbiology , Poliovirus/growth & development , Poliovirus/isolation & purification , Viral Plaque Assay
16.
Virology ; 160(1): 281-5, 1987 Sep.
Article in English | MEDLINE | ID: mdl-2820136

ABSTRACT

Disoxaril [WIN 51711, 5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3- methylisoxazole] inhibits the replication of polioviruses types 1 and 2 in HeLa cells by stabilizing the virus capsid, which results in the inhibition of the pH-dependent viral uncoating in endosomes and/or lysosomes. As shown by electron microscopy the virus entered into the cell by receptor-mediated endocytosis via coated pits and coated vesicles into endosomes irrespective of the presence or absence of the compound. Measurements of viral RNA synthesis showed that disoxaril completely inhibited the arrival of viral RNA in the cytoplasm for new RNA synthesis only when the inocula were preincubated with disoxaril for 15 min at 37 degrees at 0.3 microgram disoxaril/ml for poliovirus type 1 and 0.03 microgram disoxaril/ml for poliovirus type 2. Simultaneous addition of the compound and virus resulted in reduced inhibition of viral RNA synthesis. The inhibitory effect of the compound could be partially reversed up to 25 min p.i. if the compound was eluted from the cells.


Subject(s)
Antiviral Agents/pharmacology , Isoxazoles/pharmacology , Oxazoles/pharmacology , Poliovirus/drug effects , Capsid/drug effects , Depression, Chemical , Endocytosis , HeLa Cells/physiology , Humans , Lysosomes/physiology , Poliovirus/physiology , RNA, Viral/biosynthesis , Virus Replication/drug effects
17.
Arch Virol ; 91(3-4): 207-20, 1986.
Article in English | MEDLINE | ID: mdl-3022677

ABSTRACT

Neutralization of poliovirus type 1 was studied using radioactively labelled polyclonal IgG. With nonsaturating antibody concentrations various virus-antibody complexes were produced which were isolated by sucrose gradient centrifugation and identified by electron microscopy as virus monomers, dimers, trimers, tetramers and pentamers. The neutralization rate (n.r.) of each of the virus-antibody complexes relative to non-neutralized virus and the stoichiometry have been estimated. The monomer fraction showed that about every fifth virion was associated with one IgG molecule and neutralized. The antibody was bivalently attached. The majority of virus particles formed aggregates of different sizes, which were cross-linked by antibodies. The following neutralization rates and ratios of IgG to virus (IgG/V) were determined for the oligomers: dimers, 59.2 per cent n.r. and 0.55 IgG/V; trimers, 67.3 per cent n.r. and 0.66 IgG/V; tetramers, 79.0 per cent n.r. and 0.75 IgG/V; pentamers, 86.3 per cent n.r. and 0.98 IgG/V. Two different mechanisms of neutralization are proposed: i) an antibody-mediated mechanism specifically inhibits infectivity of the monomer virus-antibody complexes and ii) reduction of infectivity of oligomer virus-antibody complexes is caused simply by reduction of the actual number of infectious units. Immunoprecipitation of the denatured capsid proteins showed that only VP 1 was recognized by the polyclonal IgGs.


Subject(s)
Antibodies, Viral/immunology , Immunoglobulin G/immunology , Poliovirus/immunology , Antigen-Antibody Complex , Antigens, Viral/immunology , Capsid/immunology , Neutralization Tests , Poliovirus/growth & development , Viral Plaque Assay , Viral Proteins/immunology , Viral Structural Proteins , Virion/immunology
18.
J Gen Virol ; 66 ( Pt 3): 483-92, 1985 Mar.
Article in English | MEDLINE | ID: mdl-2857762

ABSTRACT

Poliovirus type 1 appeared from electron microscope studies to enter HEp-2 cells by receptor-mediated endocytosis. On adsorption the virus was evenly distributed over the cell surface, with some preference for the microvilli and their bases. Invagination of the cell surface membrane with the attached virus commenced at coated pits and led to the formation of virus-containing coated vesicles in the cytoplasm. These coated vesicles fused with intracellular vesicles to form endosomes. When cells infected with poliovirus or Mouse Elberfeld virus were treated with the weak bases chloroquine, NH4Cl or the ionophore monensin to raise the intraendosomal and intralysosomal pH above 6, virus-directed macromolecular synthesis and production of progeny were prevented. These results suggest that the virus genomes are released to the cytoplasm via endosomes and/or lysosomes by a pH-dependent process.


Subject(s)
Endocytosis , Endosomes/physiology , Lysosomes/physiology , Picornaviridae/growth & development , Poliovirus/growth & development , Ammonium Chloride/pharmacology , Cell Line , Chloroquine/pharmacology , Hydrogen-Ion Concentration , Monensin/pharmacology , RNA, Viral/biosynthesis , Virus Replication
19.
J Gen Virol ; 64 (Pt 6): 1263-75, 1983 Jun.
Article in English | MEDLINE | ID: mdl-6304233

ABSTRACT

Low doses of u.v. radiation rapidly inactivate poliovirus, and the virus is progressively converted into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. they are indistinguishable from virus in their RNA and protein content and in their sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent [3H]N-succinimidyl propionate (3H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs). The SSPs are antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but they differ physically and structurally from them. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H-antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent 3H-NSP.


Subject(s)
Poliovirus/radiation effects , Ultraviolet Rays , Centrifugation, Density Gradient , HeLa Cells , Humans , Immunodiffusion , Immunoelectrophoresis , Kinetics , Microscopy, Electron , Poliovirus/ultrastructure
20.
J Gen Virol ; 64 (Pt 4): 951-5, 1983 Apr.
Article in English | MEDLINE | ID: mdl-6300312

ABSTRACT

The preexistence of a cytoplasmic membrane complex in HEp-2 cells, induced by poliovirus when inhibited in its reproduction by guanidine, was a prerequisite for accelerated reproduction of superinfecting Mouse Elberfeld (ME) virus. Guanidine-inhibited poliovirus induced a membrane complex of 470S that was successively modified into a faster sedimenting membrane complex (up to 700S) by superinfecting ME virus and exploited for ME virus reproduction. The modified membrane complex was the site for ME virus-specific RNA polymerization characterized by the existence of in vivo and in vitro activity of ME virus RNA polymerase associated with the modified membrane complex. Proof of membrane-bound RNA polymerase and newly synthesized ME virus RNA including replicative intermediate led to the conclusion that superinfecting ME virus exploits the 'poliovirus/guanidine'-induced complex as the site of action of its replication complex.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Picornaviridae/metabolism , Poliovirus/metabolism , Carcinoma, Squamous Cell , Cell Membrane/microbiology , Cells, Cultured , Cytoplasm/microbiology , Humans , Laryngeal Neoplasms , Poliovirus/enzymology
SELECTION OF CITATIONS
SEARCH DETAIL
...