Patrice Coulon

Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice

Fixed rabies virus strains (ERA and CVS) produce a fatal paralytic disease in mice after intracerebral or intramuscular injection. Some antigenic mutants of both CVS and ERA viruses with a substitution in position 333 of the glycoprotein (arginine is replaced either by a glutamine, a glycine, or an isoleucine) are totally avirulent for adult mice whatever the dose and the route of inoculation. Here we report an exhaustive investigation of the effect of amino acid 333 on viral virulence. New antigenic mutants were isolated from either CVS, CVS derivatives, or SADBern having arginine in position 333 encoded by CGG, AGG, CGU, or AGA respectively. This study shows that when arginine is replaced by either a leucine, an isoleucine, a methionine, a cysteine, or a serine, the antigenic mutant is also totally avirulent. But when arginine is replaced by a lysine it is still pathogenic although the LD50 by the intracerebral route is higher. Furthermore 41 independent virulent revertants were isolated from four avirulent mutants (with a glycine, a glutamine, a methionine, or a serine in position 333 of the glycoprotein). Thirty-nine regained an arginine at position 333 and 2 had a lysine. From this analysis it appears that the presence of a positively charged amino acid (arginine or lysine) in position 333 of the glycoprotein is necessary for viral virulence.

Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents

The aim of the present review is to summarise available studies dealing with the respiratory control exerted by pontine noradrenergic neurones in neonatal and adult mammals. During the perinatal period, in vitro studies on neonatal rodents have shown that A5 and A6 neurones exert opposite modulations onto the respiratory rhythm generator, inhibitory and facilitatory respectively, that the anatomical support for these modulations already exists at birth, and that genetically induced alterations in the formation of A5 and A6 neurones affect the maturation of the respiratory rhythm generator, leading to lethal respiratory deficits at birth. The A5-A6 modulation of the respiratory rhythm generator is not transient, occurring solely during the perinatal period but it persists throughout life: A5 and A6 neurones display a respiratory-related activity, receive inputs from and send information to the medullary respiratory centres and contribute to the adaptation of adult breathing to physiological needs.

Phox2a Gene, A6 Neurons, and Noradrenaline Are Essential for Development of Normal Respiratory Rhythm in Mice

Although respiration is vital to the survival of all mammals from the moment of birth, little is known about the genetic factors controlling the prenatal maturation of this physiological process. Here we investigated the role of the Phox2a gene that encodes for a homeodomain protein involved in the generation of noradrenergic A6 neurons in the maturation of the respiratory network. First, comparisons of the respiratory activity of fetuses delivered surgically from heterozygous Phox2a pregnant mice on gestational day 18 showed that the mutants had impaired in vivo ventilation, in vitro respiratory-like activity, and in vitro respiratory responses to central hypoxia and noradrenaline. Second, pharmacological studies on wild-type neonates showed that endogenous noradrenaline released from pontine A6 neurons potentiates rhythmic respiratory activity via α1 medullary adrenoceptors. Third, transynaptic tracing experiments in which rabies virus was injected into the diaphragm confirmed that A6 neurons were connected to the neonatal respiratory network. Fourth, blocking the α1 adrenoceptors in wild-type dams during late gestation with daily injections of the α1 adrenoceptor antagonist prazosin induced in vivo and in vitro neonatal respiratory deficits similar to those observed in Phox2a mutants. These results suggest that noradrenaline, A6 neurons, and the Phox2a gene, which is crucial for the generation of A6 neurons, are essential for development of normal respiratory rhythm in neonatal mice. Metabolic noradrenaline disorders occurring during gestation therefore may induce neonatal respiratory deficits, in agreement with the catecholamine anomalies reported in victims of sudden infant death syndrome.

Spread of the CVS strain of rabies virus and of the avirulent mutant AvO1 along the olfactory pathways of the mouse after intranasal inoculation

Abstract After intranasal instillation in the mouse, rabies virus (CVS strain) selectively infected olfactory receptor cells. In the main olfactory bulb (MOB), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. Beyond the MOB, CVS spread into the brain along the olfactory pathways. This infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. CVS also penetrated the trigeminal system. The avirulent mutant AvOl, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as CVS. During the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the MOB and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. On the other hand, other neuronal cells permissive for CVS, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of AvO1 to penetrate several categories of neurons. From these observations, we concluded that CVS should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them.

