Search PubMed⌕ Search

Biomedical subjects

H Agut

Publications and source records attributed to H Agut.

At least 127 records · Page 7Linked to original sources
PubMed1988

A poliovirus mutant defective for self-cleavage at the COOH-terminus of the 3C protease exhibits secondary processing defects.

By in vitro recombination between the wild-type full-length infectious cDNA of poliovirus and a clone generated by the construction of a cDNA bank from a chemically derived temperature-sensitive plurimutant, we obtained a mutant cDNA with a T to C change at nucleotide 5658. This mutation replaces the isoleucine at residue 74 of the viral protease 3C by a threonine. The mutant virus recovered after transfection exhibited a small-plaque phenotype, and was deficient for viral RNA synthesis. Both these defects were more marked at 39 than at 37 degrees. The mutation was introduced into a bacterial plasmid which expresses the 3C protease along with its flanking autocatalytic cleavage sites. Analysis of the cleavage products expressed in Escherichia coli provided direct evidence that the modification impaired cleavage at the COOH-terminus of 3C. Cleavage at this same site was partially defective in mutant virus-infected HeLa cells, reducing the production of mature 3C and the viral replicase, 3D. Cleavage of P1, the precursor to the capsid polypeptides, was apparently unaffected by this defect, whereas cleavage events within the P2 region of the genome occurred inefficiently. This is indicative of differential strategies for 3C-specific cleavage events in vivo.

DNA↗
PubMed1987

Multiple mutations involved in the phenotype of a temperature-sensitive small-plaque mutant of poliovirus.

A temperature-sensitive small-plaque mutant of poliovirus type 1, ts247, has been analyzed previously. Several mutations were detected in the P3 region of the genome by analysis of proteins and by T1 oligonucleotide mapping of viral RNA. We have now studied spontaneous reversion of ts247 to the wild-type phenotype. This was found to be a two-step event, reversion to a ts+ phenotype (revertant R247-51) being distinct from acquisition of normal plaque size (revertant R247-12). The mutation responsible for the ts phenotype of ts247, implicated also in virus aggregation and heat lability, could not be detected by biochemical studies. Analysis of homotypic recombinants obtained by crossing ts247 with a guanidine-resistant derivative of a temperature-sensitive replicase mutant mapped this mutation to the P1 region or to the 5' end of the P2 region of the genome. The small-plaque phenotype of ts247 and R247-51 was correlated with an abnormality in polypeptide 3C (protease); direct sequencing of viral RNA revealed a U to C change at nucleotide 5658, which altered an isoleucine to threonine in the protease of ts247 and R247-51 but not of R247-12. Two other mutations were present in the region of the genome coding for polypeptide 3D of ts247 and of both classes of revertants. They thus seemed to play no role in the phenotype of ts247. One mutation, an A to G change at nucleotide 7135, was silent at the protein level, whereas the other, an A to G change at nucleotide 6264, determined a major amino acid change from glutamate to glycine in the viral replicase.

Genes↗
PubMed1987

Intratypic recombination of polioviruses: evidence for multiple crossing-over sites on the viral genome.

Intratypic recombinant polioviruses were isolated from cells that were coinfected with two temperature-sensitive (ts) mutants of poliovirus type 1, ts035Gr and ts247. After phenotypic characterization of these recombinants, their proteins were studied by polyacrylamide gel electrophoresis, and their genomes were analyzed by RNase T1 fingerprinting and partial nucleotide sequencing. Segregation of specific phenotypic and biochemical characteristics inherited from the parental viruses demonstrated that crossing-over could occur in at least four distinct regions of the genome. Possible mechanisms for recombination are discussed.

Base Sequence↗
PubMed1986

Simplified enzyme-linked immunosorbent assay for specific antibodies to respiratory syncytial virus.

A simplified and reliable enzyme-linked immunosorbent assay (ELISA) was applied to the detection of serum antibodies against respiratory syncytial virus (RSV). RSV-infected cells were fixed and dried on 96-well microtiter plates and kept at 4 degrees C. The titers of reference sera were determined by endpoint dilution. A linear relation was found between the titers and the logarithm of absorbance values of sera diluted to 1:1,000 (r = 0.93, P less than 0.001). Measurement of RSV antibodies was done by using a single serum dilution (1:1,000) in conjunction with a standard curve. A strong correlation was found between complement fixation and ELISA results (r = 0.89, P less than 0.001). In addition, the ELISA method exhibited higher titers and a greater sensitivity than did complement fixation, although the applicability of the assay is limited with positive serum samples of low titer.

Animals↗
PubMed1984

Recombination and rescue between temperature-sensitive mutants of poliovirus type 1.

Four different temperature-sensitive (ts) mutants derived from the Mahoney strain of poliovirus type 1, were crossed in an infectious center recombination test. Evidence for recombination was obtained in three crosses, with a different segregation of an unselected marker, resistance to guanidine, in each case. Evidence for genetic complementation between ts mutants was not found, except with one set of RNA- mutants, ts 221 and ts 035. The marked virus yield enhancement which was observed in cells mixedly infected by these two mutants resulted from a nonreciprocal rescue of ts 035 by ts 221. The effects of ts 221 input multiplicity and of guanidine inhibition of viral RNA replication on the rescue were analyzed. The results showed that yield enhancement of ts 035 in mixed infection could be correlated to the low level RNA replication of ts 221 at the nonpermissive temperature.

