Rabies virus is not cytolytic for rat spinal motoneurons <Emphasis Type="Italic">in vitro</Emphasis>

Cultures of purified rat embryonic spinal cord motoneurons were used to investigate the capacity of the neurons to survive rabies virus infection in vitro. In crude primary spinal cord cultures, neurons did not survive more than 2 days after rabies virus infection with the fixed strain Challenge Virus Standard. In contrast, virus-infected purified motoneurons resisted cytolysis for at least 7 days, as also did infected motoneurons treated with conditioned medium sampled from rabies virus-infected crude spinal cord cultures. This survival rate was also observed when motoneurons were grown in the presence of astrocytes or fibroblasts and it was not dependent on the presence of growth factors in the culture medium. Moreover, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling experiments showed that only 30% of infected motoneurons were apoptotic after 7 days of infection. In vivo, despite the massive infection of the spinal cord in infected rat neonates, the moderate number of apoptotic cells in the ventral horn suggests that only a few motoneurons were affected by this mechanism of cell death. Morphometric analyses showed that motoneurons' axon elongated at a comparable rate in virus-infected and noninfected cultures, a sign of high metabolic activity maintained in rabies virus-infected motoneurons. In contrast, hippocampus neurons were susceptible to rabies virus infection, because 70% of infected neurons were destroyed within 3 days, a large proportion of them being apoptotic. These experiments suggest that spinal cord motoneurons consist in a neuronal population that survive rabies virus infection because the viral induction of apoptosis is delayed in these neurons. They suggest also that paralyses frequently observed in rabid animals could be the consequence of dysfunctions of the locomotor network or of the spinal cord motoneurons themselves, whose parameters could be studied in vitro.     

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).     

Pathology of the human spinal ganglia in varicella-zoster virus infection

Spinal ganglia from a patient who died on the 6th day of varicella infection were examined by immunofluorescence and electron microscopy, and were compared with spinal ganglia from a patient dying on the 17th day of herpes zoster infection. In herpes zoster, typical intranuclear inclusion bodies were found in neurons, satellite cells and fibroblast-like cells of the ganglia, which contained numerous naked virus particles. In varicella, few changes were found by light microscopy but viral antigen was detected in a few neurons and satellite cells by immunofluorescence. Electron microscopy revealed scattered virus particles near the nuclear membrane of a neuron, satellite cells and capsular cells and enveloped particles in the cytoplasm of satellite cells. The particles in the nuclei were mostly naked virions with specific crescent-like inner-nuclear structure; those in the cytoplasm had complete and incomplete envelopes and showed pleomorphism. A "virus-like" intranuclear filament found in mononuclear cells in herpes zoster and a "plexiform vermicellar array" found in the nuclei of neurons in varicella are at present considered to be non-specific nuclear changes caused probably by viral infections.     

Apoptotic cell death in experimental rabies in suckling mice

A fatal encephalomyelitis developed after intracerebral inoculation of 6-day-old ICR mice with the challenge virus standard (CVS) strain of fixed rabies virus. The brains of CVS-infected mice showed widespread morphologic changes of apoptosis, which were particularly prominent in pyramidal neurons of the hippocampus and in the cerebral cortex. Evidence of oligonucleosomal DNA fragmentation was sought in situ using the TUNEL method. TUNEL staining was observed in many neurons, and rabies virus antigen was usually demonstrated with immunoperoxidase staining in similar regions. Neurons in the dentate gyrus of the hippocampus demonstrated expression of viral antigen, apoptotic changes, and positive TUNEL staining. This region normally demonstrates little infection in CVS-infected adult mice. Double labeling of neurons with TUNEL and viral antigen indicated that infected neurons actually underwent apoptosis. Increased immunoreactivity against the Bax protein was demonstrated compared to uninfected mice. Purkinje cells expressed viral antigen, but did not show significant morphologic changes of apoptosis or TUNEL staining. In contrast, neurons in the external granular layer of the cerebellum did not express viral antigen, but demonstrated greater morphologic changes of apoptosis and positive TUNEL staining than uninfected controls. Apoptotic cell death likely plays an important role in the pathogenesis of rabies virus infection in suckling mice. There was evidence of more apoptosis in the brains of suckling mice than in those of adult mice and this finding explains the greater neurovirulence of rabies virus in younger mice. Rabies virus likely induces apoptosis in vivo by both direct and indirect mechanisms. Received: 15 July 1997 / Revised: 25 August 1997 / Accepted: 8 September 1997     

Neuronal expression of foreign genes with recombinant rabies virus variants

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.     

