Rabies virus in nasal mucosa of naturally infected bats.

Rabies virus was demonstrated in the olfactory mucosa of naturally infected bats by staining with fluorescent antibody and by isolation of the virus from the nasal tissues. The olfactory mucosa is a potential portal of entry and exit for airborne rabies virus in bat caves.

From rabies to transmissible spongiform encephalopathies: an immune-mediated microbial trigger involving molecular mimicry could be the answer.

The concept of experimental allergic encephalomyelitis (EAE) being linked to both rabies post-vaccination encephalomyelitis and multiple sclerosis (MS) has raised the intriguing question whether animal studies carried out for the induction and transmission of transmissible spongiform encephalopathies (TSEs) using brain antigens including prions do have a similar immunopathogenetic mechanism. Although an essential link between autoimmunity and MS has been well established, its role in the pathogenesis of TSEs is generally lacking. However, auto-antibodies to myelin proteins and/or other neuronal antigens such as neurofilaments and prion proteins have been reported in animals with bovine spongiform encephalopathy (BSE) and scrapie as well as in patients with Creutzfeld-Jakob disease (CJD) and kuru. Acinetobacter has been suggested as a possible triggering microbial factor in the initiation of the autoimmune responses in these diseases because bacterial molecular sequences resemble brain antigens, especially in animals affected with BSE and patients with MS and CJD. These possibilities need to be evaluated further with longitudinal prospective studies carried out on larger numbers of animals or humans with such diseases. The transplantation of saline suspensions of brain homogenates will evoke immunological responses and therefore, the results in the study of MS and other neurological diseases have to be interpreted with caution.

Neurotropism of rabies virus. An in vitro study.

The relative susceptibility of neurons and glia, grown as monolayers in vitro, to rabies virus infection was explored. Established cell lines of neuronal or glial phenotype and primary cultures of cells derived from mouse dorsal root ganglia (DRC) or brain were used as homologues of the targets of rabies virus in the nervous system. Fixed rabies virus (CVS) strain was used in most experiments; other fixed rabies strains (PV, HEP, ERA) and a street rabies virus isolate were used in some. Virus-cell tropism was determined by immunofluorescence assay for rabies nucleocapsid antigen and cell permissivity was assessed by titration of virus yields. Neuronal cells always exhibited a much greater susceptibility to infection and a greater propensity to sustain viral growth. By immunofluorescence, 90-100% of neurons commonly had viral inclusion bodies, while doses of the virus three to four orders of magnitude higher still left greater than 99% of astrocytes, in brain cell cultures and 90 +/- 5% of the non-neuronal cells in DRG cultures without any obvious signs of rabies virus. Neuroblastoma cells (95 +/- 5% with viral antigens) produced viral yields about four orders of magnitude higher than glioma cells (10 +/- 5% with viral antigens). Though the overall infectivity of street virus was lower than that of fixed virus strains, a significantly higher viral tropism for neurons than for glia was maintained. Thus, primary neuronal cultures offer a means of exploring molecular events in rabies virus infection and their role in pathogenesis.

On the replication and spread of rabies virus in the human central nervous system.

Ultrastructural and immunohistochemical studies on the brains of two autopsy cases of human rabies revealed: By the peroxidase-antiperoxidase method, viral antigens were present in all eosinophilic inclusions detected in formalin fixed paraffin sections. Numerous antigenic masses, which apparently corresponded to the matrices and cylindrical particles in neurites revealed by electron microscopy, were present in the neuropil remote from neuronal perikarya. There were virions in the intercellular spaces and virus-budding from the plasma membrane into the extracellular space in the absence of a matrix, strongly indicating that rabies virus in the human central nervous system could spread through the intercellular spaces and that the replication of the virus was not necessarily accompanied by the formation of inclusion bodies. The synapse was involved in rabies as indicated by virions in the synaptic terminals. The implications of these observations are discussed in conjunction with the results of previous in vitro and animal experiments.

Axonal transport of rabies virus in the central nervous system of the rat.

