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Biomedical subjects

N Tordo

Publications and source records attributed to N Tordo.

At least 37 records · Page 2Linked to original sources
PubMed1998

Experimental rabies infection and oral vaccination in vampire bats (Desmodus rotundus).

A rabies virus variant isolated from a vampire bat (Desmodus rotundus) and characterized by genome sequencing was used for the standardization of an experimental infection in this species. The parenteral administration of 10(6) MICLD50 of this variant was capable of inducing death from rabies in 89% of animals. The mean duration of post-challenge survival was 12 days. None of the experimental rabid vampire bats showed aggressive behaviour. A vaccinia-rabies glycoprotein recombinant virus vaccine was administered orally to vampire bats on days -120, -90, -30 or -18 pre-challenge, on the same day of challenge, or on day +5 post-challenge. A significant protection was noticed only in animals vaccinated on days -18 or -30 pre-challenge. A longer period of incubation was observed in animals vaccinated 5 days post-challenge.

Administration, Oral↗
PubMed1998

Identification of a region of the rabies virus N protein involved in direct binding to the viral RNA.

In rabies virus, the ribonucleoprotein complex (RNP), the RNA genome (-) and the antigenome (+) are specifically coated by the viral nucleoprotein (N protein), forming the template for transcription and replication bythe viral RNA polymerase. This specific encapsidation starts at the 5' ends of the RNAs. To investigate domains of the N protein that govern binding specificity, we tested in vitro the ability of both full-length and truncated forms of the N protein to interact with a synthetic RNA probe corresponding to the 5' end of the antigenome. UV-LASER cross-linking, which covalently links RNA and proteins in intimate contact, showed that the entire N protein (450 aa) and the NH2-terminal 376 aa (t42) contained all of the determinants for specific interaction. It was demonstrated by affinity chromatography that a peptide near the COOH terminus of t42 (position 298352), which is located in the most conserved region of Rhabdoviridae N proteins, bound directly to the viral RNA. However, no significant sequence similarity was detected between this peptide and known RNA binding proteins in the databases. This suggests both that N proteins may possess a new type of RNA binding motif and that protein folding contributes to the architecture of the RNA binding site.

Amino Acid Sequence↗
PubMed1998

Detection of respiratory syncytial virus A and B and parainfluenzavirus 3 sequences in respiratory tracts of infants by a single PCR with primers targeted to the L-polymerase gene and differential hybridization.

A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) has been developed to identify the major agents of bronchiolitis in infants: respiratory syncytial viruses A and B (RSVA and RSVB) and parainfluenzavirus 3 (PIV3). Two primer sets (P1-P2 and P1-P3) were selected in a conserved region of the polymerase L gene. In infected cell cultures, this method detected RSVA (n = 14), RSVB (n = 13), and PIV3 (n = 13), with the exclusion of PIV1 (n = 4), PIV2 (n = 3), measles virus (n = 6), mumps virus (n = 4), influenza A virus (n = 11), and influenza B virus (n = 4). The differentiation of the amplicons by restriction fragment length polymorphism (RFLP) showed a PvuII site for PIV3 strains and an AvaII site for RSV strains, with RSVA distinguished from RSVB by BglII. The hybridization-EIA, using three internal probes specific for each virus, correlated with the immunofluorescence assay (IFA) and RFLP results. Clinical aspirates from 261 infants hospitalized with bronchiolitis were tested by IFA, viral isolation technique (VIT), and RT-PCR-EIA. RT-PCR-EIA detected RSV sequences in 103 samples (39.4%), and IFA-VIT detected RSV sequences in 109 cases (41.7%). A few samples (2.6%) were IFA-VIT positive but PCR negative, and one sample was RT-PCR-EIA positive only. RT-PCR-EIA detected PIV3 sequences in 14 of the 15 IFA-VIT-positive isolates. The two methods showed very good correlation (96.9%), but RT-PCR-EIA was clearly more efficient in typing, leaving 5% non-A, non-B isolates, while IFA failed to resolve 23% of the isolates. The two methods contradicted each other for <5% of the isolates.

Bronchiolitis↗
PubMed1997

Phage-displayed and soluble mouse scFv fragments neutralize rabies virus.

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.

Amino Acid Sequence↗
PubMed1997

The complete Mokola virus genome sequence: structure of the RNA-dependent RNA polymerase.

