[Neuronal expression of foreign genes with recombinant rabies virus variants]. / Expression neuronale de gènes étrangers à l'aide de virus rabiques recombinants.

Rabies virus variants obtained by recombinant DNA techniques enabled us to use the high neurotropism of rabies virus to express foreign genes (e.g: Chloramphenicol Acetyl Transferase gene) in neuronal cell cultures as well as in rodent brain. The foreign gene was inserted in the viral pseudogene region; this insertion did not affect the neurotropism of rabies virus, as shown by infection of neuronal cell cultures without any major cytopathic effects for several days. Stereotaxic inoculation of these rabies virus variants into rat striatum indicated that insertion of the foreign gene did not alter the viral axonal transport and the subsequent widespread brain infection. These data allow to consider rabies virus as a vector for the selective expression of foreign genes in neurons.

[Rabies is a risk for travelling children]. / Le risque de rage chez l'enfant qui voyage.

Rabies remains a dreadful disease which kills about 50,000 people per year, mostly in Asia, Africa, South America and Central Europe. Between 30% an 50% of the victims are young children. Modern rabies vaccines are safe and immunogenic. Therefore parents must be informed on the risk of rabies, and pre-exposure vaccination must be performed for children traveling often or for periods longer than one month in canine enzootic countries. Post-exposure treatment must be initiated without delay with modern vaccines wherever available, according to approved schedules. Pre-exposure vaccination is particularly useful in remote places where modern vaccines and immunoglobulins are not readily available.

Infection characteristics of rabies virus variants with deletion or insertion in the pseudogene sequence.

We investigated the infection characteristics of recombinant rabies virus variants modified in the pseudogene sequence. Infection of neuronal cell lines by the SAD W9 and SAD V* variants (respectively with deletion or insertion in this sequence) showed no significant differences as compared to the parental strain, the attenuated strain SAD B19, in infection characteristics such as number of infected cells or viral yield. The inoculation of mice by these variants resulted in similar infection patterns and pathogenicity. Stereotaxic inoculation of the different variants into the rat striatum showed that deletion or insertion did not affect the axonal virus spread, nor did insertion of a complete additional transcription unit, that could be expressed in the areas connected to the inoculation site. These results show that the pseudogene sequence is not involved in viral spread and pathogenicity and confirm the availability of this domain for targeting and expression of foreign genes into neurons.

Rabies vaccines: a review of progress towards improved efficacy and safety.

Over the years, the technology for producing human rabies vaccines has undergone many improvements. These improvements consist in the use of tissue cultures for the production of viral antigens, replacing the former nervous tissue substrate vaccines. The low virus yields in tissue cultures led to the development of the concentration and purification of virus supernatants. Another technical improvement was obtained by using microcarriers for virus production in VERO cell suspension cultures. This technique permits commercial-scale production of rabies vaccine, lowering production costs and thus extending the availability of the vaccine to a broader population in developing countries. Besides improvements in rabies vaccine production technology, the use of various vaccination regimens and routes of administration in field trials has resulted in considerable gains in our experience of postexposure treatment (PET) of this disease. The standard WHO recommended regimen for PET using concentrated and purified tissue culture vaccines consists of a 5-dose course of intramuscular injections at days 0, 3, 7, 14 and 28. Reduced vaccination regimens such as the 2-1-1 have been proven to be efficient in raising protective antibody responses. Reduction in the total volume of rabies vaccine is also possible by using the intradermal route of injection, provided the vaccine is administered at several sites. The overall consequence is a progressive shift in the worldwide use of rabies vaccines from those of nervous tissue origin to the contemporary tissue culture vaccines.

Alteration of the actin-based cytoskeleton by rabies virus.

We investigated the effect of rabies virus infection on the actin cytoskeleton using various techniques. Confocal microscopic examination of rabies virus-infected neuroblastoma cells at late stages of infection revealed a dramatic decrease in F-actin staining. The results of a fluorimetric assay with pyrenylated actin indicated that purified rabies virus nucleocapsid has no direct action on the kinetics of actin polymerization and only a weak effect on the final extent of polymerization. Video-microscopy experiments with purified components showed that rabies virus nucleocapsid inhibits the actin-bundling effect induced by dephospho-synapsin I, a neuron-specific protein which is known to exert a control on the actin-based cytoskeleton. Thus, the observed decrease in F-actin staining in infected cells might be ascribed to an indirect action of rabies nucleocapsid on the effects of actin-binding proteins such as synapsin I.

[Immunogenicity of the avirulent strain RV194-2 of the rabies virus in mice]. / Imunogenicidade da cepa avirulenta RV194-2 do vírus rábico em camundongos.

