[Crimean-Congo hemorrhagic fever: basics for general practitioners]. / La fièvre hémorragique de Crimée-Congo: l'essentiel pour le praticien.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease described in more than 30 countries in Europe, Asia and Africa. The causative agent is the Crimean-Congo hemorrhagic fever virus (CCHFV) that is a member of the genus Nairovirus of the family Bunyaviridae. CCHFV that is characterized by a high genetic variability is transmitted to humans by tick bites or contact with fluids from an infected individual or animal. The initial symptoms of CCHF are nonspecific and gradually progress to a hemorrhagic phase that can be lethal (case-fatality rate: 10 to 50%). Characteristic laboratory findings of CCHF are thrombocytopenia, elevated liver and muscle enzymes, and coagulation defects. The pathogenesis of CCHF remains unclear but might involve excessive pro-inflammatory cytokine production and dysfunction of the innate immune response. Diagnosis of CCHF is based mainly on isolation of the virus, identification of the viral genome by molecular techniques (RT-PCR), and serological detection of anti-CCHFV antibodies. There is currently no specific treatment for CCHFV infection and the efficacy of ribavirin is controversial. In absence of an effective vaccine, prevention is based mainly on vector control, protection measures, and information to increase the awareness of the population and of healthcare workers.

Mapping of monoclonal antibody epitopes of the rabies virus P protein.

Thirty-six monoclonal antibodies (MAbs) specific for the rabies virus P phosphoprotein were obtained from mice immunized with recombinant P (PV strain) produced in E. coli. All MAbs reacted against the corresponding rabies virus protein by ELISA and by Western blot analysis and revealed the presence of cytoplasmic inclusions in rabies virus infected cells. The epitopes of seven MAbs were mapped by testing their reactivity with protein fragments expressed from deletion mutants in transfected cells. Western blotting, immunoprecipitation and immunofluorescence assays were performed. These MAbs recognized epitopes in different domains of the P protein: 60% were directed against a region lying between residues 83-172 suggesting a major antigenic determinant of the rabies virus P protein in this region. Most of the antigenic sites appeared to be composed of linear epitopes. These MAbs will be useful as tools to dissect structural and functional properties of the rabies virus P protein.

Structure of the RNA inside the vesicular stomatitis virus nucleocapsid.

The structure of the viral RNA (vRNA) inside intact nucleocapsids of vesicular stomatitis virus was studied by chemical probing experiments. Most of the Watson-Crick positions of the nucleotide bases of vRNA in intact virus and in nucleoprotein (N)-RNA template were accessible to the chemical probes and the phosphates were protected. This suggests that the nucleoprotein binds to the sugar-phosphate backbone of the RNA and leaves the Watson-Crick positions free for the transcription and replication activities of the viral RNA-dependent RNA polymerase. The same architecture has been proposed for the influenza virus nucleocapsids. However, about 5% of the nucleotide bases were found to be relatively nonreactive towards the chemical probes and some bases were hyperreactive. The pattern of reactivities was the same for RNA inside virus and for RNA in N-RNA template that was purified over a CsCl gradient and which had more than 94% of the polymerase and phosphoprotein molecules removed. All reactivities were more or less equal on naked vRNA. This suggests that the variations in reactivity towards the chemical probes are caused by the presence of the nucleoprotein.

Structure of recombinant rabies virus nucleoprotein-RNA complex and identification of the phosphoprotein binding site.

Rabies virus nucleoprotein (N) was produced in insect cells, in which it forms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies virus N-RNA and from nucleocapsid in virus, we propose a low-resolution model for rabies virus N-RNA in the virus.

Neither phosphorylation nor the amino-terminal part of rabies virus phosphoprotein is required for its oligomerization.

Rabies virus (PV strain) phosphoprotein (P) was expressed in bacteria. This recombinant protein binds specifically to the nucleoprotein-RNA complex purified from infected cells. Chemical cross-linking and gel-filtration studies indicated that the P protein forms oligomers. Analytical centrifugation data demonstrated the co-existence of monomeric and oligomeric forms of rabies virus P protein and suggested that there is an equilibrium between these species. As P expressed in bacteria is not phosphorylated, this result indicates that P phosphorylation is not required for its oligomerization. Although an alignment of several rhabdovirus P sequences revealed that the amino-terminal domain of P has a conserved predicted propensity to form helical coiled coils, an amino-terminally truncated form of P protein, lacking the first 52 residues, was also shown to be oligomeric. Therefore, the amino-terminal domain of rabies virus P is not necessary for its oligomerization.

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Rabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Préhaud et al. (Virology 178, 486-497, 1990). The protein was either purified on a CsCl gradient, resulting in a mixture of nucleocapsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only. The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids. N in these structures is an 84 A long and thin molecule that is spaced at around 34 A along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA. With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar. Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4-5 nt long, again with no effect on the morphology of the nucleocapsids. Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts. Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.