The genome sequence of the rabies-related virus Mokola virus (genus Lyssavirus) has been completed by sequencing the L gene, which consists of 6384 nucleotides encoding a 2127 amino acid polymerase. Alignment of the Mokola virus L protein with other polymerases from the virus order Mononegavirales defined three domains: a divergent NH2-terminal domain, a highly conserved central domain carrying most of the functional motifs and a COOH-terminal domain with alternating conserved and divergent regions. A statistical study outlined the stringency of conservation of glycine, acidic (D, E) and basic (K, R, H) amino acids in polymerases, particularly as key residues of the conserved motifs.

Amino Acid Sequence↗
PubMed1995

Genetic polymorphism in the rabies virus nucleoprotein gene.

In an attempt to compare intrinsic and extrinsic genetic diversity of the lyssavirus genotypes, 69 rabies virus isolates from various part of the world were partially sequenced and compared to 13 representative isolates of the 6 lyssavirus genotypes. The analysis of their phylogenetic relationships, performed on the complete nucleoprotein (N) coding gene (1350 bases), established that the rabies virus isolates all belonged to genotype 1 and that at least 11 phylogenetic lineages could be identified in accordance with their geographical localization and species of origin. These lineages diverged mostly by the accumulation of synonymous mutations. Stabilizing selection, possibly related to host specificity, limits amino acid sequence and antigenic drift. Analyses were also performed either on the highly variable 400-base region coding for the amino terminus of the N protein or on the 93-base noncoding region corresponding to the 3' end of the N mRNA, the intergenic N-phosphoprotein (M1) region, and the 5' end of the M1 mRNA. These shorter nucleotide sequences were shown to provide phylogenetic data suitable for the completion of large epidemiological studies, but with less robustness. This latter noncoding sequence, despite a 3.1 times higher mutation rate than its adjacent coding N gene, followed a parallel evolutionary pattern.

Amino Acid Sequence↗
PubMed1995

An experimental rabies vaccine produced with a new BHK-21 suspension cell culture process: use of serum-free medium and perfusion-reactor system.

An experimental rabies vaccine was prepared from the BHK-21 cell line adapted to culture in suspension using bioreactors. A new serum-free medium (MDSS2) (Merten et al., Cytotechnology, 1994, 14, 47) developed for the culture of various cell lines and for the production of several biologicals, was used for cell culture and virus production. The PV-Paris/BHK-21 rabies virus strain (adapted to the BHK-21 grown in monolayer) was adapted to BHK-21 cells cultivated in suspension and in the serum-free medium. High titres of rabies virus were obtained with bioreactors equipped with a perfusion system using BHK-21 cells grown in suspension in MDSS2. Experimental vaccines were prepared and had satisfactory protective activity when tested in mice. This new and low cost technology for rabies vaccine production could be suitable for developing countries where rabies is an important health problem.

Adaptation, Physiological↗
PubMed1993

Structure and expression in baculovirus of the Mokola virus glycoprotein: an efficient recombinant vaccine.

The sequence of the glycoprotein gene of the Mokola virus, the more divergent element of the Lyssavirus genus, has been determined and the predicted protein structure compared to its counterpart in rabies vaccine strains. A global similarity of 54.3% was observed. The divergence affects particularly the rabies antigenic sites involved in the B-cell response. This provides a molecular basis for the absence of cross-protection between Mokola and rabies viruses and argues for the necessity of a specific anti-Mokola vaccine. Toward this goal, a cDNA copy of the glycoprotein gene was cloned into the baculovirus and expressed in Spodoptora frugiperda cells. A recombinant protein was produced in substantial amounts at the surface of the insect cells. Although less strongly glycosylated than the native viral glycoprotein produced in BHK-21 cells, the recombinant protein is antigenically and immunologically similar, it is recognized by specific monoclonal antibodies, and protects mice against an intracerebral challenge with Mokola virus. It therefore constitutes the first experimental genetically engineered vaccine against a rabies-related virus, and fulfills the international standard for protection.

Amino Acid Sequence↗
PubMed1993

Molecular diversity of the Lyssavirus genus.

The sequence of 5568 nucleotides of the 3' moiety of the Mokola virus genome (serotype 3 of lyssaviruses) encompassing the nucleoprotein (N), phosphoprotein, matrix protein, and glycoprotein genes is presented and compared to that of the vaccinal strains of serotype 1. It allowed us to determine consensus sequences derived from the transcriptional start/stop signals and the order of protein conservation (nucleoprotein > matrix protein > phosphoprotein) in lyssaviruses. The sequences of the N gene of a fox rabies virus isolate from France (serotype 1), Lagos bat virus (serotype 2), Duvenhage virus (serotype 4), two European bat lyssaviruses (EBL) subtype 1, and two EBL subtype 2 were also determined to study the genetic diversity throughout the whole Lyssavirus genus and reinvestigate the classification of this genus. Six clearly distinct genotypes can be distinguished according to their percentage of amino acid similarity. Genotypes 2 (Lagos bat virus) and 3 (Mokola virus) are the most phylogenetically distant from the vaccinal and classical rabies viruses of genotype 1. Genotypes 4 (Duvenhage virus) and 5 (EBL1) are closely related to each other. Genotype 6 is represented by EBL2. Compared to the N proteins of the four principal serotypes of the Vesiculovirus genus (vesicular stomatitis virus serotype New Jersey and serotype Indiana, Chandipura virus, and Piry virus), the N gene of lyssaviruses exhibits a lower genetic variability.

