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Biomedical subjects

M Girard

Publications and source records attributed to M Girard.

At least 181 records · Page 10Linked to original sources
PubMed1991

Analysis of neutralization-escape mutants selected from a mouse virulent type 1/type 2 chimeric poliovirus: identification of a type 1 poliovirus with antigenic site 1 deleted.

A chimeric type 1/type 2 poliovirus (v510), in which the antigenic site 1 (Ag1) of poliovirus type 1 (PV-1) Mahoney was replaced by the corresponding site of poliovirus type 2 (PV-2) Lansing, is known to be neurovirulent for mice and neutralized by both type 1 and type 2 monoclonal antibodies. Neutralization-escape mutants to monoclonal antibodies specifically recognizing the PV-2 sequence were obtained from v510. The nucleotide sequence and the mouse neurovirulence of mutants were determined. Amino acid substitutions obtained inside the replaced sequence, at positions 95 and 99, and outside this site, at positions 93 or 104, rendered the virus attenuated for mice. One of the escape mutants harboured a deletion of the entire substituted nonapeptide sequence in v510. This particular virus, which is a PV-1 Mahoney lacking the natural Ag1 loop, does not react with PV-2-specific monoclonal antibodies, has a ts phenotype, is heat-labile and is devoid of neurovirulence for mice.

Amino Acid Sequence↗
PubMed1991

A Gly1 to Ala substitution in poliovirus capsid protein VP0 blocks its myristoylation and prevents viral assembly.

Capsid protein VP4 of poliovirus is acylated with myristic acid via an amide linkage to its N-terminal glycine residue. Our previous studies suggested that myristic acid plays a role in poliovirus assembly and in the early events of infection. In order to understand better its role in the assembly process, we introduced a Gly1 to Ala amino acid substitution in the myristoylation signal sequence of VP4. This substitution prevented VP0 myristoylation in vivo and abolished the infectivity of genomic transcripts harbouring the mutation. These mutated RNAs were still able to replicate in the transfected cells but the assembly processes were inefficient and no mature virions could be detected.

Alanine↗
PubMed1991

Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells.

The cDNA encoding the E and NS1 proteins of the yellow fever virus (YFV) was expressed in Spodoptera frugiperda cells via the recombinant baculovirus Ac-E. NS1 as a gp100 precursor which was cleaved to generate the recombinant proteins E and NS1 similar in size, folding and antigenicity to the authentic ones. Recombinant protein E exhibited immunodominant epitopes as judged by its reactivity with YFV-neutralizing MAbs. Using the Triton X-114 phase separation system, authentic and recombinant E proteins as well as the gp100 precursor exhibited hydrophobic properties similar to those of integral membrane proteins. Recombinant protein E was found neither in the extracellular medium nor on the cell surface, suggesting that it did not migrate within the secretory pathway of insect cells. Analysis of protein NS1 expressed in primate and insect cells revealed that the newly synthesized 48K NS1 glycoprotein was converted to a heat-labile gp72 homo-oligomeric form. This phenomenon did not require the presence of carbohydrate groups. Using the Triton X-114 phase separation system, the oligomeric form of NS1 was shown to be associated with cellular membranes although it appeared less hydrophobic than protein E and gp100. A small fraction of YFV NS1 oligomers were transported throughout the secretory pathway to be shed into the extracellular medium of primate cells. YFV NS1 oligomers migrated from the endoplasmic reticulum to the Golgi complex, whereas their N-oligosaccharides of the high-mannose type are processed to the complex-mannose type. Protein NS1 expressed by recombinant baculovirus-infected insect cells was not found in the extracellular medium but associated with the plasma membrane of the cells. Two recombinant NS1 forms were detected in insect cells: a major one with an apparent Mr of 48K and a minor one of 47K in which N-linked glycans were probably processed to a trimannosyl core without further elongation. Thus, it appears that the transport strategy as well as the N-glycosylation of NS1 in insect cells infected with recombinant baculovirus were different from those of the NS1 in primate cells infected with YFV.

Animals↗
PubMed1991

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).

Adsorption↗
PubMed1991

Intracellular pH on adrenergic-stimulated cAMP and cGMP production in rat pinealocytes.