RV194-2 rabies virus, an avirulent mutant of CVS strain, induces an inapparent infection limited to the central nervous system (CNS) in adult mice inoculated intracerebrally. This fact suggest that immune response of the host is able to eliminate the virus in CNS. For this reason, we have studied the induction of interferon and the humoral immune responses in BALB/c mice after RV194-2 inoculation. These mice presented high levels of interferon in the plasma and in the brain, with elevated levels of neutralizing antirabies antibodies. The 2-5A synthetase, an enzyme marker of interferon action, was analyzed in the brain of inoculated animals. Its enhancement in parallel to the interferon production in the brain, showed biochemical evidence that this interferon is active. Forty five days after RV194-2 virus inoculation, mice were protected against a challenge with the CVS virulent strain. The results presented herein show that RV194-2 strain has a high level of immunogenicity.

Induction of immunoreactive interleukin-1 beta and tumor necrosis factor-alpha in the brains of rabies virus infected rats.

Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) are important cytokines in the development of brain inflammation during pathological process. During rabies virus infection, the level of these proinflammatory cytokines are enhanced in the brain. In the present study we determined the cellular localization of these two cytokines by immunocytochemistry in brains of rats infected with rabies virus, at different time-intervals of the disease (day 1, 3, 4, 5 and at final stage day 6 post-infection (p.i.)). Cellular identification of IL-1 beta (irIL-1 beta) and TNF alpha (irTNF alpha) immunopositive cells was studied using a polyclonal antibody against these cytokines and against glial fibrillary acidic protein (GFAP) to detect astrocytes and GSA-I-B4 isolectin to detect microglial cells and/or infiltrating macrophages. In brains of control and early infected rats, irIL-1 beta was only detected in fibers located in the hypothalamus, supraoptic and tractus optic nuclei and infundibular nucleus. From day 4 onwards until day 6 p.i., enhanced irIL-1 beta was found and identified either in activated ameboid and/or infiltrated macrophages (amygdala, thalamus, internal capsula, subtantia nigra, septal nuclei and around blood vessels), or in activated ramified cells (hypothalamus and periventricular nucleus, piriformis and cingulate cortex, hippocampus). IrTNF alpha was observed in the brains of rats at a final stage of disease (day 5 and 6 p.i.): in the hypothalamus, the amygdala, the internal capsula, the thalamus, the septal nuclei, the hippocampus, the habenular nuclei and around the blood vessels. Ir-TNF alpha was detected in round cells identified as ameboid microglia and/or infiltrated macrophages. A marked activation of microglial and astroglial cells was observed mainly in the hypothalamus, the thalamus and hippocampus and around the blood vessels, at day 4 p.i. and later, revealing a high central inflammatory reaction in brains of rabies virus infected rats. These results showed that IL-1 beta and TNF alpha are produced in the brain both by local microglial cells and infiltrating macrophages during rabies infection. Thus, these cytokines may play an important role in coordinating the dramatic inflammatory response associated with the rabies-encephalopathy as well as in the neural modification and alteration of brain functions.

Ionizing radiation modulates the spread of an apathogenic rabies virus in mouse brain.

Ionizing radiation has been shown to affect a broad range of viral diseases including neurotropic infections through an immunosuppression mechanism. In the present study we have investigated the effect of ionizing radiation on the characteristics of neurotropic infection by rabies virus, which has the unusual feature of infecting almost exclusively neurons. In order to analyze better the effect produced, the study concerned the spread of an apathogenic rabies virus variant in mouse brain. Irradiation was shown to increase both the intensity and duration of the infection in a reversible and dose-dependent manner and was effective in whole-body irradiation and in head-protected body irradiation, whereas cephalic irradiation had no effect. These results underline the role played by the immune system in the regulation of neurotropic virus infections in the brain and show that phenomena such as viral clearance and time-course of a neurotropic viral infection may be significantly modified by ionizing radiation, even for viruses whose infection involves only neurons.

Rabies: interactions between neurons and viruses. A review of the history of Negri inclusion bodies.

The first clear-cut description of a virus-nerve cell interaction was made by Adelchi Negri in 1903 with the detection of cytoplasmic bodies (Negri bodies) in subsets of neurons in the brain from rabies-infected animals. A biographical sketch of Negri is given here; he was born in Perugia, Italy, in 1875 and died in Pavia in 1912. In 1900 Negri became assistant to Camillo Golgi, who encouraged him to study rabies-infected brains with histological techniques. The report of intraneuronal bodies described by Negri as specific for rabies stimulated an intense debate both concerning their diagnostic value and their nature. The diagnostic value was finally determined in a study by Negri's wife, Lina Negri-Luzzani, in 1913, while the viral nature of the bodies had to await the introduction of electron microscopy and immunohistochemistry. However, the true significance of the Negri bodies is still mysterious, since they only develop in subsets of infected neurons and occur mainly after infection with wild, so-called 'street', virus strains and not after infection with strains passaged in the laboratory, so-called 'fixed' strains.