Amino Acid Sequence↗
PubMed1993

Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice.

A mouse model was designed to test in vivo the efficacy of rabies immune globulins and specific neutralizing monoclonal antibodies to prevent European bat lyssavirus 1 infection. Human or equine rabies immune globulins previously found to contain variable amounts of neutralizing bat lyssavirus crossreactive antibodies were passively transferred to mice receiving intramuscularly a lethal dose of bat lyssavirus type 1. Immune globulins did not protect mice well against bat lyssavirus 1 whereas they reduced the mortality caused by rabies virus. In contrast, mice inoculated with bat lyssavirus 1 or rabies virus survived when passively immunized with bat lyssavirus 1 specific monoclonal antibody (mAb 8-2). This monoclonal antibody, an IgG2 alpha, recognized an epitope located in the antigenic site IIa of rabies glycoprotein. A mutation replacing the lysine 198 by glutamate in a rabies variant abrogated sensitivity to this neutralizing antibody. Because of its broad neutralizing spectrum against wild virus isolates, including European bat lyssaviruses, this monoclonal antibody should be a good candidate for rabies immune globulin replacement. It could improve efficacy of rabies vaccination, used either alone or in conjunction with human rabies immune globulins or monoclonal antibody cocktail to supplement their lack of crossreactivity to European bat lyssavirus 1.

Animals↗
PubMed1993

Beta-propiolactone treatment impairs the biological activity of residual DNA from BHK-21 cells infected with rabies virus.

The effects of beta-propiolactone (BPL), an alkylating and virus inactivating agent, on the structural and in vitro biological properties of different DNA preparations from BHK-21 cells were investigated. Both uninfected and rabies virus-infected cells were used. Purified cellular DNA (celDNA) was used as the reference, and supernatants from infected cells were treated with BPL. For structural and biological studies three types of DNA preparation were tested: celDNA; purified DNA from cell (infected or uninfected) supernatant (pcsDNA) with or without BPL treatment; and residual cell DNA present in purified rabies virus (inactivated or not) preparations. Rabies infection and BPL (diluted 1:4000) treatment induced modifications in the structure of the three DNA types, including strand breaks and nicks. The damage to the DNA structure by BPL modifies the biological properties of the pcsDNA appraised by its ability to serve as the template in vitro for different polymerases. When rabies virus was inactivated with BPL diluted 1:1000 the DNA damage increased dramatically: small double-stranded DNA fragments (50-200 base pairs) were generated which could not function as templates for polymerases.

Animals↗
PubMed1993

The rabies virus genome: an overview.

The replication strategy, genome organization and extent of variation within the genome of the genus Lyssavirus is briefly reviewed. Strategies used in the approach to genome studies are discussed.

Genetic Variation↗
PubMed1993

Taxonomy and evolutionary studies on lyssaviruses with special reference to Africa.

Currently, the Lyssavirus genus is composed of four serotypes and the ungrouped European bat lyssaviruses (EBL). Using previously described PCR amplification and direct sequencing methods, the N genes of 70 representative lyssaviruses collected from 40 different countries were examined. From the results, a phylogenetic tree with six branches (genotypes) was constructed: genotypes 1-4 correlated with the classification of serotypes 1-4 and in addition EBL 1 and EBL 2 were identified as two further genotypes (5 and 6). Analysis of the 51 rabies (genotype 1) viruses in the study identified nine distinct groups which could be correlated with their geographical origins. A comparison of the six genotypes with four principal Vesiculovirus serotypes showed that their relationship was closer than that of even the two most closely related Vesiculovirus serotypes. Antigenic site sequences of isolates from vaccine/treatment failures were similar to vaccine and field strains, suggesting that failures were not due to genetic variation at the N protein level.

Africa↗
PubMed1992

Molecular epidemiology of rabies virus in France: comparison with vaccine strains.

