Unit: Molecular Genetics of Bunyaviridae
Director: Bouloy Michèle
The main projects of the Unit deal with Rift Valley fever virus, an arbovirus which represents a model to study an hemorrhagic fever. The aim is to understand the mechanisms involved in pathogenesis in the mammalian host by identifying determinants of virulence and their cellular partners. Infection of mosquitoes or mosquito cells is asymptomatic. Studies on the molecular bases of the effects of virus infection in these two hosts constitutes a complementary axis of research. A better understanding of these mechanisms will allow production of vaccines and molecules with therapeutic effects.
Other activities in the Unit aim at producing recombinant antigens for diagnostic tests utilized by laboratories of the reference center and institutes of the Pasteur International network. These non pathogenic recombinant antigens are very useful tools for the diagnosis of BSL4 viruses or viruses, like hantaviruses, which grow poorly in tissue cultures.
Role of Interferon and NSs in pathogenesis
Rift Valley fever virus is an arbovirus of the Bunyaviridae family (Phlebovirus genus) transmitted by mosquitoes. It is endemic in sub-saharian Africa where it provokes many epizootics/epidemics. Recently it spreads to Middle East. Ruminants are severely affected with a high mortality rate, abortions and teratogenesis. In humans, infection can lead to a fatal hemorrhagic fever with an acute hepatitis. The role of the non structural protein NSs has been determined only very recently. Studies on this phosphoprotein which forms filamentous structures in the nucleus whereas every step of the viral cycle occurs in the cytoplasm, represent our main axis of research. Genetic analysis of Clone 13, a naturally avirulent mutant, which possesses a large internal deletion in the NSs gene helped to show that NSs plays a major role in virulence and to demonstrate that NSs prevents the host antiviral response by blocking type I interferon production. Analysis of the mechanism utilized by NSs to act as antagonist of interferon production indicates that NSs is in fact a general inhibitor of cell transcription which interacts with p44 a sub-unit of the general transcription factor TFIIH. By sequestering p44 and XPB, another sub-unit of TFIIH in the nuclear filament of NSs, the cellular concentration of TFIIH is drastically reduced, making the cell unable to transcribe its RNAs. Other partners are being studied.
Studies on transcription and replication with minigenomes.
To develop a system for reverse genetics, we established a basic transcription system in which viral proteins and RNAs were expressed from cDNA sequences cloned in plasmids. In a first step, the system was tested with a minigenome consisting in a viral like-RNA carrying the non coding sequences of the L, M or S genomic segments and the Chloramphenicol acetyl transferase gene in the antisense orientation. These RNA molecules were synthesized from plasmids under the control of the cellular Pol I promoter. Plasmids carrying the S-CAT, M-CAT and L-CAT sequences were constructed and these genome-like RNAs were efficiently recognized and transcribed by the viral complex composed of the L polymerase and the N nucleoprotein.
Infection of mosquitoes
As all arboviruses, the transmission of RVFV requires the involvement of an invertebrate host (the mosquito) and a vertebrate host (animal or human). Among factors which could explain RVF emergence, host ecologies are undoubtedly implicated. As mosquitoes colonize breeding sites which are subjected to temporal fluctuations of water availability, bionomics and genetic structure of populations are consequently, affected. Among the mosquito vectors of RVFV, two species play a crucial role in transmission: Aedes vexans, a selvatic species involving in enzootic cycles and Culex pipiens, an urban species described as the major vector in Egyptian outbreaks. We tended to define the ability of vectors to transmit RVFV and to evaluate the genetic organization of populations in the field. For such purpose, we have infected mosquitoes with different viral strains and established a survey of mosquito populations using population genetic approaches. Thus we have succeeded in infecting RVF vectors to evaluate vector competence and isolated and characterized genotypic markers, microsatellites for population genetics. Collaborations are setting with institutes from the Pasteur International Network.
Recombinant antigen for serological diagnosis
Several Bunyaviruses replicate poorly in cell cultures (Hantavirus) or they must be handled in a high security level laboratory (Crimean-Congo hemorrhagic fever virus). During infection with Bunyaviruses, the nucleorprotein N is the major antigen which induces a strong humoral response. We developed an ELISA to detect antibodies using a recombinant form of Puumala hantavirus nucleoprotein. This antigen allows the diagnosis of this non fatal hemorrhagic fever which circulates in the North and Est of France as well as to detect viral infections in bank voles, the rodents carrying the virus.
Fig 1. The RVFV NSs protein forming a nuclear filament in infected Vero cells
Keywords: hemorrhagic fever, arbovirus, zoonosis, hantavirus, pathogenesis