Alain Ermine

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.

The anterograde transport of rabies virus in rat sensory dorsal root ganglia neurons.

We have previously described the capacity of neurites extending from cultured rat sensory dorsal root ganglia (DRG) neurons to transport rabies virus through axoplasm in the retrograde direction. Here we report the infection of cultured neurons derived from the DRG and the subsequent anterograde transport of rabies virus from the infected cell somas through the extending neurites to its release into the culture supernatant. Viral transport was monitored by titration of the virus yield in the external compartment. Both early and late transport mechanisms of rabies virions were identified. The first one occurred a few hours post-infection and was undetectable 6 h later, before the initiation of viral replication. The velocity of this first wave of infective virions was in the range of 100 to 400 mm/day. The early viral transport was probably the result of a direct translocation of infective virions from the somatic site of entry to the neuritic extensions and subsequent release into the culture medium without replication in the cellular perikaryon. The second virus transport peak was detected 48 h post-infection. In this case, the virions detected in the neuritic compartment were presumably the progeny of the inoculated virus which had replicated in the perikaryon before the viral transport occurs. Using a four-compartment culture device we were able to demonstrate, simultaneously, retrograde and anterograde transport of the virus. The presence of antirabies serum in contact with the exposed neurites did not inhibit either the retrograde or the anterograde transport mechanisms. The viral release from the neuritic extensions after the fast anterograde transport was evaluated to be in the range of 150 to 300 infectious virions per bundle of neurites per day.

Rapid diagnosis of rabies infection by means of a dot hybridization assay

Dot hybridization was used to detect specific rabies RNA in brains, either from experimental infection in mouse or from brain material to be processed for routine diagnosis. 32P cDNA probes were employed to identify minute amounts of specific viral RNA. Purified RNA was obtained after phenol extraction. The RNA was fixed on nylon membranes and hybridized with a pool of M13 inserts complementary to 200-400 nucleotides of each rabies gene and mRNA. Hybridized, labelled probes were detected by autoradiography. There was strong cross-hybridization between fixed rabies and street rabies virus RNA, which enable the detection of field strains for diagnosis purpose using a fixed rabies (PV strain) cDNA. A positive response was obtained with as little as 80 ng of brain RNA material from a fixed rabies-infected mouse. Detection of viral RNA was still specific 1 week after death, the brain material being left at room temperature. A total correlation was found when the samples were examined in parallel using a fluorescent rabies-specific antibody and by virus isolation on murine neuroblastoma cells. These data show that the use of rabies-specific cDNA probes in a dot-blot hybridization assay has great potential for the diagnosis of rabies.

Polymerase chain reaction amplification of rabies virus nucleic acids from total mouse brain RNA

In an attempt to improve the sensitivity of the rabies genome hybridization test, PCR amplification was used following reverse transcription of rabies RNA extracted from infected brain. Presence of amplified DNA is demonstrated with either cDNA synthesized from the antigenomic primer or from antimessenger primer.

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.