[Immunoprophylaxis of rabies: current recommendations]. / L'immuno-prophylaxie de la rage: actualisation des recommandations.

Pre-exposure rabies vaccination should comprise 3 injections (day 0, day 7, day 28) followed by boosters 1 year later then every 5 years. Populations who are particularly exposed due to occupation, regular contact with animals in endemic areas during leisure activities or holidays should be vaccinated, especially if access to post-exposure treatment is difficult. Post-exposure treatment should comprise 5 injections (day 0, day 3, day 7, day 14, day 28) which must be given with specific immunoglobulins on day 0 if there are penetrating wounds. In persons whose prior vaccination status is well-documented and correctly maintained, post-exposure treatment may be limited to 2 injections on day 0 and day 3. The vaccine is given is intramuscular injection in the deltoid region. Immunoglobulins are used at the dose of 20 IU/kg for human immunoglobulins and at 40 IU/kg for horse immunoglobulins. The injections are infiltrated around the lesions and the remaining quantity injected in the gluteus muscle. Seroconversion must be monitored by assaying neutralizing antibodies (titre > or = 0.5 IU/ml with the RFFI Test) in vaccinated populations who regularly exposed. Monitoring can also be useful after post-exposure treatment in certain specific cases (immunosuppressed subjects, treatment protocol incorrectly or incompletely applied). An antibody titre under 0.5 IU/ml requires an immediate vaccine injection.

Alteration of interleukin-1 alpha production and interleukin-1 alpha binding sites in mouse brain during rabies infection.

We have evaluated the effect of rabies virus infection on interleukin-1 alpha (IL-1 alpha) production and its receptors in mouse brain. Study of virus dissemination in the central nervous system (CNS) showed a massive infection of main brain structures from day 4 post infection (p.i.) up to the agony stage on day 6 p.i. At the same time, IL-1 alpha concentrations increased in cortical and hippocampal homogenates, whereas no change was detected in serum. In non-infected mice, IL-1 alpha binding sites were observed in the dentate gyrus, the cortex, the choroid plexus, the meninges and the anterior pituitary. During rabies virus infection, a striking decrease in IL-1 alpha binding sites was observed on day 4 p.i. with a complete disappearance on day 6 p.i., except in the pituitary gland where they remained at control level. In conclusion, concomitantly with the early rabid pathological signs, brain IL-1 alpha production and IL-1 alpha binding sites are specifically and significantly altered by brain viral proliferation. These results indicate that IL-1 alpha could be involved in the brain response to viral infection as a mediator and could participate in the genesis of the rabies pathogeny.

[Evolution of human anti-rabies vaccines from Pasteur to the present]. / L'évolution des vaccins antirabiques humains de Pasteur à nos jours.

Since the first immunization of man against rabies in 1885 by Louis Pasteur, antirabies vaccine has been continuously improved. Treatment failures, clinical infections by the fixed virus, neuroparalytic accidents in connection with myelin were progressively eliminated. Vaccines can be standardized and accurately controlled. After the original spinal cord, adult or newborn animals brains, embryonated eggs, primary tissue cultures, diploid and permanent cell lines have been used for the vaccine production. Today, safe and potent vaccines are available. New products might be developed from the technology of genetic recombinants.

Receptors for interleukin-1 in the central nervous and neuroendocrine systems. Role in infection and stress.

The functional interactions between the Immune (IS) and the Central Nervous Systems (CNS) are clearly indicated by the fact these systems are sharing mediators and receptors. Interleukins and more specifically Interleukin-1 (IL-1) have been shown to be powerful regulators of both system activity which suggested IL-1 receptors in the CNS. IL-1 receptors, similar to type I lymphocyte receptors, have been characterized in murine nervous structures (dentate gyrus of the hippocampus and frontal cortex), in vascular structures (vessels, choroid plexus) and in a neuroendocrine structure (anterior pituitary). Stimulation of the immune system and of IL-1 synthesis by bacterial product (intra peritoneal injection of LPS) induced a marked decrease of IL-1 receptor levels in the CNS. Under the same conditions pituitary receptors were unaffected indicating the autonomy of brain functioning. This decrease is in relation with an increase in local IL-1 synthesis as indicated by the increase of IL-1 mRNA in the brain tissue. During viral infection (rabies virus) very similar results are observed. Brain receptors are decreasing in the brain at day 4 post infection while IL-1 concentration is increasing in the brain tissue. Pituitary receptors are not modified during the evolution of the disease. Stress and glucocorticoid treatment are strong inhibitors of immune functions by inhibiting IL-1 synthesis. Neither treatment modified brain receptors suggesting that IL-1 synthesis is not modulated by glucocorticoids in the CNS as in the immune system. However an increase in pituitary receptor level was observed in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon production and immune response induction in apathogenic rabies virus-infected mice.