Stereotaxic inoculation of rabies virus into specific nuclei in the central nervous system has been used for the investigation of the central neural transport mechanisms of viral information. The infection was monitored by specific fluorescence and peroxidase studies and the titration of viral infectivity in dissected brain areas. Twenty-four hours after inoculation into the striatum, cortex, or substantia nigra, infected neurons were detected only in cells from areas and nuclei which were related to the site of inoculation. The distribution of infected neurons showed that retrograde axoplasmic flow plays a determining role in the transport of rabies virus 24 hours after delivery of virus to specific target nuclei. Local destruction of neurons by kainic acid at the site of viral inoculation did not prevent the uptake and subsequent retrograde axonal transport of virus. There was an overall correlation between the major neural connections of the inoculated areas (e.g. the striatum) and the infected areas 24 hours later (e.g. the substantia nigra).

"Paramyxovirus-like" intranuclear inclusions occurring in the nervous system in diverse unrelated conditions.

Filamentous intranuclear inclusions similar to the "paramyxovirus-like" intranuclear inclusions described in active multiple sclerosis (MS) were observed incidentally in four patients who had diverse disorders such as rabies, mannosidosis, metachromatic leukodystrophy, cerebral aneurysm and ischemic infarcts. The observation is in support of the mounting evidence that the "virus-like" intranuclear inclusions are not specific for MS and may occur frequently in a variety of diverse conditions. Moreover, neither virological nor immunological evidence as to the viral nature of the intranuclear inclusions has been presented to date, and the intranuclear inclusions have been observed mostly in degenerating or autolyzed cells in biopsied or autopsied tissue samples. In view of all the circumstantial evidence, it is suggested that the intranuclear "paramyxovirus-like" inclusions may represent an alternation of nuclear chromatin common to antemortem degeneration and postmortem autolysis in a variety of cells, the nature of which remains to be elucidated.

Airborne rabies encephalitis: demonstration of rabies virus in the human central nervous system.

A veterinarian contracted rabies in the course of laboratory work with homogenates of rabid goat brain. Epidemiologic study determined a respiratory mode of transmission. After a fulminant encephalitic illness, formed rabies virions were identified in the synaptic zones of the olfactory glomeruli. Identification, isolation, experimental disease production, and tissue cytopathic effects of virus recovered from the brain fulfilled Koch's postulates in this unusual instance of virus disease of the nervous system.

Immunohistological demonstration of serum proteins and structural and viral antigens in paraffin sections of nervous tissues.

A brief outline is given of applications of immunohistological techniques to the study of normal and diseased nervous tissue. Protease treatment of paraffin sections usually enhances sensitivity and reliability both of IF and PAP techniques. Sensitivity of immunohistological examination of paraffin sections is comparable to that of virus detection by normal virological techniques in animal rabies and slightly superior to EM search for virions in SSPE and PML. Immunostaining for MBP appears to be the most sensitive method for myelin, especially for demonstration of very thin myelin sheaths, which are important in studies of myelogenesis and cortical myeloarchitecture. Prolonged fixation in formalin clearly diminishes or abolishes immunoreactivity. Compacted myelin stains less well for MBP than preparative myelin artefacts and the surface of myelinated fibers. GFAP production is enhanced when glioma cells invade surrounding mesenchymal structures. The chance finding of GFAP-like immunoreactivity in a cancer metastasis casts doubt on the astroglial specificity of GFAP.

Intermittent excretion of rabies virus in the saliva of a dog two and six months after it had recovered from experimental rabies.

A dog inoculated with a rabies virus isolate from the saliva of an apparently healthy Ethiopian dog was followed for more than 9 months. Saliva and blood specimens were collected three times weekly and cerebrospinal fluid weekly. Saliva samples collected on days 42 and 169 after the dog's recovery produced fatal rabies infections in mice inoculated intracerebrally.

Experimental rabies in the vampire bat.

Vampire bats were inoculated intramuscularly and subcutaneously with varying doses of rabies virus to simulate bites by rabid animals in nature. Daily saliva samples were then taken from these animals to determine whether they excreted virus and for how long. Vampire bats appear to react to rabies virus as do other animals, with variable incubation periods, some excretion of virus in the saliva, but no prolonged excretion "carrier state."

Detection of rabies virus RNA in the central nervous system of experimentally infected mice using in situ hybridization with RNA probes.

