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  Arbofhem


  Director : Florence COLBERE-GARAPIN par intérim (fcolbere@pasteur.fr)


  abstract

 

Research programs have been conducted in the laboratory to characterize viral and cellular mechanisms involved in the pathogenicity of flaviviruses (Family Flaviviridae) and phleboviruses (family Bunyaviridae). A particular emphasis has been made on four major public health concerns : flaviviroses yellow fever, dengue, West Nile encephalitis, and phlebovirosis Rift Valley fever. Yellow fever, dengue, and Rift Valley fever viruses cause acute human diseases associated with hemorrhagic manifestations.



  report

cale

1. Virus-cell molecular interactions involved in the pathogenicity of flaviviruses (Marie Flamand, Philippe Desprès & Armelle Delécluse)

1.1 - Analysis of the biological properties of flavivirus nonstructural protein NS1 (S. Alcon, F. Coulibaly, M.-T. Drouet & M. Flamand)

NS1, the non-structural glycoprotein of flaviviruses, is essential for virus viability. It has been shown to participate in virus replication although no proper biological activity has been related to it. In addition to the cell-associated form of the protein, NS1 may be secreted in a host-dependent fashion from mammalian and not vector-derived insect cells. We have been particularly interested in characterizing the regulatory mechanism of NS1 secretion. In dengue type 1 virus-infected green monkey kidney Vero cells, we found that NS1 is secreted as a major soluble form, characterized by biochemical and biophysical means as a unique hexameric species. Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells as suggested by the repressive effect of N-glycan processing inhibitors swainsonine and deoxymannojyrimicin. To investigate the biological significance of NS1 secretion in vivo, the level of circulating NS1 was analyzed in the serum of patients infected with Den-1 using an ELISA sandwich assay. NS1 can be detected in most if not all patients, during the entire clinical phase of the disease, from day 0 corresponding to the onset of fevers till day 6. None of the patients remain positive in antigen in the convalescent phase, presumably once NS1 is no longer produced by infected host cells or becomes entirely complexed to antibodies. The fact that NS1 circulates during the acute phase of the disease in the human blood stream suggests that the protein may be a soluble effector of biological relevance to the pathophysiology of the disease. (Coll. : M. Arborio, S. Dartevelle, A. Falconar, J. Krinsje-Locker, J. Lepault, F. Nato, F. Rey, & A. Talarmin).

1.2 —Mechanisms of flavivirus-induced apoptosis (A. Catteau & P. Desprès)

The characteristic response of cells to viral infection is initiation of apoptotic cell death. Flaviviruses can provoke apoptosis in target cells such as liver cells, neurons, and endothelial cells. The underlying molecular processes triggering apoptotic pathway are not well characterized, but the knowledge regarding the viral determinants that influence apoptosis in response to DEN virus infection is improving. The efficiency of DEN virus replication modulates apoptosis. The study of viral factors associated to apoptosis has revealed that the envelope E protein and viral RNA helicase domain of the NS3 protein share determinants that have an apparent effect on the induction of apoptosis process by altering virus morphogenesis and replicative functions. We have investigated whether DEN virus-mediated apoptosis can be ascribed to specific viral products. Apoptotic induction was mapped to the DEN envelope glycoproteins prM (intracellular precursor of virion-associated M protein) and E. We have addressed the question whether their cytotoxic effects were linked to the presence of death-mediating sequences. Our investigation has linked the transport of the M protein in the secretory pathway to the induction of apoptosis. The role of the M protein in pathogenicity of flavivirus remains to be understood.

1.3 - The neuropathogenicity of West Nile virus

(Transverse Research Program 21 ; team leader P. Desprès) (S. Alcon, F. Coulibaly, P. Desprès, M.-T. Drouet, M. Flamand, M.-P. Frenkiel & M. Lucas)

With the recent expansion of a new variant of West Nile (WN) virus, it has become a serious public health concern in the Middle East, Europe, and more recently the USA. WN virus can cause a human disease with meningo-encephalitis as a severe complication. We used WN virus infection of mice as a model to investigate the importance of host genetics in the pathogenicity of this virus. The resistance/sensitivity phenotype of mice was completely correlated with the occurrence of a point mutation in the gene encoding the 2'-5'-OligoAdenylate Synthetase L-1 isoform, an antiviral molecule which is interferon-induced and double-stranded RNA-activated. Our objective is to explore the genetic variability of the candidate gene, and test whether this variability may explain part of the differential susceptibility to infection and pathological manifestations in individuals exposed to flaviviruses. (Coll.: J.-L. Guénet, P.-E. Ceccaldi & C. Julier).

 

1.4 - Molecular basis of mosquito-flavivirus interactions

(Transversal Research Program 23 ; coordinator: Armelle Delécluse)

Relationships between mosquito vectors and the viruses they transmit are crucial in the maintenance and transmission of several infectious diseases (dengue, yellow fever, West-Nile,…). Understanding these interactions would allow development of innovative control methods to prevent disease transmission.

1.4.1 - Identification of molecular determinants involved in vector competence (V. Mayau & A. Delécluse)

Mosquito populations display variable susceptibility to flaviviruses development, termed ‘vector competence'. Competence reflects the different barriers encountered by the virus from its entry into the mosquito midgut to its release in the saliva. Various factors such as the presence of specific mosquito receptors and/or differential viral replication in the mosquito might be involved in the competence. We are developing several approaches (surface plasmon resonance, in situ hybridization…) to identify the factors responsible for vector competence to dengue, yellow fever and West-Nile viruses.

