Activity report

The majority of emerging or re-emerging infectious diseases that pose a significant threat to human health are caused by arthropod-borne viruses (arboviruses). Flavivirus-, bunyavirus- and alphavirus-related diseases provide some of the most important examples of emerging arboviral diseases of global significance. The mission of the Flavivirus-Host Molecular Interactions (FHMI) lab is to undertake basic virological research on mosquito-borne RNA virus diseases. The investigators of FMHI have established excellent collaborations with individual virology groups throughout Institut Pasteur, within France and abroad, from which several notable achievements have resulted. Our research program on arboviruses has a number of themes, the majority of which is supported by the Transverse Research Programs (PTR) at the Institut Pasteur, the Agence Nationale pour la Recherche (ANR), the European FP programs and R&D partnerships. During the period 2008-11, the FHMI lab hosted the NRC for Arboviruses that is involved in surveying and diagnosing arbovirus infections.

 

The FHMI’s research program is geared towards understanding how host cell/virus interactions define the pathogenicity of emerging arboviruses. To date, arboviruses studied include chikungunya (CHIK), dengue (DEN), Japanese encephalitis (JE), West Nile (WN), and Yellow Fever (YF) viruses. The purpose of the virological research is several fold: first, mechanisms of virus replication, a comprehensive study on molecular basis of virus virulence, host cell/virus interactions, and immune evasion strategies of arboviruses towards innate immunity. Second, to apply the knowledge gained from the basic research to develop more sensitive and specific molecular tools for arbovirus detection as well as vaccines and therapy to prevent or fight virus infection. In 2012, Dr Valérie Choumet has joined the FMHI lab to study entomological virology, focusing on the interplay between host and arboviruses (bunyavirus Rift Valley Fever virus and CHIK virus) at the invertebrate-vertebrate interface.

 

Investigators of the FHMI lab have basic research interests on the molecular mechanisms of virus replication, mechanisms by which arboviruses manipulate the host factors, and interactions between arboviruses and antiviral innate immunity. They have also put emphasis on understanding the molecular basis of arboviral pathogenicity using molecular clones of CHIK, YF, and WN viruses. The knowledge of virus replication mechanisms has provided a rational basis for the selection of host and viral factors to be used in diagnosis, immunotherapy, and vaccinology. The major achievements in 2011 are the following:

 

Cellular factors that contribute to arbovirus life cycle. Depletion of Class II Arf proteins Arf4 and Arf5 leads to inhibition of flavivirus secretion suggesting an important role in late stages of virus life cycle. Release of YF and DEN viruses but not WN virus was altered by depletion of Arf4 and 5. Interaction between DEN virus structural glycoprotein prM (intracellular precursor of virion-associated M protein) and Arfs has been observed. The objective is now to determine the molecular basis of this interaction in order to provide new mechanistic insights into how Class II Arf proteins Arf4 promote flavivirus replication (coll.: Institut Pasteur of Hong-Kong). The first identification of a cellular interactor for the 40-residue long ectodomain of flavivirus M (ectoM) protein that displays pro-apoptotic activity has been obtained. Yeast-two-hybrid screen using human cDNA library was used to assess a panel of ectoM from flaviviruses transmitted by mosquitoes or ticks, as well as GST pull-down assays. Interaction between human dynein light chain (DLC) Tctex-1 and ectoM has been observed for all flaviviruses tested, except YF virus. Silencing Tctex-1 inhibited flavivirus progeny production in human cells. Human DLC Tctex-1 might play an important role in late stages of flavivirus replication through its ability to interact with ectoM (N. Pardigon; coll.: Unité Génomique Virale et Vaccination). Extensive use of high-throughput yeast two-hybrid to characterize interactions between CHIK virus and human proteins identified a limited number of cellular protein partners to viral nsP2 protein. Among them, the heterogeneous nuclear ribonucleoprotein K (hnRNP-K), the ubiquilin 4 (UBQL4), and the tetratricopeptide repeat protein 7B (TTC7B) might play a major role in the efficiency of CHIK virus replication in vitro (coll.: Unité Génomique Virale et Vaccination).