In rat pinealocytes, activation of alpha 1-adrenergic receptors leads to increases in intracellular pH (pHi). In this study, the role of pHi on adrenergic regulation of cyclic nucleotide accumulation was investigated using ammonium chloride, which increased pHi, and sodium propionate, which reduced pHi. Ammonium chloride significantly enhanced the norepinephrine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) responses, while it selectively potentiated the isoproterenol (ISO)-stimulated cGMP response. Reduction of pHi by sodium propionate reduced the norepinephrine-stimulated cGMP accumulation by 70%, and its effect on the ISO-stimulated cGMP response was stimulatory. Treatment with sodium propionate effectively neutralized the enhancing effects of ammonium chloride on the adrenergic-stimulated cAMP and cGMP responses. These effects of sodium propionate and ammonium chloride on cyclic nucleotides appeared to reflect altered rate of synthesis, and they were also in part secondary to changes in intracellular Ca2+. Our findings indicate that the receptor-mediated changes in pHi may play an integral part in the adrenergic regulation of cAMP and cGMP production in rat pinealocytes.

Ammonium Chloride↗
PubMed1991

[HMPAO radionuclide of temporal cortex in the study of deafness].

A new method of dynamic exploration of central auditory pathway is presented, using measurement of cortical temporal perfusion variations, during auditory stimulation (tonal stimulation in normal hearing patients, electrical stimulation with promontory test in profoundly deaf patients). This study shows a perfusion increment in normal hearing and deaf patients with positive promontory test, which doesn't exist in a patient with negative promontory test. Further development of this study could help for optimization of post implantation auditory results.

Acoustic Stimulation↗
PubMed1991

[Respective importance of vertical and horizontal transmission of hepatitis B in the Australian archipelago (French Polynesia). Preliminary results of the vaccination of newborns].

A seroepidemiological survey of hepatitis B was conducted in 1988 in the Austral archipelago (French Polynesia) in a randomly selected sample of 957 persons, has showed an infection rate (at least one positive marker) with HBV of 64.1% for the entire archipelago. Among women of child-bearing age, HBs Ag carriage rate was 11.45% of whom 19.1% were so HBe Ag carriers. Among infants HBs Ag carriage rate was 2.1% and among juveniles it was 10.6%. In this area horizontal transmission of HBV is essential. An immunization program was implemented in August 1988 using a recombinant vaccine (GenHevac BTM). At one year of the program, 97% of the children had received at least one dose of vaccine, 90% had HBs-antibodies titer higher than 10 mUI/ml, 96% had seroconverted for at least one of the two antibodies (anti-HBs and/or anti-preS2) and any of them was neither HBs Ag carrier nor anti-HBc IgM antibodies carrier. Finally, among 7 neonates whose mother were positive for both HBs Ag and HBe Ag, 6 were immunes and the last one, aged of 1 month (one dose received only), was not HBs Ag carrier. These preliminary results show that vaccination with GenHevac BTM to be immunogenic and effective in field conditions.

Adolescent↗
PubMed1990

Characterization of the 5' and 3' ends of viral messenger RNAs isolated from BHK21 cells infected with Germiston virus (Bunyavirus).

The 3' ends of the S and M messenger RNAs isolated from BHK21 cells infected with Germiston virus were analyzed by mapping with RNase T2 or nuclease S1. The transcription termination signal was found to be located approximately 115 and 80 nucleotides upstream from the 3' end of the S and M genomic RNA templates, respectively. Both mRNAs were found to possess several adenosine residues at their 3' ends, but were not polyadenylated. They have acquired at their 5' end a heterologous 12- to 18-nucleotide-long sequence, which is not coded for by the virus. Sequencing of the 5' terminal region from single molecules cloned into pBR327 revealed that these primers are rich in C and G residues and possess a U or a C adjacent to the viral sequence.

Animals↗
PubMed1990

The 15 amino acid residues preceding the amino terminus of the envelope protein in the yellow fever virus polyprotein precursor act as a signal peptide.

The 15 amino acids which precede the sequence of the envelope (E) protein in the yellow fever virus (YFV) polyprotein precursor have been proposed to function as a signal peptide for the E protein (P. Desprès A. Cahour, C. Wychowski, M. Girard and M. Bouloy; Ann. Inst. Pasteur/Virol., 139, 59-67, 1988). To confirm this hypothesis, recombinant SV40 genomes were constructed in which the sequence of the E protein, or that of the poliovirus VP0 capsid polypeptide were placed immediately downstream of and in frame with the sequence of the putative signal peptide, under the control of the late SV40 promoter. The E protein expressed by the hybrid virus SV-E was recognized by two neutralizing monoclonal antibodies directed against the YFV envelope protein. In this construct, the E protein was deleted of its C-terminal transmembrane zone. Therefore, as expected, the protein appeared to be efficiently transported along the exocytic pathway and excreted into the cell culture medium. In addition, when the putative signal peptide was fused in frame with poliovirus polypeptide VP0, the expressed chimeric polypeptide was targeted to the endoplasmic reticulum where it underwent glycosylation.

