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.

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.

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.

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.

[Genetic diversity of Lyssavirus]. / Diversité génétique des 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.

The signature of balancing selection: fungal mating compatibility gene evolution.

A key problem in evolutionary biology has been distinguishing the contributions of current and historical processes to the maintenance of genetic variation. Because alleles at self-recognition genes are under balancing selection, they exhibit extended residence times in populations and thus may provide unique insight into population demographic history. However, evidence for balancing selection and extended residence times has almost exclusively depended on identification of transspecific polymorphisms; polymorphisms retained in populations through speciation events. We present a broadly applicable approach for detecting balancing selection and apply it to the b1 mating type gene in the mushroom fungus Coprinus cinereus. The comparison of neutral molecular variation within and between allelic classes was used to directly estimate the strength of balancing selection. Different allelic classes are defined as encoding different mating compatibility types and are thus potentially subject to balancing selection. Variation within an allelic class, where all alleles have the same mating compatibility type, provided an internal standard of neutral evolution. Mating compatibility in this organism is determined by the complex A mating type locus, and b1 is one of several redundantly functioning genes. Consequently, we conducted numerical simulations of a model with two subloci and varying levels of recombination to show that balancing selection should operate at each sublocus. Empirical data show that strong balancing selection has indeed occurred at the b1 locus. The widespread geographic distribution of identical b1 alleles suggests that their association with differing A mating types is the result of recent recombination events.

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.

The divergence-homogenization duality in the evolution of the b1 mating type gene of Coprinus cinereus.

The A mating type locus of the fungus Coprinus cinereus is a complex, multigenic locus which regulates compatibility and subsequent sexual development. Genes within the A locus such as the b1 gene studied here exhibit extreme sequence variation. In this work, we asked how b1 alleles have evolved high levels of variation and, at the same time, conserved function. We compared sequence variation in 17 alleles characterized as belonging to seven different compatibility classes. Comparison of sequence variation between representatives of these seven classes shows that different regions of the b1 gene have been subject to varying levels of substitution, recombination, and structural/functional constraints. The N-terminal region of the encoded protein, which has been previously demonstrated to govern self/nonself recognition, exhibited hypervariability with levels of amino acid identity as low as 41%. We used a novel analysis of neutral mutations accumulating in this gene to rule out the possibility that the N-terminal region is hypermutable. In contrast, the C-terminal region displayed heterogeneous levels of variation, with functional motifs being better conserved. In fact, there is a duality in the b1 gene between variability and conservation; recombination events have homogenized the C-terminal region, while recombination events are undetectable in the N-terminal region. The ability to regulate sexual development is maintained in all of the mating compatibility alleles studied, and these data suggest that some functional motifs may tolerate high levels of substitution.

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.

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.

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.