Neuroinvasiveness of pseudorabies virus injected intracerebrally is dependent on viral concentration and terminal field density.

Pseudorabies virus (PRV), a neurotropic swine alpha herpesvirus, has been used extensively for transneuronal analysis ofmultisynaptic circuitry after peripheral injection. In the present analysis, we examined the influence of viral concentration and neuronal architecture on the invasiveness, replication, and transynaptic passage of an attenuated strain of PRV (PRV-Bartha) injected into rat striatum. Different concentrations of PRV-Bartha were injected into the striatum at a constant rate of infusion (10 nl/minute), and animals were killed 50 hours later. Viral concentration was manipulated by either altering the volume of the inoculum (100, 50, 20 nl) or by diluting the inoculum within a constant volume of 100 nl. Immunohistochemical localization of infected neurons revealed dramatic differences in the progression of infection that were dependent directly on the concentration of injected virus. In every case, the pattern of infection was consistent with preferential uptake of virions by axon terminals and retrograde transynaptic passage of virus from the injection site. The known topographically organized corticostriatal projections permitted a precise definition of the zone of viral uptake. This analysis demonstrated that the "effective zone of viral uptake" (i.e., the zone within which viral uptake led to productive replication of virus) varied in relation to the concentration of injected virus, with the highest concentration of PRV invading terminals within a 500 microm radius of the canula. Concentration-dependent changes in the progression of retrograde transynaptic infection also were observed. The highest concentration of virus produced the most extensive infection. The distribution of infected neurons in these cases included those with known afferent projections to striatum as well as those that became infected by retrograde transynaptic infection. Lesser concentrations of PRV-Bartha produced an increasingly restricted infection of the same circuitry within the same postinoculation interval. It is noteworthy that neurons known to elaborate dense striatal terminal fields were less sensitive to reduction in viral concentration than those giving rise to terminal fields of lesser density. Collectively, the data indicate that the onset of viral replication after intracerebral injection of PRV is directly dependent on virus concentration and terminal field density at the site of virus injection.     

Presence of specific antigens in neuronal cells infected with fixed and street rabies virus strains

Summary The presence of rabies specific antigens is investigated after infection with different rabies virus strains in neural cell lines and in the central nervous system of laboratory rodents. In fixed rabies infected cells, the rabies glycoprotein is found to be present 48 h after infection, whereas in hamsters this protein was found 5 days after an intracerebral inoculation. In contrast, rabies glycoprotein was not detectable in any of the street rabies-infected cell system by the fluorescent antibody test, although nucleoprotein was present, showing that infection occurred in these cells. Rabies glycoprotein was also undetectable in the central nervous system (CNS) of athymic nude mice which is known to be very sensitive to street rabies infection and to contain large quantities of viral material. Our results suggest that the smaller amount of rabies glycoprotein synthesized during street rabies infection are of consequence for the pathogenesis of rabies disease. The immunopathology of street rabies virus infection is certainly modulated by the failure of the viral glycoprotein to be present in large quantities on the surface of the infected cellular membrane as in the case of fixed rabies.Supported by grants from the Institut Pasteur and from INSERM (CRE 78.4.146.1 and ATP 50.77.82.009)     

Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis

Nonfatal paralysis, induced by the attenuated Pasteur strain of rabies virus, is characterised by local and irreversible flaccid paralysis of the inoculated limbs. We characterised the spread and localisation of virus in the CNS of infected mice, determined the nature of cell injury and examined the role of the immune response. Data indicate that infection of BALB/c mice induced paralysis in 60% of infected mice, the others recovering without sequelae. In both groups of mice, virus was detected in restricted sub-populations of neurons from the brain and spinal cord, and intensity of the neuropathology correlated with levels of rabies RNA and apoptotic infected neurons. However, apoptosis of neurons and paralysis were not due to a direct deleterious effect of the virus, but induced by a T-dependent immune response, as evidenced by their absence in nude mice. Paralysed and asymptomatic mice developed a similar rabies virus-specific IgG2a antibody response, thus excluding the role of any modification of the humoral immune response. In contrast, three events were critically associated with the development of neurological symptoms: the amount of virus in the CNS, the level of apoptosis in both infected neurons and uninfected surrounding cells and the progressive parenchymal infiltration of CD4+ and CD8+ T cells at the site of infection. These data suggest that during nonfatal rabies infection, the levels of viral replication and primary degeneration of infected neurons by apoptosis could be responsible for the infiltration of T lymphocytes capable of inducing secondary degeneration of neural cells.     