1.4.2 - Evolution of dengue genome within the mosquito

(V. Juárez-Pérez, V. Mayau & A. Delécluse)

Evolutionary studies of dengue virus have revealed that its genetic diversity is increasing. Evolution of the virus genome could occur during its replication within the mosquito, Aedes aegypti. Mutations in a single genome or recombination events between separate genome might account for this evolution. We are currently studying the mutation rate of the dengue virus during its replication in the mosquito and investigating the possibility of genome recombination.

2- Rift Valley fever virus (M. Bouloy)

For the first time in 2000, RVFV was introduced in Middle East, causing a severe outbreak in Yemen and Saudi Arabia. This enveloped virus possesses a tripartite genome of negative polarity, the S segment utilizing an ambisense strategy to code for two proteins, the N nucleoprotein and the nonstructural protein NSs, the function of which was unknown. Our goal has been to determine the role of this protein.

2.1 - The NSs protein (A. Billecocq & P. Vialat)

The NSs protein is mainly located in the nuclei of infected cells where it forms filaments. Analysis of several mutants expressed via the Semliki forest replicon showed that the domain composed of the 10 C-terminal amino acids is involved in the formation of the filament but not the nuclear localisation. This domain comprises the two serine residues which are phosphorylated by Casein kinase II. These two amino acids do not play an important role for the filament formation since mutants in which one or both of the serines were changed into alanine still continue to form nuclear filaments. It is not known whether this filament is composed only of NSs or whether cellular proteins are associated . The implication of actin was tested but was not detected in the infected cell nuclei.

Clone 13 was isolated from a natural isolate from a benign human case in Republic of Central Africa. This virus possesses a large deletion in the NSs open reading frame and is avirulent for mice which represent a good animal model. The truncated NSs protein is hardly detected in Clone 13 infected cells : it does not form filament but remains in the cytoplasm where it is degraded by the proteasome. Reassortants containing a mixed genome from Clone 13 and virulent strain were used to show that the S segment carries a major marker of virulence which is deficient in Clone 13. Although NSs appears as a non essential protein in tissue culture, it plays an important role in animal since it blocks type I interferon production.

2.2 - Transcription-replication of viral genome (N. Le May &N. Vahsen)

A novel system reconstituted from plasmid expressed components was developed to asses transcription and replication. Synthesis of the RNA template is dependant on the pol I promoter which produces uncapped and unpolyadenylated transcripts, a property shared with RVFV genome. The L and N proteins which form the transcription complex will be expressed from CMV-based plasmids. This system will allow to establish the conditions for reverse gentics. In addition, other studies showed that the N protein forms dimers which could be involved in the switch between transcription and replication.

2.3 — Diagnostic tools (A. Billecocq, D. Coudrier, H Zeller, J.M. Reynes & M Bouloy)

Diagnosis of infections by hantaviruses circulating in France relies mainly on serological tests. The N protein was expressed via the semliki forest virus replicon and appeared as an efficient antigen for IgM and IgG detection as well as for immunofluorescence. Epidemiological studies on hantaviruses carried by rodents near Pnomh Penh in Cambodia showed that the genome of these viruses belong to the Seoul-like type and are transmitted by rats. A real time RT-PCR method was established for the diagnosis of Puumala virus and Rift Valley fever virus infection in collaboration with D Garin in CRSSA, Grenoble.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

BADELLA Jacqueline jbadella@pasteur.fr

MILLIOT Brigitte bmilliot@pasteur.fr

BILLECOCQ Agnès, chargée de recherche IP abilleco@pasteur.fr

BOULOY Michèle chef de laboratoire IP mbouloy@pasteur.fr

DELECLUSE Armelle chargée de recherche IP armdel@pasteur.fr

DESPRES Philippe chargé de recherche IP pdespres@pasteur.fr

FLAMAND Marie chargée de recherche IP mflamand@pasteur.fr

JUARES-PEREZ Victor chercheur contractuel vicjua@pasteur.fr

LUCAS Marianne chercheur contractuel mlucas@pasteur.fr

ALCON Sophie, PhD student (Paris 7 Univ.)

CATTEAU Adeline, PhD student (Paris 6 Univ.)

COULIBALY Fasséli, PhD student (Paris 11 Univ.)

GARCIA Stéphan, PhD student (Marseille Univ.)

GAULIARD Nicolas, DEA student (Paris 7 Univ.)

LE MAY Nicolas, PhDstudent (Paris 7 Univ.)

VAHSEN Nicola PhD student (Heidelberg Univ.)

DROUET Marie-Thérèse, Technician, mtdrouet@pasteur.fr

FRENKIEL Marie-Pascale, Technician mpfrenk@pasteur.fr

MAYAU Véronique, Technician, vmayau@pasteur.fr

MEGRET Françoise, Ingeneer (> 07/01), fmegret@pasteur.fr

OLLIVIER Noëlle, Maintenance Technician ollivier@pasteur.fr

PALMYRE Jocelyne, Maintenance Technician

ROTSEN Rolande, Maintenance Technician

TAMIETTI Carole, Maintenance Technician

VIALAT Pierre, Technician pvialat@pasteur.fr


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