A molecular epidemiological study of the rabies virus currently prevalent in France was carried out by directly sequencing polymerase chain reaction-amplified genes. The rabies virus pseudogene psi was chosen as the most divergent genomic area, and as such the best 'clock' for measuring virus evolution. Sequence comparisons between 12 wild rabies virus isolates indicated strong conservation whatever the host and wherever the virus had been isolated. This holds true for a unique wild reservoir, the fox. On the other hand, a good correlation between genetic and geographical criteria indicates a slow evolution of the wild virus in parallel with the spatio-temporal progression of the epizootic. In contrast to their intrinsic homogeneity (about 2% divergence), the wild isolate sequences showed a marked divergence from those of vaccine seed strains (about 14.7%). This finding invites world-wide molecular epidemiological studies, particularly in countries in which vaccination failures have been reported.

Animals↗
PubMed1992

Inhibition of rabies virus transcription in rat cortical neurons with the dissociative anesthetic ketamine.

In a previous study (B. P. Lockhart, H. Tsiang, P. E. Ceccaldi, and S. Guillemer, Antiviral Chem. Chemother. 2:9-15, 1991), we demonstrated an antiviral effect of the general anesthetic ketamine for rabies virus in neuronal cultures and in rat brain. This report describes an attempt to determine at what level ketamine acts on the rabies virus cycle in rat cortical neuron cultures. Immunofluorescence and [35S]methionine labelling of infected neurons showed that ketamine (1 to 1.5 mM) inhibited viral nucleoprotein and glycoprotein syntheses. Northern (RNA) blots of total RNA from drug-treated neurons, hybridized with 32P-labelled oligonucleotide probes for rabies virus nucleoprotein, matrix protein, and glycoprotein genes, showed a marked reduction (5- to 11-fold) in the levels of rabies virus mRNAs, relative to those in untreated neurons. No significant change in the levels of cellular beta-actin mRNA were detected in ketamine-treated cells. A similar antiviral effect was observed with MK-801; however, no inhibition of rabies virus synthesis was observed with the general anesthetic chloral hydrate. The antiviral effect was not complete; a time-dependent recovery of viral transcription and rabies virus protein synthesis was observed, but no infectious virus was released into the culture supernatant. The lack of any modification of cellular protein or mRNA synthesis by ketamine suggests an antiviral mechanism acting at the level of rabies virus genome transcription.

Animals↗
PubMed1992

Antigenic and molecular characterization of bat rabies virus in Europe.

The predominant role of Eptesicus serotinus in the epizootic of bat rabies in Europe was further outlined by the first isolation of the rabies virus from this species in France. The distribution of the virus was studied in naturally infected E. serotinus bats at the time of death and suggested that the papillae of the tongue and the respiratory mucosa may play a role in virus production and excretion. The analysis of 501 French rabies virus isolates from various animal species by antinucleocapsid monoclonal antibodies indicated that transmission of the disease from bats to terrestrial animals is unlikely. The antigenic profile of two isolates from French bats corresponded to that of European bat lyssavirus type 1 (EBL1). Comparisons of 12 different isolates from bats with antinucleocapsid and antiglycoprotein monoclonal antibodies and by direct sequencing of the polymerase chain reaction amplification product of the N gene indicated that EBL1, EBL2, Duvenhage virus (serotype 4 of lyssavirus), and the European fox rabies virus (serotype 1) are phylogenetically distant. They formed four tight genetic clusters named genotypes. EBL1 was shown to be antigenically and genetically more closely related to Duvenhage virus than to EBL2. We propose that EBL1 and EBL2 constitute two distinct genotypes which further serologic characterization will probably classify as new serotypes. We also report a simple method for the rapid characterization of EBL based on the digestion of the polymerase chain reaction product of the N gene by three restriction endonucleases.

Animals↗
PubMed1991

PCR technique as an alternative method for diagnosis and molecular epidemiology of rabies virus.

We have investigated the PCR amplification technique of viral nucleic acids as an alternative protocol for diagnosis and epidemiological studies of rabies virus. A primer set mapping in the nucleoprotein cistron allowed a specific and sensitive amplification of infected brain material, fulfilling the diagnosis requirements. One hundred samples checked by Southern or dot-blot analysis using both radioactive and non-radioactive probes showed identical results in parallel with routine techniques. For molecular epidemiological studies we selected another set of conserved primers flanking the highly evolutive pseudogene (psi gene) region. This set was found to be efficient for all tested fixed rabies virus strains or wild rabies virus isolates as well as the rabies-related Mokola virus. We describe a progressive characterization of the strain that could be extended from rapid typing by a limited panel of restriction enzymes, to the ultimate identification of the nucleotide sequence by an original direct sequencing technique of amplified segments.

Animals↗