Amino Acid Sequence↗
PubMed1990

Critical role of neighbouring sequences on the immunogenicity of the C3 poliovirus neutralization epitope expressed at the surface of recombinant bacteria.

The C3 neutralization epitope of poliovirus type 1 (PV-1) is a continuous epitope comprised within residues 93-103 of capsid protein VP1. These residues form a loop at the surface of the virus particle. The authors compared the immunogenicity of two peptides which contain this epitope, when presented at the surface of Escherichia coli by genetic insertion in the outer membrane protein LamB. One peptide was 13 residues long (VP1:93-103) and the other one contained flanking sequences increasing its size to 35 residues (VP1:86-115). Mice and rabbits were immunized with recombinant bacteria expressing the corresponding LamB-VP1 hybrid proteins. Antibodies against synthetic peptides, against native and heat denatured viral particles, as well as neutralizing antibodies were monitored. In this mode of presentation the shortest form of the epitope was more immunogenic. We provide evidence that the conformation of the epitope is different in the two hybrid LamB proteins and discuss possible consequences for immunogenicity.

Amino Acid Sequence↗
PubMed1990

Immunogenicity of the human immunodeficiency virus (HIV) recombinant nef gene product. Mapping of T-cell and B-cell epitopes in immunized chimpanzees.

The nonstructural nef gene product of human immunodeficiency virus (HIV), p27, is a regulatory "early phase" protein produced by HIV-infected cells. As a possible negative regulator of transcription, it has been suggested that p27 may be involved in the control of HIV proviral latency. Immune reactivity to p27 may result in early destruction of HIV-replicating cells before viral assembly or of latently infected cells. It appeared, thus, of interest to investigate the immunogenicity of the molecule in chimpanzees immunized against HIV antigens. Two of the six chimpanzees that were injected with soluble recombinant p27 in association with other HIV proteins, displayed significant and sustained T-helper lymphocyte proliferative responses to p27 and to the other antigens. Using a set of synthetic peptides spanning the entire p27 sequence, two T-cell epitopes could be located: one within the last 20 amino-acids of the C terminus of the molecule, the other around the region of residues 118-122. Sera from the same animals also reacted to p27 in a radioimmunoassay as well as to some of the peptides in enzyme-linked immunosorbent assay. Sequential B-cell epitopes could thus be determined as being located in the regions of amino acids: 17-35, 52-66, and 185-205. The results obtained with peptides spanning the region between amino acid residues 65 and 172 indicate that at least two additional B-cell epitopes were present in the region comprised between amino acid 65 and 146. Interestingly, the extreme C terminus of the molecule encompasses both immunodominant T- and B-cell epitopes. Taken together, these observations should prove useful for the rational design of a HIV vaccine.

Amino Acid Sequence↗
PubMed1990

Cleavage specificity of the poliovirus 3C protease is not restricted to Gln-Gly at the 3C/3D junction.

The 3C protease of poliovirus is distinguished from that of all other picornaviruses in that it only cleaves at Gln-Gly amino acid pairs within the viral polyprotein. To determine whether this strict cleavage specificity is an intrinsic property of the poliovirus 3C protease, amino acid substitutions were introduced at one of the Gln-Gly cleavage sites. Oligonucleotide-directed site-specific mutagenesis of an infectious poliovirus type 1 (Mahoney strain) cDNA was used to change the Gln-Gly site at the 3C/3D junction of the polyprotein into Gln-Val, Gln-Ala, Gln-Ser or Gln-Pro. The effects of these substitutions were studied in vivo after transfection of primate cells by the mutated cDNAs. The Gln-Gly to Gln-Pro substitution was lethal for virus growth, and the corresponding altered 3CD polypeptide expressed in insect cells using a recombinant baculovirus vector did not appear to undergo autocleavage. The Gln-Gly to Gln-Val change was also lethal, although production of virus was occasionally observed as a result of reverse mutations. Mutants with Gln-Ala and Gln-Ser sequences were viable, indicating that these dipeptides can be cleaved by the poliovirus protease in vivo. However, processing at the 3C/3D junction occurred relatively inefficiently in the case of the Gln-Ser virus. Furthermore, the Gln-Gly to Gln-Ala substitution seemed to result in an additional cleavage event within the N-terminal part of polypeptide 3D.

3C Viral Proteases↗