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.     

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)     

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)     

Furious and paralytic rabies of canine origin: Neuroimaging with virological and cytokine studies

Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus, hypothalamus, brainstem, and spinal cord of paralytic dogs. More widespread changes of less intensity were seen in furious dog brains. During the late stage of infection, brains from furious and paralytic rabid dogs were similarly infected and there were less detectable cytokine mRNAs. These results suggest that the early stage of furious dog rabies is characterized by a moderate inflammation (as indicated by MRI lesions and brain cytokine detection) and a severe virus neuroinvasiveness. Paralytic rabies is characterized by delayed viral neuroinvasion and a more intense inflammation than furious rabies. Dogs may be a good model for study of the host inflammatory responses that may modulate rabies virus neuroinvasiveness.     

Differential use of the nicotinic receptor by rabies virus based upon substrate origin

To determine the role that the neuronal nicotinic acetylcholine receptor plays in the adsorption process of rabies virus (RV), adult dorsal root ganglion dissociated cultures were exposed to nicotinic agonists before being inoculated. The fixed strain of RV Challenge Virus Standard-11 (CVS-11) was used after being passaged in two different ways, in baby hamster kidney (BHK) cells and in adult mouse brain (MB). Carbachol and nicotine reduced the percentage of CVS-MB infected neurons, yet none of the agonists tested changed the proportion of CVS-BHK infected neurons. This result suggests that the RV phenotype changes depending on its replication environment and neuronal nicotinic acetylcholine receptors are preferentially used for infection by RV strains adapted to adult mouse brain but not to fibroblasts.     

Latent herpes simplex virus infection of mice. Infectious virus in homogenates of latently infected dorsal root ganglia.

C-57 albino weanling mice were latently infected with herpes simplex virus (Mp strain, type 1) by inoculation of 10(4) plaque forming units in the right hind footpad. The virus was demonstrable in explant cultures of the sacral dorsal root ganglia of these mice for as long as 18 months following inoculation. In addition, the virus was detectable when homogenates of these latently infected ganglia were placed on to differentiated organotypic cultures of fetal mouse dorsal root ganglia for as long as 8 months following inoculation of the mice. Virus was not demonstrable in these homogenates when they were placed on the Hela cells. The results suggest that during herpes simplex virus latent infection in mice there is continuous synthesis of infectious virus, probably in a highly localized area, which is detectable if a sensitive indicator substrate, such as these organotypic cultures, is used.     

Differences in the activation of the GFAP gene promoter by prion and viral infections

The expression of glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. After surgical injury or viral infections, an increase in this protein reflects reactive gliosis in the brain. We analyzed the activation of the GFAP gene in transgenic mice using a prion and two different viruses (rabies and Theiler viruses). Inoculation of the transgenic mice with the C506M3 mouse prion strain resulted in activation of the GFAP-lacZ transgene. Expression of the GFAP transgene increased concomitantly with the expression of GFAP in astrocytes from the infected mice. In contrast, infection with rabies or Theiler's virus had no effect on the expression of the GFAP transgene, showing that the glial reactions to these infectious agents involved different mechanisms. These findings indicate that the activation of the endogenous GFAP gene as a consequence of viral infection could involve different regulatory pathways than activation as a result of prion infection. The first 2 kb upstream from the start codon of the GFAP gene seems to provide enough activation domains to produce efficient activation of the reporter gene in prion-infected mice.     

SPONTANEOUS RECOVERY FROM THE ENCEPHALOMYELITIS IN MICE CAUSED BY STREET RABIES VIRUS

Recovery from rabies was studied in an experimental model. Young adult mice were inoculated in a hindlimb footpad with street rabies virus (fox salivary gland isolate). In a group of 62 mice, 97% developed clinical rabies with paresis of the extremities and spasticity, and 37% recovered with neurological sequelae. There was an acute inflammatory reaction in the brainstem and grey matter of the spinal cord, and degeneration of myelinated axons in the white matter of the cord and in the dorsal roots. Rabies virus antigen was found in the central nervous system of all mice examined between day 5 and 13, and also in trigeminal and dorsal root ganglia. Surviving mice had neutralizing antibodies in serum and brain tissue, and 90% survived an intracerebral challenge with the CVS strain of fixed rabies virus. Spontaneous recovery from rabies encephalomyelitis was demonstrated with evidence of viral replication and pathological changes in the central nervous system.     