Rapid sequence evolution of street rabies glycoprotein is related to the highly heterogeneous nature of the viral population

The sequence of the glycoprotein gene of a street rabies virus was determined directly using fragments of a rabid dog brain after PCR amplification. Compared with that of the prototype strain CVS, this sequence displayed 10% divergence in overall amino acid composition. However only 6% divergence was noted in the ectodomain suggesting that structural constraints are exerted on this portion of the glycoprotein. A human strain isolated on cell culture from the saliva of a patient with clinical rabies had only five amino acid differences with the canine isolate, an indication of their close relatedness. These differences could have originated during transmission from dog to dog, or from dog to man, or during isolation on cell culture; they are nonetheless indicative of a genetic evolution of street rabies virus. This evolution was further evidenced by the selection of cell-adapted variants which displayed new amino acid substitutions in the glycoprotein. One of them concerned antigenic site III where arginine at position 333 was replaced by glutamine. As expected this substitution conferred resistance to a site IIIa monoclonal antibody (MAb), but surprisingly did not abolish neurovirulence for adult mice. However, a decrease in the neurovirulence of the cell-adapted variant in the presence of a site IIIa specific MAb was noted, suggesting that neurovirulence was due to a subpopulation neutralizable by the MAb. Simultaneous presence of both the parental and variant sequences was indeed evidenced in the brain of a mouse inoculated with the cell-adapted variant; during multiplication in the mouse brain, the frequency of the parental sequence rose from less than 10% to nearly 50%, indicating the selective advantage conferred by arginine 333 in nervous tissue. Altogether these results were suggestive of an intrinsic heterogeneity of street rabies virus. This heterogeneity was further demonstrated by the sequencing of molecular clones of the glycoprotein gene, which revealed that only one-third of the viral genomes present in the brain of a rabid dog had the consensus sequence. Two-thirds of the clones analyzed displayed from one to three amino acid substitutions. Such heterogeneous populations have been referred to as quasispecies, a concept which implies heterogeneous populations kept together in a dynamic equilibrium. This equilibrium could be rapidly displaced, giving the virus the capacity to adapt easily to new environmental conditions.

The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice.

The sequential distribution of transneuronally infected neurons was studied in the olfactory pathway of mice after unilateral inoculation of the challenge virus standard (CVS) strain in the nasal cavity. A first cycle of viral multiplication was observed in a subpopulation of receptor cells scattered in the main olfactory epithelium and in the septal organ. No viral spread from cell body to cell body was reported even in later stages of infection. The second round of viral replication which took place in the ipsilateral main olfactory bulb at 2 and 2.5 days post-inoculation (p.i.), involved second order neurons and periglomerular cells, known to be directly connected with the axon terminals of receptor cells. Also reported as a result of a second cycle of viral replication, was surprisingly the spread of CVS at 2 and 2.5 days p.i. in bulbar interneurons located in the internal plexiform layer and in the superficial granule cell layer, as well as that of 2 ipsilateral cerebral nuclei, the anterior olfactory nucleus and the horizontal limb of the diagonal band. From day 3, a rapid spread of CVS was suggested by detection of virus in all ipsilateral direct terminal regions of the second order neurons and in most tertiary olfactory projections. The locus coeruleus, a noradrenergic nucleus which sends direct afferents to the olfactory bulb, never appeared immunoreactive. In spite of a certain inability of CVS to infect some neuron types, the virus appears relevant to provide new information regarding the complex network of olfactory-related neurons into the CNS.

Vaccination against rabies : construction and characterization of SAG2, a double avirulent derivative of SADBern

A double avirulent mutant was isolated from the SADBern strain of rabies virus by two successive selection steps using neutralizing anti-glycoprotein monoclonal antibodies. Both mutations affect the triplet coding for amino acid 333 of the glycoprotein. The resulting virus, called SAG2, has a glutamate coded by GAA in position 333 instead of an arginine. This new codon differs by two nucleotides from all the arginine triplets. SAG2 is avirulent in adult mice by intracerebral and intramuscular routes and it protects mice against a challenge by the CVS strain. This double mutant is still avirulent after three successive passages in suckling mouse brain or after ten successive cycles of multiplication in cell culture. Because it is protective and genetically stable, SAG2 represents an improvement on SAG1 which is already used for oral vaccination of foxes in Switzerland and France. It could also be a candidate for oral vaccination of dogs against rabies.