Rabies virus is usually demonstrated in human or animal tissues using antigen-detection or viral isolation techniques. Rabies virus RNA can be demonstrated in paraffin-embedded tissues using in situ hybridization. Negative (-) sense 35S- and 3H-labeled RNA probes, specific for rabies virus nucleocapsid protein mRNA, were used for the detection of rabies virus RNA in the nervous system of mice experimentally infected with fixed and street strains of rabies virus. In situ hybridization signals were compared with rabies virus antigen demonstrated with immunoperoxidase staining. Rabies virus RNA and antigen were also demonstrated in the same neurons using a double-labeling technique. In situ hybridization has potential applications as a diagnostic test for rabies and in studies of rabies pathogenesis.

Continuous delivery of colchicine in the rat brain with osmotic pumps for inhibition of rabies virus transport.

Rabies virus is a neurotropic agent which spreads in the CNS via axonal transport. Previous studies had shown that this axonal transport through the brain could be inhibited by stereotaxic administration of colchicine; however, this inhibition was reversible. We describe here a method to enhance the duration of this colchicine-mediated inhibition by delivering the drug continuously in the rat brain with osmotic pumps.

Electrophysiological and sleep alterations in experimental mouse rabies.

Changes in the spontaneous brain electrical activity and sleep organization were investigated in 5 mice strains during the evolution of experimental fixed rabies infection. Cortical electrodes were chronically implanted for continuous EEG recording and spectral analysis until death. Three evolutionary phases were individualized. The initial phase exhibited alterations of sleep stages, REM sleep disappearance, pseudoperiodic facial myoclonus and first clinical signs. The mature phase was characterized by a generalized EEG slowing (2-4 cycles/s). The terminal phase occurring with extinction of hippocampal rhythmic slow activity showed a flattening of cortical activity. The brain electrical activity ceased about 30 min before the cardiac arrest. Paroxysmal activities appeared during the course of the disease as bursts of rhythmic slow waves, pseudoperiodic spikes or occasionally ictal epileptic discharges. Spectral analysis revealed a progressive and characteristic clustering of the EEG frequency band power values. The spread of infection in the CNS was monitored by specific immunofluorescence studies which revealed the presence of rabies virus antigen in the pons, the cerebellum, the thalamus and the cortex during the initial phase. The pyramidal field of the hippocampus was infected during the mature phase but the gyrus dentatus was never infected even at the terminal phase. These studies show that particular neuronal functions are impaired during rabies virus infection suggesting that neuronal alterations may be involved in the pathogenic mechanisms leading to lethality.

Regional distribution of rabies viral antigen in central nervous system of human encephalitic and paralytic rabies.

We studied the distribution of rabies viral antigen in the brain and spinal cord of 7 patients with rabies by immunohistochemical techniques. Four patients presented with encephalitis, the remaining 3 had paralysis. Neither the rabies viral antigen distribution nor inflammation paralleled clinical presentations. Patients who had survival times of 7 days or less (4/7) had a greater amount of antigen-positive neurons in brainstem and spinal cord regardless of the clinical type. Neuroglial cells were also found to contain rabies antigen. Our findings suggest that virus localization may not account for the difference in clinical manifestations.

Neuronal cell cultures as a model for assessing neurotoxicity induced by encephalitic viruses.

Primary dispersed and organotypic cultures were prepared from selected brain areas and spinal cords of rat (Sprague-Dawley) and mouse (SJL/OLA(F) Ness-Ziona) fetuses and neonates. Following fiber regeneration, synapse formation and myelination, cultures were infected with one of the following viruses: Rabies CVS-21 strain, Sindbis Alphavirus, West-Nile Flavivirus and Theiler Murine Encephalomyelitis virus. Light and electron microscopical studies showed clear differences in the target cells for virus infection; time of viral replication and in the intensity and specificity of the cytopathic effects induced by these viruses. Thus, Sindbis and Theiler viruses induced severe cytotoxicity and demyelination due to rapid viral replication in both neurons and all glial cell types. Rabies and West-Nile viruses, on the other hand, replicated mainly in neurons and at a much slower rate, causing only mild damage to the cells and the myelin sheath. A very specific alignment of West-Nile virions was observed along the interperiod lines of the myelin sheath in several myelinated axons. This peculiar arrangement of the virions, entrapped between the myelin lamellae may lead to a novel concept in the understanding of viral infection.

Further studies on rabies virus isolated from healthy dogs in Nigeria.