Serine residues at positions 162 and 166 of the rabies virus phosphoprotein are critical for the induction of oxidative stress in rabies virus infection

Our previous work in a mouse model of experimental rabies showed neuronal process (dendrites and axons) degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 (CVS) strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases mitochondrial complex I activity and reactive oxygen species (ROS) production. Expression of a peptide from amino acid 139–172 of the CVS phosphoprotein (P) increased complex I activity and ROS generation similar to expression of the entire P. Site-directed mutational analyses illustrated the importance of the 145–151 and 157–169 regions of P and that serine residues at 162 and 166 are important single amino acid sites. Two CVS recombinant viruses with serine to alanine mutations at positions 162 (A162r) and 166 (A166r) did not increase complex I activity or ROS generation and also did not induce axonal swellings or inhibit axonal growth in DRG neurons. RABV infection is a mitochondrial disorder initiated by interaction of the RABV P and complex I; S162 and S166 are critical sites in the P for this interaction. The resulting mitochondrial dysfunction produces oxidative stress in neurons causing acute degenerative changes affecting neuronal processes resulting in a severe and fatal clinical disease. This information will be important for the future development of novel therapies for rabies.     

Pathologic and virologic characterization of neuroinvasion by HSV-2 in a mouse encephalitis model

Herpes simplex virus type 2 (HSV-2), a ubiquitous human pathogen associated with genital infections, is neurotropic. It establishes latent infections in local dorsal root ganglia from which it reactivates causing recurrent lesions and frequent episodes of viral shedding. Herpes simplex virus type 2 can also be transmitted from mother to child during birth, causing major neonatal complications including encephalitis. Animal models of HSV-2 genital infection are well described and used for testing of therapies; little is known about animal models of HSV-2-induced encephalitis. We analyzed the pathologic and immunohistochemical features of the nasal rostrum and brain tissue and correlated them with viral distribution in a mouse model of HSV-2 encephalitis induced by intranasal infection and examined viral replication in the brain tissue using quantitative polymerase chain reaction and traditional plaque assay. Our results suggest that the primary route for HSV-2 neuroinvasion after intranasal infection is via the trigeminal pathway, ultimately leading to infection of the brainstem and meningoencephalitis.     

Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus

Rabies virus multiplication was investigated in cultured primary rat myotubes and neurons. The susceptibility of these two cell types to fixed rabies challenge virus strain (CVS) was monitored by fluorescence and virus titration. Differentiated rat myotubes were susceptible to rabies virus infection, and showed an increasing accumulation of viral material from day one to day four. However, these cells did not release infective viral particles, nor did they accumulate infectious virions in the cytoplasm. In contrast, infected neurons released large amounts of infectious particles. Electron microscopy observation of infected myotubes showed minor alterations and the presence of typical viral inclusions in the cytoplasm without mature virions assembling viral membranes. Competition binding experiments show that alpha-bungarotoxin inhibits rabies virus infection from 10(-5) to 10(-7) M, whereas lower toxin concentrations failed to have any effect. These data do not confirm the hypothesis of a fixed rabies virus amplification step at the site of the viral entry. On the other hand, the high susceptibility of peripheral neurons to rabies virus infection is an argument for the direct uptake of virions by these cells. The restrictive viral multiplication in the myotubes is an alternative explanation for the local persistence of rabies virus at the site of inoculation.     