Animals↗
PubMed1984

Biochemical characterization of poliovirus type 1 temperature-sensitive mutants.

Four temperature-sensitive mutants selected upon chemical mutagenesis of the poliovirus type 1 Mahoney strain [H. Agut, T. Matsukura, C. Bellocq, M. Dréano, J. C. Nicolas, and M. Girard, Ann. Virol. (Inst. Pasteur), 132E, 445-460 (1981)]. were analyzed by T1 oligonucleotide mapping. Three mutants (ts 203, ts 221, and ts 035) had T1 fingerprints identical to that of wild-type virus while mutant ts 247 exhibited two differences on oligonucleotides that mapped in the region of the genome coding for the replicase (polypeptide 4b) and for the protease (polypeptide 7c) of the virus. Cells were infected with each of the four mutants separately, labeled with [35S]methionine, and the labeled polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. Mutants and wild-type virus polypeptides showed a similar electrophoretic pattern except for the replicase and the protease of ts 247 which showed abnormal apparent molecular weights. The labeled proteins were subjected to two-dimensional isoelectrofocusing and SDS-polyacrylamide gel electrophoresis. Polypeptides 4b (replicase) and 2 (the common precursor to polypeptides 7c and 4b) of ts 247 and ts 035 exhibited distinct charge alterations when compared to the corresponding wild-type polypeptides. These alterations were also found on polypeptide 6a in the case of ts 247 and on polypeptide 6b in the case of ts 035, both polypeptides resulting from an alternate cleavage of polypeptide 2.

Animals↗
PubMedDate not supplied

Detection of HHV-6 by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect human herpes virus 6 (HHV-6) sequences in tissue culture. A pair of primers was synthesized and used to amplify a conserved region of the genome. Amplified products were detected either by visualization of UV illuminated ethidium bromide stained gel or, by hybridization with a specific radiolabeled oligonucleotide. As little as 5 fg of HHV-6 could be detected in infected cells, making this assay suitable for diagnostic purposes.

Base Sequence↗
PubMedDate not supplied

Evaluation of a new non-isotopic sandwich hybridization for the detection of HIV1-specific PCR products.

A new non-isotopic sandwich hybridization assay was developed to detect human immunodeficiency virus type 1 (HIV1) provirus amplified by the polymerase chain reaction. The sensitivity and specificity of this new technique using 96-well microplates as the support for the sandwich hybridization procedure and stable enzyme-linked oligomer as the detection probe were compared with those of Southern hybridization using a 32P-labelled oligomer probe. Three laboratories studied 437 peripheral blood mononuclear cell samples from 294 different subjects including both HIV1-seropositive and -seronegative individuals. The non-isotopic assay exhibited a sensitivity of 99.5% and a specificity of 99.1% when compared with the Southern procedure. In addition, the non-isotopic assay gives clear numeric data, is safe when handling, and is especially adapted to large-scale analyses.

Base Sequence↗
PubMedDate not supplied

Dose-dependent systemic human immunodeficiency virus infection of SCID-hu mice after intraperitoneal virus injection.

SCID mice were engrafted with human foetal liver, thymus and lung. Human cells were subsequently detected among peripheral blood leukocytes for 81% of tested animals and in tissue implants for 100% of tested animals. SCID-hu mice received intraperitoneal injections of human immunodeficiency virus type 1 (HIV1) at from 20 up to 20,000 median tissue culture infectious doses (TCID5). HIV1 infection was detected by means of cell culture and polymerase chain reaction both in blood and implants, up to 58 days after infection. The rate of infection was dependent upon the inoculated dose: the frequency of thymus infection ranged from 14% with 20-500 TCID50 up to 100% with 20,000 TCID50. HIV1 infection was detected less frequently in blood leukocytes than in thymus. Thymus virus load ranged from 40 to 50,000 HIV1 provirus copies per million cells and was not correlated with either infectious dose or viraemia. Thymus T-cell depletion was observed mainly in the CD1+4+8+ immature thymocyte compartment. The same rate of SCID-hu mouse infection was obtained using three different primary HIV1 isolates, suggesting that infection was not restricted to a few particular virus strains. The systemic infection of SCID-hu mice following intraperitoneal virus injection mimics some traits of human HIV infection and provides a promising, novel approach for future investigations in this field.

Animals↗
PubMedDate not supplied

In vitro determination of antiviral activity of MS8209, a new amphotericin B derivative, against primary isolates of HIV1.

MS8209, an amphotericin B derivative, was previously reported to be an inhibitor of HIV1 replication in vitro. In the present study, we determined the 50 and 90% in vitro inhibitory concentrations of MS8209 for 9 HIV1 isolates including both zidovudine-sensitive and zidovudine-resistant isolates and the reference strain Lai, using the peripheral blood mononuclear cell (PBMC) assay. We also evaluated the sensitivity of HIV1 replication to MS8209 during primary isolation from PBMCs. An inhibitory effect of MS8209 in PBMC infection was observed either when the drug was only present during the adsorption step or when the drug was initially absent but maintained throughout the culture period; the combination of these two approaches provided the highest inhibition rate. These results indicate that MS8209 can inhibit the replication of HIV1 isolates in PBMCs and suggest that it mainly acts by blocking the virus entry into cells.

