Henri Tsiang

Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study.

Rabies virus (RV), a highly neurotropic enveloped virus, is known to spread within the CNS by means of axonal transport. Although the envelope spike glycoprotein (G) of cell-free virions is required for attachment to neuronal receptors and for virus entry, its necessity for transsynaptic spread remains controversial. In this work, a G gene-deficient recombinant RV (SAD delta G) complemented phenotypically with RV G protein (SAD delta G+G) has been used to demonstrate the absolute requirement for G in virus transfer from one neuron to another, both in vitro, in neuronal cell cultures (cell line and primary cultures), and in vivo, in murine animal models. By using a model of stereotaxic inoculation into the rat striatum, infection is shown to be restricted to initially infected cells and not transferred to secondary neurons. In mouse as in rat models of infection, the limited infection did not cause any detectable symptoms, suggesting that G-deficient RV recombinants might be valuable as non-pathogenic, single-round vectors for expression of foreign genes.

Involvement of Gangliosides in Rabies Virus Infection

The role of gangliosides in rabies virus infection of chick embryo-related (CER) cells was investigated. Cultured cells were pretreated with neuraminidase to render the cells transiently non-susceptible to viral infection. Incubation of these desialylated cells with gangliosides allowed them to incorporate exogenous gangliosides and they recovered their susceptibility to rabies virus infection. Infection of CER cells was monitored by specific fluorescence 24 h after virus inoculation. The use of individual purified gangliosides or mixtures of two gangliosides to restore cellular susceptibility to viral infection showed that GT1b and GQ1b were the most effective. The disialogangliosides were also active, principally GD1b, whereas GM1, GM3 were poorly active and GD3 inactive. Incubation of rabies virus with gangliosides prior to virus infection reduced the percentage of infected cells. The results indicate that highly sialylated gangliosides are part of the cellular membrane receptor structure for the attachment of infective rabies virus. However, it is possible that other glycoconjugates such as glycoproteins or glycolipids also participate as components of a receptor structure for rabies virus.

Mechanism of Rabies Virus Entry into CER Cells

The early steps of rabies virus (CVS) infection in vitro were studied in chicken embryo-related (CER) cells. The infection was monitored by looking for specific intracytoplasmic viral inclusions using anti-rabies fluorescein isothiocyanate at 24 h after the addition of virus. The attachment of rabies virus to CER cells was shown to be inhibited by pretreatment of the cells with neuraminidase. These cells recovered their susceptibility to rabies virus infection 6 h after removal of the enzyme. Treatment of CER cells with neuraminidase after the viral attachment step did not inhibit infection. The subsequent delivery of infectious virions into acid prelysosomal vacuoles or lysosomes was studied using lysosomotropic agents. Ammonium chloride and chloroquine were used to prevent the virus fusion step thus preventing infection. Both drugs were shown to inhibit the early steps of infection, NH4Cl having a much earlier effect than chloroquine. The two drugs had no effect on the attachment step nor did NH4Cl inhibit virus multiplication. The use of metabolic inhibitors (2-deoxy-D-glucose and sodium azide) shows that the entry of rabies virus into CER cells does not require the involvement of cellular energy processes. In electron microscopy studies, the presence of rabies virus particles was detected in coated pits and coated vesicles as well as in uncoated vesicles, and later in lysosomes. These data indicate that the mechanism by which rabies virus enters CER cells is probably through adsorptive endocytosis and does not require the participation of cellular metabolic active processes.

Induction of immunoreactive interleukin-1β and tumor necrosis factor-α 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.

Pathophysiology of Rabies Virus Infection of the Nervous System

Publisher Summary The clinical course of human rabies virus infection can be separated into five stages: incubation, prodrome, neurological stage, coma, and death. Encephalitic rabies is reported to occur in the majority of human cases. Hyperactivity is the major sign of encephalitic rabies, appearing as anxiety, nervousness, and mental confusion, alternating with periods of lucidity and preserved intelligence. Typical encephalitic rabies is diagnosed by autonomic dysfunction, hydrophobia and aerophobia, and periods of agitation, hyperexcitation, confusion, hyporeactivity, and drowsiness. During paralytic rabies in humans, seizures are common and fever is usually high and constant. The pathology of rabies virus infection in the central nervous system has been reviewed and the only gross pathological lesion is congestion of the meningeal vessels; a mild cerebral edema is also observed. Some of the histological features of rabies virus infection are (1) a perivascular accumulation of leukocytes, primarily in the spinal cord and brainstem, (2) neuronal degeneration and neuronophagia, and (3) glial proliferation. Ganglionic lesions and cranial nerve lesions of the same kind are also said to be good indications of rabies virus infection. In humans, great variability ranging from no histological lesions to widespread inflammatory changes associated with neuronal degeneration and neuronophagia is observed.

Rabies virus infection and transport in human sensory dorsal root ganglia neurons

Cultured human sensory neurons are directly susceptible to CVS rabies virus infection and produce virus yields of 10(5) p.f.u./ml; infection can persist for more than 20 days without any sign of c.p.e. The use of a compartmentalized two-chamber culture system, with access to either the cell soma or neuritic extensions, permitted the study of viral retrograde transport, which occurs at between 50 and 100 mm/day. Neurons of human origin were more susceptible to virus infection than rat neurons and the axonal transport of rabies virus was more efficient. Electron microscopy allowed virus transport and infection of human dorsal root ganglia neurons to be observed.

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.

Role of phospholipids in rhabdovirus attachment to CER cells

SummaryThe effect of phospholipases on rhabdovirus attachment to CER cells and the competitive binding of phospholipids and viruses to cells were studied. Results obtained indicate that, although to a different extent, some phospholipids represent a binding site for both Vesicular Stomatitis Virus (VSV) and rabies virus.

Rabies virus selectively alters 5-HT1 receptors 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.

Sleep alterations in experimental street rabies virus infection occur in the absence of major EEG abnormalities

Brain electrical activity and sleep organization were investigated in chronically implanted mice during street rabies virus infection. Continuous EEG recordings showed no gross electrical abnormalities until a few hours before the fatal issue. In contrast, alterations of sleep stages were observed at an early stage during the course of rabies virus infection, at a time when clinical signs were absent. Quantification by spectral analysis showed that the main feature was the early decrease of REM-sleep stages and the increase of the duration of waking stages. Neuromuscular disorders which could occur early were also observed during the disease. Comparison of these data with those obtained from fixed rabies virus infection shows that in the latter the EEG recordings demonstrated early alterations and a progressive deterioration with disappearance of both sleep and waking stages, which were replaced by a pathological sleep stage. In order to evaluate the potential role of the host-specific immune response in promoting brain electrophysiological alterations, EEG recordings and spectral analysis were also performed in cyclophosphamide-treated mice. Street rabies virus-infected and immunosuppressed mice showed identical physiopathological changes as those observed in immunocompetent mice. The implication of these viral-induced electrophysiological alterations in the context of the pathogenic mechanisms of rabies virus is discussed.