Rabies viruses isolated from healthy dogs, were passaged in mice and adapted to cell culture. After 5-7 passages, isolated viruses were subjected to monoclonal antibody (Mab) characterization with a panel of 36 anti-nucleocapsid (NC) and 40 anti-glycoprotein (G) MAbs. The four viruses showed positive fluorescence with all NC hybridomas except MAb 422-5, confirming them as true rabies virus isolates. The anti-G MAb reactivity pattern was the same in the four isolates indicating that they belong to the same antigenic group, but were antigenically distinct from the Flury LEP rabies vaccine virus which is widely used throughout Nigeria for canine vaccination, and from other previously characterized street lyssaviruses from Nigeria.

The rabies viruses of bats.

In the 1930s rabies was shown to affect blood-, insect- and fruit-eating bats. We have prepared anti-nucleocapsid monoclonal antibodies (MAbs) using Mokola and bat (Lagos, Duvenhage and Denmark) rabies viruses as immunogens. With these MAbs we have examined rabies viruses from vampire, insectivorous and frugivorous bats from the Americas, Africa, Europe and the Soviet Union and have compared them with isolates from terrestrial species including man. As well as confirming the findings of others with viruses of African and American bat origin, the results revealed the presence of a second biotype in European bats and demonstrated the presence of serotype 1 as well as serotype 4 viruses in bats of the Soviet Union.

[Molecular and cellular aspects of the pathogenesis of rabies]. / Aspects moleculaires et cellulaires de la pathogenie dans la rage.

The true causes of the beginning of the symptoms and the eventual mortality of rabies are not very well known. Immunopathological factors certainly intervene, but they only complete a process of neuronal alteration. Contrary to other viruses which lyse the infected cells, the rabies virus does not necessarily destroy the neurones which are the preferential target cells. On the contrary, RNA synthesis is stimulated during CNS infection by the rabies virus. Synthesis is not stimulated by the multiplication of the rabies virus in the cell lines of fibroblastic or neuronal origin, which suggests the existence of regulations on neuronal level functions in the CNS. Nevertheless, the protein virus synthesis seemed equally submitted to specific neuron regulations or neuronal populations. The traces of a neuronal dysfunction during infection by the rabies virus have been investigated, using as a probe, variations of muscarinic acetyl choline receptors in the CNS. The presence of these receptors is revealed by the use of an antagonistic ligand of acetyl choline, quinuclidinyl benzylate. We were able to show that as regards the rat, the appearance of the first signs of paralysis is correlated with the diminution of the receptor binding. Thus, it seems that the presence of the virus in the CNS provokes neuronal alteration which can in itself condition the viral multiplication and eventually modulate the viral pathogenesis expression.

[Progression of the rabies virus in the visual system of the rat]. / Etude de la progression du virus rabique dans le système visuel du rat.

The CVS rabies virus, inoculated in the anterior chamber of the eye, is transported from the retina to the central nervous system only along the accessory optic tract and invades transsynaptically its terminal nuclei. On the other hand the retino-geniculo-cortical system is affected much later. Thus the virus shows a special affinity for a well defined neuronal system and behaves as a precise tracer of its intracerebral connections.

Rabies virus pathogenicity and challenge. Influence of the method of preparation, the route of inoculation, and the species. Comparison of the characteristics of the modified, fixed and wild strains.

The challenge we carried out, which regularly brought about the death of the control animals, led us to study the different factors influencing the pathogenicity of the rabies virus. So, the method of preparation is important. The strain NYC, prepared from the salivary glands is extremely pathogenic for dogs; however, when it is prepared from mouse brains after nine passages have been made, it proves to be already partially modified. One then notices a death-rate which is less severe, even when larger quantities of virus are employed, and also the presence of some aberrant phenomena. (The survival of some of the infected animals and the nature of their inapparent forms of infection were confirmed by serology.) The significance of the route of inoculation in the different species of animals was studied. The injection in the crotaphytes was reserved for dogs, the cervical muscles for cats and the masseters for sheep. Paradoxically, cats prove to be most resistant to the challenge under our conditions. Finally the numerical data, allowing us to compare the different strains, modified, fixed or wild, was established. The data was based on the incubation period and on the differences between the titres obtained via intramuscular routes and intracerebral routes in the mice.