Influence of acetylcholinesterase on embryonic spinal rat motoneurones growth in culture: a quantitative morphometric study

Rat spinal motoneurones sampled at day embryonic 15 were purified using a Nycodenz gradient and cultured in defined medium, during 7 days, on glass coverslips coated with poly‐l‐lysine and laminine. Purified acetylcholinesterase (AChE), ecothiopate, BW 284C51 and fasciculin II, inhibitors of either the catalytic or peripheral site of AChE, were added to the defined medium. Morphological changes of spinal motoneurones were measured using a statistical quantitative morphometric method, allowing the determination of various parameters such as the number of neurites and bifurcations, the length of neurites, the surface and spreading index. Presence of AChE in the medium (4 units/mL) increases the surface and the total length of neurites and axons without any change in the spreading index. When spinal motoneurones were cultured on AChE coated substrate, neurones rapidly migrate and form clusters. Addition of ecothiopate (10–6m) in the medium, which selectively blocks the catalytic site of AChE, leads to a slight increase in the number of primary neurite and a decrease of the spreading index during the three first days in culture. BW 284C51 (10–5m) which blocks the catalytic site but also affect the peripheral one, significantly reduces the number of primary neurites and increases the number of bifurcations. Fasciculin II, a potent blocker (10–9m) of the AChE peripheral site induces a decrease of both primary neurites and bifurcations with a significant increase of the length and growth velocity of the axon, giving a drastic enhancement of the spreading index. These phenomena are discussed in terms of catalytic and non‐catalytic function of AChE, including the involvement of the enzyme in adhesive processes, occurring during growth and differentiation of spinal motoneurones.

Multiple cerebellar zones are involved in the control of individual muscles: a retrograde transneuronal tracing study with rabies virus in the rat

To identify cerebellar regions that are involved in the control of limb muscles, rabies virus was injected into the tibialis anterior (TA), the gastrocnemius (GC) or, for comparison, into the flexor digitorum (FD) muscles of the rat. Progression of retrograde transneuronal infection at supraspinal levels was assessed after variable time spans and was divided into three groups. Initially, infected neurons were observed in the reticular formation, lateral vestibular nucleus, red nucleus and motor cortex (group 1). Group 2 was characterized by labelling within the cerebellar nuclei as well as of two vermal strips of Purkinje cells (PCs). Double‐labelling with zebrin enabled identification of these strips as the lateral part of the A1‐ and B‐zone. For TA both zones were ipsilateral, whereas for GC the A1 strip predominated contralaterally. Group 3 infections showed additional labelling of multiple, in part bilateral, identifiable strips of PCs in vermis, paravermis and hemisphere. FD injections resulted in less robust labelling of vermal strips and more pronounced labelling within paravermal and hemispheral zonal regions. Only sporadic labelling in corresponding regions of the inferior olive and no labelling of cortical interneurons or granule cells was observed. Prolonged infection was seen to result in degeneration of PCs and possibly of motoneurons. We conclude that vermal, paravermal as well as hemispheral zones of the cerebellar cortex converge upon motoneurons that innervate a particular muscle. In addition, individual zones may control motorpools of different muscles and thus contribute to muscle synergies.

Changes to interneuron-driven striatal microcircuits in a rat model of Parkinson's disease

Striatal interneurons play key roles in basal ganglia function and related disorders by modulating the activity of striatal projection neurons. Here we have injected rabies virus (RV) into either the rat substantia nigra pars reticulata or the globus pallidus and took advantage of the trans-synaptic spread of RV to unequivocally identify the interneurons connected to striatonigral- or striatopallidal-projecting neurons, respectively. Large numbers of RV-infected parvalbumin (PV+/RV+) and cholinergic (ChAT+/RV+) interneurons were detected in control conditions, and they showed marked changes following intranigral 6-hydroxydopamine injection. The number of ChAT+/RV+ interneurons innervating striatopallidal neurons increased concomitant with a reduction in the number of PV+/RV+ interneurons, while the two interneuron populations connected to striatonigral neurons were clearly reduced. These data provide the first evidence of synaptic reorganization between striatal interneurons and projection neurons, notably a switch of cholinergic innervation onto striatopallidal neurons, which could contribute to imbalanced striatal outflow in parkinsonian state.