Pathogenic parental rabies virus strain CVS (challenge virus standard) and its apathogenic variant RV194-2 were shown to differ in their ability to induce interferon (IFN) and immune response of the host. After intracerebral inoculation, IFN and antibody production was higher in the RV194-2 virus-infected mice than in the CVS infection. The enhancement of 2-5A synthetase activity, an IFN-mediated enzyme marker, showed biochemical evidence that IFN is active in both apathogenic and pathogenic infections. On the other hand, spontaneous proliferation in vitro of thymocytes and splenocytes from CVS virus-infected mice was strongly inhibited in contrast to the RV194-2 infection. In the CVS infection, the thymocyte proliferation was more affected than the splenocyte proliferation. However, in the RV194-2 infection, the thymocyte proliferation was higher than of the splenocytes. These results suggest a better performance of T-cell response to the RV194-2 infection than to the CVS infection. This fact can be critical for an enhancement of antibody production in the apathogenic infection and subsequent virus clearance from the brain of RV194-2 virus-infected mice.

Modification of tritiated gamma-amino-n-butyric acid transport in rabies virus-infected primary cortical cultures.

The role of brain neurotransmitter transport processes in rabies virus infection of neurons was examined. The uptake and release of gamma-amino-n-butyric acid (GABA) in rabies virus-infected embryonic rat cortical neurons was assayed using tritiated ligands. A 45% reduction of [3H]GABA uptake was observed 3 days post-infection, when a maximum level of infectious particle release occurs. At this time, kinetic analysis revealed significant changes in Vmax, whereas no changes were found in Km values in comparison with the control values. K+ and veratridine-induced [3H]GABA release was increased in infected cultures (98% and 35%, respectively) as compared with control values. The results obtained from rabies virus-infected cultures provide some preliminary evidence of the involvement of GABA in the pathogenesis of rabies.

Alteration of potassium-evoked 5-HT release from virus-infected rat cortical synaptosomes.

Potassium-evoked 3H-serotonin (5-HT) release from rat cerebrocortical synaptosomes was performed after peripheral inoculation with fixed rabies virus CVS (challenge virus strain). At the onset of clinical symptoms, the rats were sacrificed, synaptosomes were prepared from dissected cortices and assayed for K+ evoked 5-HT release. The results show a decrease in evoked 5-HT release from virus-infected synaptosomes. Alterations in serotoninergic transmission in rabies virus infected brain cortex indicate a possible involvement in the triggering of pathogenetic mechanisms relating to the clinicopathological manifestations of the viral disease.

Rabies virus selectively alters 5-HT1 receptor subtypes in rat brain.

Rabies virus infection in man induces a series of clinical symptoms, some suggesting involvement of the central serotonergic system. The results of the present study show that, 5 days after rabies virus infection in rat, the total reversible high-affinity binding of [3H]5-HT in the hippocampus is not affected, suggesting that 5-HT1A binding is not altered. 5-HT1B sites identified by [125I]cyanopindolol binding are not affected in the cortex 3 and 5 days after the infection. Accordingly, the cellular inhibitory effect of trifluoromethylphenylpiperazine (TFMPP) on the [3H]acetylcholine-evoked release, presumably related to 5-HT1B receptor activity, is not modified 3 days after infection. In contrast, [3H]5-HT binding determined in the presence of drugs masking 5-HT1A, 5-HT1B and 5-HT1C receptors, is markedly (50%) reduced 3 days after the viral infection. These results suggest that 5-HT1D-like receptor subtypes may be affected specifically and at an early stage after rabies viral infection.

Rabies RNA synthesis, detected with cDNA probes, as a marker for virus transport in the rat nervous system.

The kinetics of viral RNA synthesis in different parts of the rat brain, infected with fixed or street rabies virus strains, is correlated with their anatomical neuronal connections with the masseter muscles, using hybridization with rabies cDNA probes. Viral RNA synthesis is first detected in the brain-stem and in the pons where the direct anatomical projection of the masseter muscle nervous arborization into the sensory and motor nuclei is located, through the trigeminus nerve. Rabies RNA detection is delayed in the other regions of the rat brain depending on the time course of virus transport from the trigeminal nuclei through multiple nervous connections.