 

Pathogenicity of european strains of WN virus in vivo and in vitro. Until recently, there was no available molecular clone of a European/Mediterranean strain of WN virus. The Middle-Eastern WN virus strain IS-98-ST1 has proved to be highly virulent for birds and mammals, in particular in mouse models for WNV infection. A molecular clone of IS-98-ST1 has been generated that enables the analysis of selected mutations or novel genomic changes appearing in European emerging strains, in the context of increasing numbers of outbreaks and severity of infection in the Mediterranean basin and Eastern Europe ((N. Pardigon; FP-7 program EuroWestNile).

 

Antiviral effect of OAS3 against arboviruses and mechanisms of viral subversion towards OAS3. Expression of OAS3 results in inhibition of viral growth in human epithelial cells infected by CHIK virus. The major characteristic of the anti-alphaviral effect of OAS3 is the absence of viral RNA accumulation within the infected cells with CHIKV. To circumvent antiviral innate immunity, alphaviruses have developed strategies by which cellular machinery is devoted to their own advantage. Analysis of a CHIK virus variant showing a remarkable resistance towards OAS3 showed that a single amino acid change in E2 is sufficient to enhance viral replication through a more robust phosphorylation of PKR and eIF2a in human muscle cells (ANR ARBOAS; coll. Unité Génétique Fonctionnelle des Maladies Infectieuses). Expression of OAS3 in human hepatoma cells inhibits replication of wild-type strain Asibi of YF virus. A variant of Asibi showing resistance towards OAS3 has been identified. Few amino acid substitutions were detected and they are currently being analyzed in the context of a molecular clone of Asibi that has been recently obtained in the lab.

 

Vaccine candidates against DEN or CHIK virus based on a measles vector. A tetravalent dengue antigenic construct, acronym MVDVax, expressing in tandem the antigenic domain EDIII from the four DEN serotypes fused with ectoM induced neutralizing antibodies against the four DEN viruses (ANR MVDVax; coll.: Unité Génomique Virale et Vaccination). MVDVax vaccine will be qualified if it succeeds at reducing viral load in a preclinical macaque model in terms of neutralizing antibody titers as well as level of viral load reduction. (coll: CEA, Fontenay-aux-Roses). A recombinant MV vector expressing the three structural proteins from CHIK virus strain La Réunion-2006 has been generated. Immunization of mice with the recombinant MV induces anti-CHIK virus neutralizing antibodies that protect against a lethal challenge with CHIK virus. The vaccine candidate against CHIK virus based on MV vector is now included in a process of production of a GMP lot and a preclinical and then clinical development are foreseen in collaboration with the SME THEMIS Biologicals Gmbh, Vienna, Austria (coll.: Unité Génomique Virale et Vaccination).

 

The National Reference Center (NRC) for Arboviruses (also Collaborating Centre for WHO and OIE) at the Institut Pasteur, Paris (2008-11) carried out the tasks defined by the Institut de Veille Sanitaire (InVS) in France. The expertise of the NRC is requested in a number of specialized work groups around the issues posed by the spread of arboviruses in Europe with a constant adaptation of the means of expertise required for an effective risk assessment. It is particularly requested that the NRC: (i) provides all expertise to diagnose arbovirus infection and identify the infecting pathogen, (ii) develops and/or validates the serological and molecular kits for viral diagnosis, (iii) contributes by an extensive network of collaborations with the National, European and International Agencies for Public Health to the surveillance and alarm by reporting any unusual events such as increase of the number of suspected cases, changes in clinical forms of viral diseases, and introduction of new viruses in Europe. Since 2006, the NRC plays a key role in the survey of dengue, West Nile, Toscana and Chikungunya in Southern France where the mosquito vector Aedes albopictus has successfully disseminated. The first autochthonous human cases of dengue and Chikungunya had been identified in the French Riviera in 2010 by the NRC. 

 

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Figure 1. Confocal analysis of human muscle cells infected with Chikungunya virus. Immunofluorescence staining of viral replication complexes (red) and viral proteins (green) from H. Gad et al.)

 

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Figure 2. Confocal analysis of Hela cells infected with West Nile virus. Immunofluorescence staining of the E glycoprotein (left) and Tctex-1 (right) (J-B. Brault)

 

Updated on 13/02/2014

Contact

Flavivirus-Host Molecular Interactions Unit

Institut Pasteur

25, Rue du Docteur Roux

75724 Paris Cedex 15 FRANCE

 

Tel + (33) 1 45 68 87 23

Fax + 33 (0)1 40 61 37 74

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