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N Tordo

Publications and source records attributed to N Tordo.

At least 19 recordsLinked to original sources
PubMed2006

Rabies: epidemiological tendencies and control tools.

Rabies encephalitis still generates 50,000 human deaths/year. It is due to neuron infection by lyssaviruses. Seven genotypes (GT) are currently distinguished within the Lyssavirus genus which segregate in two phylogroups (PG). This classification is constantly evolving due to isolation of new lyssaviruses within bat populations. Functional differences exist between GTs in term of neurotropism, pathogenesis, induction of apoptosis, immunogenicity, and their molecular basis are starting to be elucidated. Lyssavirus vectors are mammals, preferentially from the Carnivora and Chiroptera orders. Phylogenetic reconstruction strongly supports that lyssaviruses evolved in chiropters long before the emergence of carnivoran rabies which very likely occurred through host-switchings from bats to carnivores. If dog rabies control is possible by vaccination and population control, if oral vaccination demonstrated its potential to eliminate rabies from a terrestrial wildlife reservoir (fox in Western Europe), it is unrealistic today to clear lyssaviruses from bats, while bat rabies is a growing concern for both public and animal health. As bat transmit divergent lyssavirus GTs which are not well prevented by available vaccine strains, there is a need to increase the protection spectrum of vaccines. DNA-based immunization with plasmids expressing chimeric G proteins (fusion of two halves from different GTs) was shown to be effective in inducing a complete immune response and to broaden the spectrum of rabies vaccines toward lyssavirus vaccines. Further, the lyssavirus G protein can carry foreign epitopes/antigens in the perspective of multivalent vaccines against various zoonoses of carnivores.

Animals↗
PubMed2005

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.

Animals↗
PubMed2001

Production and neurotropism of lentivirus vectors pseudotyped with lyssavirus envelope glycoproteins.

We investigated the production efficiency and the gene transfer capacity in the central nervous system of HIV-1-based vectors pseudotyped with either the G protein of the Mokola lyssaviruses (MK-G), a neurotropic virus causing rabies disease, or the vesiculo-stomatitis G protein (VSV-G). Both envelopes induced syncitia in cell cultures. They were incorporated into vector particles and mature virions were observed by electron microscopy. Vector production was two- to sixfold more efficient with VSV-G than with MK-G. For equivalent amounts of physical particles, vector titration was 5- to 25-fold higher with VSV-G than with MK-G pseudotypes on cultured cells, and in vivo gene expression in mouse brain was more intense. Thus, VSV-G pseudotypes were produced more efficiently and were more infectious than MK-G pseudotypes. Tropism for brain cells was analyzed by intrastriatal injections in rats. Both pseudotypes preferentially transduced neurons (70-90% of transduced cells). Retrograde axonal transport was investigated by instilling vector suspensions in the rat nasal cavity. Both pseudotypes were efficiently transported to olfactive neuron bodies. Thus, although coating HIV-1 particles with rabdhovirus envelope glycoproteins enables them to enter neuronal cells efficiently, pseudotyping is not sufficient to confer the powerful neurotropism of lyssaviruses to lentivirus vectors.

Animals↗
PubMed2001

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada.

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Animals↗
PubMed2001

Host switching in Lyssavirus history from the Chiroptera to the Carnivora orders.

Lyssaviruses are unsegmented RNA viruses causing rabies. Their vectors belong to the Carnivora and Chiroptera orders. We studied 36 carnivoran and 17 chiropteran lyssaviruses representing the main genotypes and variants. We compared their genes encoding the surface glycoprotein, which is responsible for receptor recognition and membrane fusion. The glycoprotein is the main protecting antigen and bears virulence determinants. Point mutation is the main force in lyssavirus evolution, as Sawyer's test and phylogenetic analysis showed no evidence of recombination. Tests of neutrality indicated a neutral model of evolution, also supported by globally high ratios of synonymous substitutions (d(S)) to nonsynonymous substitutions (d(N)) (>7). Relative-rate tests suggested similar rates of evolution for all lyssavirus lineages. Therefore, the absence of recombination and similar evolutionary rates make phylogeny-based conclusions reliable. Phylogenetic reconstruction strongly supported the hypothesis that host switching occurred in the history of lyssaviruses. Indeed, lyssaviruses evolved in chiropters long before the emergence of carnivoran rabies, very likely following spillovers from bats. Using dated isolates, the average rate of evolution was estimated to be roughly 4.3 x 10(-4) d(S)/site/year. Consequently, the emergence of carnivoran rabies from chiropteran lyssaviruses was determined to have occurred 888 to 1,459 years ago. Glycoprotein segments accumulating more d(N) than d(S) were distinctly detected in carnivoran and chiropteran lyssaviruses. They may have contributed to the adaptation of the virus to the two distinct mammal orders. In carnivoran lyssaviruses they overlapped the main antigenic sites, II and III, whereas in chiropteran lyssaviruses they were located in regions of unknown functions.