Pathogenesis of experimental rabies in mice: an immunohistochemical study

Summary The spread of rabies virus in the central nervous system of mice was examined after hindlimb footpad and intracerebral inoculation of the CVS strain of fixed rabies virus. All mice developed paralytic rabies. After intracerebral inoculation there was early simultaneous infection of neurons in the cerebral cortex and pyramidal neurons of the hippocampus, and later there was spread to the cerebellum. After high-dose intracerebral inoculation there was early infection ependymal cells lining the lateral ventricles and neurons adjacent to the central canal of the spinal cord, suggesting that rabies virus entry into the CNS occurs, at least in part, by a cerebrospinal fluid pathway. The sequence of involvement was different after hindlimb footpad inoculation. Infection became established in the cerebellum on day 5, in the cerebral cortex on day 6, and in the hippocampus on day 8. CA3 was initially affected, CA1 became infected 2 days later, and there was much less involvement of the dentate gyrus. Hippocampal infection occurred late relative to the rest of the brain after peripheral inoculation, but not after intracerebral inoculation. The hippocampus is not a good location for the detection of early brain infection after peripheral inoculation, although it may be involved when a natural rabies vector has the ability to transmit infection. These findings also raise questions about the mechanisms for the limbic dysfunction observed in clinical rabies.Supported by grant MA-10068 from the Medical Research Council of Canada     

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.     

Ultrastructural localization of rabies virus antigens in infected trigeminal ganglion of hamsters

Summary The trigeminal ganglion of hamsters infected with CVS strain of rabies virus was investigated by electron microscopy with peroxidase-labeled antibody. Major localization of virus antigens were the following three types of structures; rabies virion, cytoplasmic inclusion, and ribosomal granules consisting of Nissl body. Rabies virions in neurons were mostly found within the cluster of such ribosome-rich regions suggesting a close relationship between the two in the synthesis of virus antigen. The cytoplasmic inclusion appeared to be derived from ribosomal or allied particles with virus-specific antigens. The significance of results obtained is briefly discussed.     

The distribution of challenge virus standard rabies virus versus skunk street rabies virus in the brains of experimentally infected rabid skunks

Summary The proposal that the bizarre behavioral changes which occur during rabies infection are due to selective infection of limbic system neurons was further studied in skunks (a species important in naturally occurring disease). A detailed immunohistochemical study of brains of skunks experimentally infected with either Challenge virus standard (CVS) or street rabies virus revealed only trace amounts of viral antigen in many limbic system neurons and marked differences in viral distribution between street and CVS virus. These data were collected during early stage rabies when behavioral changes occur. Areas which contained heavy accumulations of street rabies virus but low amounts of CVS rabies virus were the neuronal perikarya and processes of the dorsal motor nucleus of the vagus, midbrain raphe, hypoglossal and red nuclei. In contrast, large accumulations of CVS virus were found in the Purkinje cells of the cerebellum, the habenular nuclei and in pyramidal cells throughout the cerebral cortex, while corresponding areas in all street virus-infected skunks contained minimal antigen. These findings were very consistent for animals of the same experimental group and between skunks inoculated both intramuscularly and intranasally with skunk street virus. Skunks inoculated intramuscularly with CVS rabies virus failed to develop rabies. Since, in this model, street virus infection generally produces furious rabies and CVS infection results in dumb rabies, we speculate that the behavioral changes which occur in these two different clinical syndromes are due to the heavy and specific accumulation of virus in different regions of the CNS. These results show that regions other than those of the limbic system may also be involved in the pathogenesis of behavior changes in rabid animals.Supported by an MRC fellowship (NLS)     