Amphotericin B↗
PubMedDate not supplied

Difference in permissiveness of human fibroblast cells to variants A and B of human herpesvirus-6.

Human herpesvirus-6 (HHV6) is a lymphotropic virus genetically related to human cytomegalovirus (CMV) and for which two variants, A and B, have been distinguished. Human CMV is usually cultivated with human fibroblasts (HF). The lack of cell lines useful for HHV6 isolation and propagation led us to investigate whether HHV6 variants A and B could infect HFs as CMV does. Isolates of HHV6 variants A and B were used to infect MRC-5 HFs. HHV6 infection was detected by means of immunoperoxidase assay using three specific monoclonal antibodies. HHV6-specific antigens were detected in 88 and 38% of cases after infection with variants A and B, respectively. The highest number of HHV6-antigen-positive cells was obtained at 4-5 days p.i. The titre of HHV6 stocks was determined in parallel by immunoperoxidase assay on HFs and by observation of cytopathic effect using serial dilutions on peripheral blood mononuclear cells (PBMC). The number of infectious particles inducing the appearance of antigen-positive HF cells was consistently lower than the titre of virus stocks, expressed as TCID50. The amount of HF-associated HHV6 DNA was measured using limiting dilution PCR assay; it was significantly increased during 4-day infection in the case of variant A but not variant B. The yield of virus from infected HFs was demonstrated only for variant A by the serial propagation of virus from HFs to PBMCs and by the increase in cell-free HHV6 DNA in HF culture supernatant. Our results show that HHV6 can reproducibly infect HFs, albeit at a low level, and that HFs are more permissive to variant A than to variant B, as reported previously for PBMCs and human T-cell lines.

Antigens, Viral↗
PubMedDate not supplied

DNA hybridization for detection of human cytomegalovirus in bronchoalveolar lavage and pharynx biopsy.

A simplified hybridization procedure was used for detection of cytomegalovirus (CMV) in human specimens. The probe was a 32P-labelled cloned DNA fragment of CMV strain AD169. This probe did not hybridize to DNA from uninfected cells or other herpesviruses (herpes simplex virus, Epstein-Barr virus). Specific hybridization was obtained with unselected bronchoalveolar lavage specimens, but the sensitivity of the test (33%) was lower than that of culture (80%) and immunofluorescence (60%) assays which are routinely performed in our laboratory. The detection procedure was also carried out with pharynx biopsy specimens which had been kept frozen at -70 degrees C. CMV DNA was detected in 14% of tumour specimens and only in 1.7% of control specimens (p less than 0.05). The indications of DNA hybridization for CMV diagnosis are discussed.

Bronchoalveolar Lavage Fluid↗
PubMedDate not supplied

In vitro sensitivity of human herpesvirus-6 to antiviral drugs.

We studied the sensitivity of human herpesvirus-6 (HHV-6) to 4 antiviral drugs known to be effective in the treatment of infections with other human herpesviruses and human immunodeficiency virus. HHV-6 was grown in peripheral blood lymphocytes, and virus multiplication was quantified by evaluation of the cytopathic effect by molecular hybridization and indirect immunofluorescence assay. The 50% and 90% inhibitory concentrations (IC50 and IC90) were determined for each drug. The results obtained by the 3 different quantification techniques were found to correlate, and enabled us to conclude that HHV-6 replication was readily inhibited by foscarnet or ganciclovir. In contrast, inhibition of HHV-6 replication was observed only at high concentrations of acyclovir, and was not detected at the tested concentrations of zidovudine.

Antiviral Agents↗
PubMedDate not supplied

Preliminary characterization of cis interactions between mutations of poliovirus genome.

The effects of three distinct point mutations in the P3 genomic region of poliovirus type 1 (Mahoney strain) have previously been analysed. A U to C change at position 5658, which modifies the proteinase 3C, is responsible for a small plaque phenotype and defective RNA replication. An A to G change at position 7256, which modifies the replicase 3D, is responsible for temperature sensitivity. A C to U change at position 7348, which is silent at the protein level, has no apparent phenotypic effect. These three mutations were introduced into a single poliovirus genome by recombinant cDNA techniques. The resulting plurimutant virus, vHA507, was temperature-sensitive and exhibited a small plaque phenotype. Both these characters were accentuated compared to those of the viruses in which the mutations were on separate genomes. Spontaneous ts+ revertants of vHA507 arose by true reversion of the replicase mutation. The small plaque phenotype and defective RNA replication were partially suppressed in the case of these revertants, despite the fact that the proteinase mutation was still present on their genomes. This suppression was assumed to be due to the presence of the silent mutation at position 7348. This system provides a simple model of cis interactions within the poliovirus genome.

DNA-Directed RNA Polymerases↗