Selective vulnerability of dorsal root ganglia neurons in experimental rabies after peripheral inoculation of CVS-11 in adult mice

The involvement of dorsal root ganglia was studied in an in vivo model of experimental rabies virus infection using the challenge virus standard (CVS-11) strain. Dorsal root ganglia neurons infected with CVS in vitro show prolonged survival and few morphological changes, and are commonly used to study the infection. It has been established that after peripheral inoculation of mice with CVS the brain and spinal cord show relatively few neurodegenerative changes, but detailed studies of pathological changes in dorsal root ganglia have not previously been performed in this in vivo experimental model. In this study, adult ICR mice were inoculated in the right hindlimb footpad with CVS. Spinal cords and dorsal root ganglia were evaluated at serial time points for histopathological and ultrastructural changes and for biochemical markers of cell death. Light microscopy showed multifocal mononuclear inflammatory cell infiltrates in the sensory ganglia and a spectrum of degenerative neuronal changes. Ultrastructural changes in gangliocytes included features characteristic of the axotomy response, the appearance of numerous autophagic compartments, and aggregation of intermediate filaments, while the neurons retained relatively intact mitochondria and plasma membranes. Later in the process, there were more extensive degenerative neuronal changes without typical features of either apoptosis or necrosis. The degree of degenerative neuronal changes in gangliocytes contrasts with observations in CNS neurons in experimental rabies. Hence, gangliocytes exhibit selective vulnerability in this animal model. This contrasts markedly with the fact that they are, unlike CNS neurons, highly permissive to CVS infection in vitro. Further study is needed to determine mechanisms for this selective vulnerability, which will give us a better understanding of the pathogenesis of rabies.     

Cell specificity and efficiency of the Semliki forest virus vector- and adenovirus vector-mediated gene expression in mouse cerebellum

Establishing efficient gene transfer and expression in post-mitotic neurons is important in understanding the genetic basis of neural circuits with cellular complexity. This study evaluates the properties of exogenous green fluorescent protein (GFP) expression mediated by the Semliki forest virus (SFV) and adenovirus (Ad) vectors in dissociated and slice cultures of the mouse cerebellum. Infection with SFV-GFP resulted in early-onset and high-level GFP expression in about 90% of Purkinje cells and in about 40% of granule cells in dissociated cultures at 1 day after infection. Two days after infection, GFP-positive cells showed signs of SFV-derived cytotoxicity. Ad-GFP infected almost all astrocytes and granule cells in dissociated cultures, and showed a steady increase in GFP fluorescence with a plateau at around 2 days post-infection. Ad vector-mediated GFP expression lasted for several weeks with no significant cell damage. In the slice cultures, both viral vectors mainly infected astroglial cells, but also showed a similar cell preference as that in dissociated cultures. These data indicate that the use of different viral vectors and infection conditions offers a powerful means of expressing exogenous genes in cerebellar cultures with different cell-type specificity and timing and duration of expression.     

Replication of the herpes simplex virus type 1 RL1 mutant 1716 in primary neuronal cell cultures--possible relevance to use as a viral vector

The herpes simplex virus type 1 (HSV-1) RL1 deletion mutant 1716 has properties that make it a promising candidate as a viral vector for gene therapy in the human nervous system. These properties include its ability to spread along neural pathways and establish a latent infection in post-mitotic neurons, while retaining a non-virulent phenotype in vivo and an inability to cause a lytic infection in stationary or fully differentiated cells. In this study, we used viral replication assays and indirect immunofluorescence to investigate the ability of 1716 to bind to, enter, express genes and produce progeny virus in dissociated neuronal cell cultures prepared from rat hippocampal, medial septal and dorsal root ganglion (DRG) tissues and in primary rat astrocyte cultures. Both heterogeneous cultures and those that had been enriched for neurons were employed. Following both low and high multiplicities of virus infection, the behaviour of 1716 was compared with its wild-type parent HSV-1 strain 17 in these cultures. It was found that the growth of 1716 was significantly impaired compared to wild type HSV-1, with these differences being magnified at lower multiplicities of viral infection as well as in neuron-enriched cultures: this impairment is likely to be due to decreased replication, as immunofluorescence assays showed that 1716 bound to, entered and expressed genes in all neuronal cell types and astrocytes with similar efficiency to the wild type virus. This ability of 1716 to enter and express genes in different neuronal populations demonstrates its potential suitability as a viral vector.     