Inhibition of the transport of rabies virus in the central nervous system

The effect of colchicine, an inhibitor of axonal transport, on the spread of rabies virus in the central nervous system was investigated using Wistar rats. Colchicine was inoculated into the striatum at various times before and after inoculation of rabies virus into the same site. Rats were killed at various times after viral inoculation and the spread of rabies virus was monitored by rabies immunofluorescence of selected areas of brain. The most effective inhibitory effect was obtained by colchicine treatment applied two days before virus inoculation. Under these conditions, no fluorescent foci could be detected until day 3 post-infection whereas control rats exhibited infected cells as soon as two days post-infection. This inhibitory effect is reversible and the general consequence seems to be a delay in the rate of viral spread. However, five days after the virus challenge, some major brain areas were still partially preserved from infection (striatum, frontal cortex, pyriform cortex). Ten days after colchicine treatment, the microtubules have recovered their capacity to transport the virus. At the onset of paralysis, the general pattern of infection in brain sections from colchicine-treated rats was not significantly different from that of control rats. This inhibitory effect on the transport of rabies virus can be prolonged by administration of additional colchicine.     

The blood-brain barrier in the cerebrum is the initial site for the Japanese encephalitis virus entering the central nervous system

Japanese encephalitis (JE) virus is a member of the encephalitic flaviviruses and frequently causes neurological sequelae in a proportion of patients who survive the acute phase of the infection. In the present study, we molecularly identified viral infection in the brain of mice with rigidity of hindlimbs and/or abnormal gait, in which JE virus particles appeared within membrane-bound vacuoles of neurons throughout the central nervous system. Deformation of tight junctions (TJs) shown as dissociation of endothelial cells in capillaries, implying that the integrity of the blood-brain barrier (BBB) has been compromised by JE virus infection. BBB permeability evidently increased in the cerebrum, but not in the cerebellum, of JE virus-infected mice intravenously injected with the tracer of Evans blue dye. This suggests that the permeability of the BBB differentially changed in response to viral infection, leading to the entry of JE virions and/or putatively infected leukocytes from the periphery to the cerebrum as the initial site of infection in the central nervous system (CNS). Theoretically, the virus spread to the cerebellum soon after the cerebrum became infected.     

Experimental rabies virus infection in Artibeus jamaicensis bats with CVS-24 variants

An experimental model of rabies was established in the fruit-eating bat species Artibeus jamaicensis. The infections caused by CVS-N2c and CVS-B2c, which are both stable variants of CVS-24, were compared after inoculation of adult bats in the right masseter muscle. CVS-N2c produced neurologic signs of rabies with paresis, ataxia, and inability to fly, while CVS-B2c did not produce neurologic signs. Bats were sacrificed and the distribution of rabies virus antigen was assessed in tissue sections with immunoperoxidase staining. Both viruses spread to the brain stem and bilaterally to the trigeminal ganglia by days 2 to 3. CVS-N2c had disseminated widely in the central nervous system (CNS) by day 4 and had involved the spinal cord, thalamus, cerebellum, and cerebral cortex. CVS-B2c had infected neurons in the spinal cord on day 5 and in the cerebellum, thalamus, and cerebral cortex on day 6. Infected pyramidal neurons of the hippocampus were observed on day 5 in CVS-N2c infection, but infected neurons were never noted in the hippocampus in CVS-B2c infection. CVS-N2c infected many more neurons and more prominently involved neuronal processes than CVS-B2c. CVS-N2c spread more efficiently in the CNS than CVS-B2c. Morphologic changes of apoptosis or biochemical evidence of DNA fragmentation were not observed in neurons with either virus after this route of inoculation. The different neurovirulent properties of these CVS variants in this model were not related to their in vivo ability to induce apoptosis.     

Mumps virus alters aggregation of acetylcholine receptors in cultured rat skeletal muscle cells

Cultured myoblasts, but not myotubes, from rat skeletal muscles were infected with the RW strain of mumps virus. Such myoblasts then fused to form myotubes containing viral antigen. The infected myotubes showed a significant decrease in the number of dorsal, linear acetylcholine receptor (AChR) aggregates as determined by FITC-conjugated alfa-bungarotoxin. Infected myotubes co-cultivated with spinal cord cells showed no increase in the number of dorsal, linear AChR aggregates, compared to normal, uninfected myotubes. In addition, an increased proliferation of the myoblasts, which remained uninfected in the infected cultures, was noted. This may indicate a release of a growth stimulating factor from the virus containing cells. This study shows that mumps virus infection can lead to an altered receptor organization in a morphologically preserved cell.     