Adaptation, Physiological↗
PubMed2001

Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains.

Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein-reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template.

Amino Acid Sequence↗
PubMed2001

Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity.

The genetic diversity of representative members of the Lyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), the European bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and the Australian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.

Amino Acid Sequence↗
PubMed2000

Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein.

Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.

Animals↗
PubMed2000

DNA-based immunization against Lyssaviruses.

Rabies is a fatal encephalomyelitis. Most cases occur in developing countries and are transmitted by dogs. Because of their high cost, cell culture vaccines have not totally replaced the unsafe brain-derived vaccines which are still used in many developing countries. Moreover, there will be a need for vaccines against rabies-related viruses against which classical vaccines are not always effective. DNA vaccines would, therefore, be a valuable alternative for the production of cheaper rabies vaccines against a larger spectrum of viruses. In this review we report published data on DNA-based immunization with sequences encoding rabies and rabies-related virus antigens.

Animals↗
PubMed2000

Bat lyssavirus infections.

Bats, which represent approximately 24% of all known mammalian species, frequently act as vectors of lyssaviruses. In particular, insectivorous bats play an important role in the epidemiology of rabies and some rabies-like viruses, while the haematophagous vampire bats are the major wildlife vector for rabies in Latin America. In contrast, the role of fruit bats (flying foxes) in the epidemiology of the recently discovered Australian bat lyssavirus is only just emerging. Information on the pathogenesis of lyssaviruses in bats is scarce. However, in general, mortality in bats infected via a natural route appears to be low, and seroconversion occurs in many of those that survive. While transmission of rabies from an infected bat may be via a bite, other routes are apparently also possible. Methods for the diagnosis of bat lyssavirus infections in bats and terrestrial mammals (including humans) are similar to the classical procedures for rabies. Measures for the prevention and control of these diseases are also similar to those for rabies, although additional innovative methods have been tested, specifically to control vampire bat rabies.

Animals↗
PubMed2000

[Genetic diversity of Lyssavirus].

Rabies is a zoonosis of which the archaeological agents belong to the Lyssavirus species. Seven genotypes are known to exist. The isolates are grouped according to a) their geographical origin, b) historical context, c) type of vector. In parts of the world where several epidemic cycles exist, practical tools have been developed in order to distinguish between them more easily. Vaccine strains--all of which have been elaborated from genotype 1--offer little or no protection against the other genotypes. It is thus necessary to work on extending vaccine coverage.

Genetic Variation↗
PubMed2000

DNA-based immunisation against rabies and rabies-related viruses: towards multivalent vaccines.

Prototypes of multivalent DNA vaccines against lyssaviruses (LV: rabies and rabies-related viruses) and other viruses were developed using chimaeric LV glycoprotein (cLVG) DNA and cLVG DNA carrying foreign epitopes. cLVG is composed of the N-terminal half of an LV genotype (GT) containing antigenic site II, the C-terminal half of GT containing antigenic site III, as well as the transmembrane and cytoplasmic domains of the same or a different GT. Both antigenic sites induced virus neutralizing antibodies (VNAb). Foreign B and T cell epitopes inserted between the two halves of cLVG correspond to the B cell C3 neutralisation epitope of poliovirus VP1 protein and to the H2d CMH I restricted T cell epitope of the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV). In mice and dogs homogenous rabies virus G DNA induced protection against wild-type rabies virus whereas cLVG protected against lyssaviruses. cLVG DNA carrying foreign epitopes induced VNAb against LV and poliovirus and protection against LCMV. The results obtained clearly demonstrate the potential usefulness of cLVG for the development of multivalent vaccines against viral diseases, including rabies and zoonoses.

Animals↗
PubMed1999

Immunization of dogs with a DNA vaccine induces protection against rabies virus.

Rabies is a fatal encephalomyelitis which is transmitted to man, mostly by dogs in developing countries. This zoonosis can be prevented by vaccination of humans before or after exposure. However, a more radical approach is possible, involving the elimination of the principal vector/reservoir by vaccinating dogs. The vaccine must be effective, safe and inexpensive. Mass production of plasmids is possible and DNA-based immunization with a plasmid encoding the antigen responsible for inducing protection seems to be more cost-effective than classical techniques involving cell culture. Beagles were immunized by intramuscular (i.m.) injection with a plasmid encoding the rabies virus (PV strain) glycoprotein. Neutralizing antibodies against both wild-type rabies virus and European Bat Lyssaviruses (EBL1 and EBL2) were detected after a single injection and a boost, but levels of neutralizing antibodies against EBL1 were low. Moreover, all vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain. This is one of the first studies to demonstrate that dogs can be protected by DNA vaccines, and opens important perspectives for rabies control.

Animals↗
PubMed1999

Is there an advantage to including the nucleoprotein in a rabies glycoprotein subunit vaccine?