Intramuscular AAV delivery of NT‐3 alters synaptic transmission to motoneurons in adult rats

We examined whether elevating levels of neurotrophin‐3 (NT‐3) in the spinal cord and dorsal root ganglion (DRG) would alter connections made by muscle spindle afferent fibers on motoneurons. Adeno‐associated virus (AAV) serotypes AAV1, AAV2 and AAV5, selected for their tropism profile, were engineered with the NT‐3 gene and administered to the medial gastrocnemius muscle in adult rats. ELISA studies in muscle, DRG and spinal cord revealed that NT‐3 concentration in all tissues peaked about 3 months after a single viral injection; after 6 months NT‐3 concentration returned to normal values. Intracellular recording in triceps surae motoneurons revealed complex electrophysiological changes. Moderate elevation in cord NT‐3 resulted in diminished segmental excitatory postsynaptic potential (EPSP) amplitude, perhaps as a result of the observed decrease in motoneuron input resistance. With further elevation in NT‐3 expression, the decline in EPSP amplitude was reversed, indicating that NT‐3 at higher concentration could increase EPSP amplitude. No correlation was observed between EPSP amplitude and NT‐3 concentration in the DRG. Treatment with control viruses could elevate NT‐3 levels minimally resulting in measurable electrophysiological effects, perhaps as a result of inflammation associated with injection. EPSPs elicited by stimulation of the ventrolateral funiculus underwent a consistent decline in amplitude independent of NT‐3 level. These novel correlations between modified NT‐3 expression and single‐cell electrophysiological parameters indicate that intramuscular administration of AAV(NT‐3) can exert long‐lasting effects on synaptic transmission to motoneurons. This approach to neurotrophin delivery could be useful in modifying spinal function after injury.     

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.     

Expression of the interferon-alpha/beta-inducible bovine Mx1 dynamin interferes with replication of rabies virus

Rabies is a fatal anthropozoonotic viral infection of the central nervous system that remains a serious public health problem in many countries. As several animal cases of spontaneous survival to infection were reported and because type 1 interferons were shown to protect against the virus, it was suggested that innate resistance mechanisms exist. Among the antiviral proteins that are synthesized in response to interferon-alpha/beta stimulation, Mx proteins from several species are long known to block the replication of vesicular stomatitis virus (VSV). As both VSV and rabies virus belongs to the Rhabdoviridae family, this study was started with the aim to establish whether the anti-VSV activity of a mammalian Mx protein could be extended to rabies virus. This question was addressed by inoculating the virus onto a bovine Mx1 or human MxA-expressing Vero cell clone. Plaque formation was unambiguously blocked, and viral yields were reduced 100- to 1000-fold by bovine Mx1 expression for both SAG2 and SADB19 viral strains. In opposition, only SAG2 strain could be inhibited by the expression of human MxA protein. The effect of both proteins expression was then evaluated at the viral protein expression level. Again, boMx1 was able to repress protein expression in both strain, whereas only SAG2 proteins were inhibited in human MxA-expressing cells. These results suggest that protection conferred by interferon-alpha/beta against rabies could be, at least partially, attributable to the Mx pathway. Alternatively, bovine Mx1 could be unique in its ability to repress rabies virus which, if confirmed in vivo, would open an avenue for the development of new antirabies therapeutic strategies.     

Multiple neural cell types are infected in vitro by border disease virus

Border disease (BD) of sheep results from a congenitally acquired nonarbotogavirus infection which causes a highly selective central nervous system (CNS) pathological lesion consisting of diffuse decreased myelination without inflammation or neuronal destruction. Thus, a selective disruption of oligodendroglial function appears to occur. In order to investigate the in vitro cell tropism of BD virus, primary cultures derived from fetal and adult ovine CNS and peripheral nervous system were inoculated with BD virus. Infected cell types were determined by dual immunofluorescent labeling for viral and cell type specific antigens. Infection of all the major cell types represented in these cultures, including oligodendrocytes, astrocytes, fibroblasts, dorsal root ganglion neurons and Schwann cells was found. Oligodendrocytes were only infected earlier and appeared to remain infected longer than astrocytes and fibroblasts. Infectious virus was produced by all cultures and continued to be produced even after the disappearance of nearly all immunocytochemically detectable viral antigen within cells. These studies suggest that the selective dysfunction of the oligodendrocyte in BD is not based on a selective viral tropism.