Rabies encephalomyelitis: clinical, neuroradiological, and pathological findings in 4 transplant recipients

BACKGROUND: Three patients received solid organ transplants from a common donor and were subsequently discharged from the hospital following an uneventful hospital course. Within 30 days, all 3 organ recipients returned to the hospital with varying symptoms that progressed to rapid neurological deterioration, coma, and death. OBJECTIVE: To describe the clinical, neuroradiological, and pathological findings of rabies virus infection in organ transplant recipients infected from a common donor. DESIGN: Case series involving a common donor and 3 organ recipients ascertained through review of clinical course and autopsy findings. A fourth case was determined by review of pending autopsy cases in which death occurred within the same time interval. Portions of postmortem central nervous system and organ tissues were frozen and formalin-fixed. Fluids and tissues were also collected for cultures, serology, and molecular studies. Postmortem fluids and tissues and antemortem fluids and tissues from all 4 transplant recipients and serum and banked lymphocyte or spleen cells from the donors were sent to the Centers for Disease Control and Prevention for further evaluation. SETTING: Transplant unit of an urban teaching hospital. RESULTS: Antemortem cerebrospinal fluid analysis for 3 of the 4 recipients was consistent with a viral etiology. Neuroimaging and electroencephalogram studies were suggestive of an infectious encephalitis or a toxic encephalopathy. Initial laboratory testing did not demonstrate an infectious etiology. Postmortem histologic analysis, immunohistochemistry, electron microscopy, and direct fluorescence antibody testing revealed rabies virus infection. Serological testing done postmortem confirmed rabies virus infection in the common donor. CONCLUSIONS: These cases demonstrate a risk for transmitting rabies virus infection through solid organ and tissue transplantation, and this diagnosis should be considered in any rapidly progressing neurological disease.     

Theiler's virus replication in isolated Schwann cell cultures

Theiler's murine encephalomyelitis viruses causing both fatal encephalitis (GDVII virus) and chronic demyelinating disease (WW virus) are capable of replicating in isolated Schwann cell cultures. Light microscopy combined with immunohistochemical staining of viral antigens revealed that large numbers of Schwann cells infected with the two viruses show cytopathic effect (rounding) and contain viral antigens. Electron microscopy of virus-infected Schwann cells shows that the morphological alterations that the cells undergo following infection by the two virus isolates are different. In the early stages of GDVII and WW virus infection, different inclusion bodies are formed in the cells cytoplasm. At late stages of the infection GDVII virions are found in all infected cells and are arranged in crystalline arrays around inclusion bodies. In contrast, in WW virus-infected Schwann cells only in few cells virions were observed and they appeared aligned between two membrane units.     

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.     

Ultrastructure of herpes simplex virus infection of the nervous system of mice

Summary The nervous system and small intestine of mice infected with herpes simplex virus were examined by electron microscopy from the viewpoint of virus-host interaction.The host cells examined included the neuron, astrocyte, oligodendrocyte, and Schwann cell. The susceptibility of the latter was not less than that of the neuron. The endothelial cell, perineural fibrocyte and smooth muscle cell were also host cells. Replication of herpes virus in the nervous system was proven to be identical to that occurringin vitro; initial reproduction of nucleocapsids in the nucleus and subsequent maturation at the nuclear membrane with envelope formation, followed by discharge into the cytoplasmic reticular cavities and finally release from the host cell. Inconsistency in the distribution of virus particles and viral antigen was chiefly concerned with the host cell nucleus and the glial cytoplasm.Herpes virions, though few, were identified in the axons of peripheral nerves, and in the periaxonal space of myelinated fibres in the brain and the nerve ganglia. Virions were present in tiny vesicles in the perikarya or as naked particles. In the distal parts of peripheral nerve, there was marked dissociation in the amount of virions between Schwann cells and the axon. The significance of the endoneural space and the axon in the neural speread of infection is discussed briefly.