The PV rabies (genotype 1) G and N proteins were produced by recombinant baculoviruses in insect cells. We tested the ability of recombinant antigens to synergistically induce an immune response and, particularly, to broaden the spectrum of Lyssavirus-neutralizing antibodies produced. Cell-free preparations of recombinant proteins caused an immune response. Recombinant rabies G protein (RRG) from infected cell extract or supernatant induced virus neutralizing antibodies (VNAb) against rabies (CVS virus (genotype 1) and in a less extent against European Bat Lyssavirus-1 (EBL-1:genotype 5) Recombinant rabies N protein (RRN) induced antibodies that reacted with the rabies virus ribonucleoprotein (RNP) and primed mice for both the production of VNAb induced by inactivated and purified rabies virus and the protection conferred by RNP. RRN also had an adjuvant effect on VNAb production induced by RRG when the two recombinant proteins were physically associated either encapsulated in liposomes or subjected to ultrasound treatment. However, there was no increase in production of VNAb directed against EBL-1 although classical vaccines (genotype 1) induce partial protection against this virus. thus, beside its adjuvant effect there is some doubt as to whether including rabies N protein in a rabies subunit vaccine containing the recombinant G protein would be advantageous.

Animals↗
PubMed1999

Dynamics of rabies virus quasispecies during serial passages in heterologous hosts.

To understand the mutations and genetic rearrangements that allow rabies virus infections of new hosts and adaptation in nature, the quasispecies structure of the nucleoprotein and glycoprotein genes as well as two noncoding sequences of a rabies virus genome were determined. Gene sequences were obtained from the brain and from the salivary glands of the original host, a naturally infected European fox, and after serial passages in mice, dogs, cats and cell culture. A relative genetic stasis of the consensus sequences confirmed previous results about the stability of rabies virus. At the quasispecies level, the mutation frequency varies, in the following order: glycoprotein region (21.9 x 10(-4) mutations per bp), noncoding sequence nucleoprotein-phosphoprotein region (7.2-7.9 x 10(-4) mutations per bp) and nucleoprotein gene region (2.9-3.7 x 10(-4) mutations per bp). These frequencies varied according to the number, type of heterologous passages and the genomic region considered. The shape of the quasispecies structure was dramatically modified by passages in mice, in which the mutation frequencies increased by 12-31 x 10(-4) mutations per bp, depending on the region considered. Non-synonymous mutations were preponderant particularly in the glycoprotein gene, stressing the importance of positive selection in the maintenance and fixation of substitutions. Two mechanisms of genomic evolution of the rabies virus quasispecies, while adapting to environmental changes, have been identified: a limited accumulation of mutations with no replacement of the original master sequence and a less frequent but rapid selective overgrowth of favoured variants.

Animals↗
PubMed1999

Chimeric lyssavirus glycoproteins with increased immunological potential.

The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6).

Amino Acid Sequence↗
PubMed1998

DNA-based immunization for exploring the enlargement of immunological cross-reactivity against the lyssaviruses.

DNA-based immunization was used for studying the cross-reactivity of lyssavirus neutralizing antibodies and for exploring the induction of a wider range of protection against lyssaviruses. In order to immunize mice with homogeneous and chimeric genes of glycoproteins (G) from two divergent lyssaviruses, we used for the first time a new plasmid (pCI-neo) known to be a highly efficient vector for in vitro expression. The homogeneous plasmids pGPV and pGMok encoded the Pasteur virus (PV: genotype 1-GT-) and Mokola virus (Mok: GT 3) G, respectively. The chimeric pGMokPV encoded the NH2 part of GMok and the COOH part of GPV. These plasmids elicited full protection against intracerebral challenges with various lyssaviruses and a range of antigen-specific and non-specific immune responses. Virus neutralizing antibody (VNAb) levels were dose dependent and a single intramuscular (i.m.) injection of plasmids was sufficient to induce continuous high levels of VNAb. Production of antigen-specific T helper (Th), cytotoxic T cells (Tc) and non-specific natural killer cells was observed. Cross-reactivity studies showed that VNAb are obtained by immunizing with: (i) pGPV against GT 1 (classical rabies), GT 4 (Duvenhage: Duv), GT 5 (European Bat Lyssavirus: EBL-1) and GT 6 (European Bat Lyssavirus: EBL-2); (ii) pGMok against GT 2 (Lagos Bat: LB) and GT 3 (Mokola: Mok); (iii) pGMokPV against all GTs except GT 4 which is weakly neutralized. Therefore, the DNA-based immunization with the chimeric pGMokPV, could be very interesting to enlarge protection to all the lyssaviruses. According to the cross-reactivity of VNAb induced by the G genes, the lyssavirus GTs could be classified into two groups: the first including GT 1, 4, 5 and 6; the second including GT 2 and 3.

Animals↗