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  Director : Sylvie van der WERF (svdwerf@pasteur.fr)


  abstract

 

The activities of the unit are focused on molecular genetics of positive and negative strand RNA viruses (influenza viruses, picornaviruses, hepaciviruses). They include analysis of the molecular mechanisms of expression and replication of the viral genomes and of their use as expression vectors, study of virus host interactions and pathogenesis, and of the evolution and genetic variability of the viral genomes.



  report

cale

I. "MOLECULAR GENETICS OF RESPIRATORY VIRUSES" directed by Sylvie van der WERF

a) Molecular genetics of influenza virus transcription/réplication complexes (Nadia NAFFAKH, Pascale MASSIN, Bernadette CRESCENZO-CHAIGNE, Monika MARASESCU)

We pursued the identification of molecular determinants of the transcription/replication complex involved in the type-specificity and species-specificity of influenza viruses by making use of a transient expression system in eucaryotic cells which directs the synthesis of the three subunits of the polymerase complex (PB1, PB2, PA) together with the nucleoprotein NP, and allows to monitor the process of transcription/replication of a pseudo-viral RNA carrying a reporter gene. Having thus shown that the nature of nucleotides and the stability of the secondary structure at the extremities of the viral RNA were important determinants of type specificity, we showed that the type C polymerase complex exhibits a more stringent specificity as compared to the type A complex. Furthermore, analysis of the RNA species synthesized from the pseudo-viral RNA templates revealed that the type A polymerase complex preferentially initiates the synthesis of mRNA wheras the type C complex preferentially initiates that of cRNA. In order to evaluate the impact of preferential mRNA versus cRNA initiation during virus multiplication, analysis of the effect of the mutations when introduced by reverse genetics in the context of an infectious virus was undertaken.

Analysis of polymerase complexes derived from avian or human viruses showed that PB2 residue 627 was a determinant of cold-sensitivity of the polymerase complex, suggesting that the reduced ability of a polymerase complex to replicate the viral genome at 33°C might contribute to the inability of avian viruses to efficiently replicate in humans. We also established that the nature of residue 118 of PA determines the reduced cold-sensitivity of the A/Hong Kong/156/97(H5N1) virus, and could thus have contributed to the ability of this virus of avian origin which was reponsible for a fatal human case of influenza to replicate in the upper respiratory tract of humans. Furthermore, we showed that the efficiency with which the polymerase complex recognizes the promoter sequences at the extremities of the viral RNA varies depending on the virus strain from which the polymerase complex is derived, and in particular on the nature of PB2 residue 627.

b) Viral vectors and vaccinology (Nicolas ESCRIOU, Alexandre VIEIRA-MACHADO, India LECLERCQ, Sylvie GERBAUD, Christophe BATEJAT)

Genetic immunization with recombinant replicons in the form of naked RNA

We have previously shown that replicons derived from the genome of Mengo virus (MV) that express the influenza nucleoprotein (NP) are able, upon injection in the form of naked RNA into mice, to elicit a protective immune response towards a challenge infection with homologous influenza virus. We undertook to extend this observation and demonstrated the ability of recombinant MV replicons that express sequences of the NP of the lymphocytic choriomeningitis virus (LCMV) to protect mice against a lethal challenge with LCMV. Furthermore, we developped replicons derived from the MV genome that could direct the expression of a glycosylated protein such as the influenza hemagglutinin (HA), according to an approach similar to the one previously used for poliovirus replicons,

Production of transfectant influenza viruses with a dicistronic segment by means of reverse genetics

Transfectant influenza viruses harboring a dicistronic segment derived from segment 6 encoding the neuraminidase (NA) were produced by reverse genetics. The strategy used consists in a duplication of the 3' non-coding sequences of the genomic RNA segment, thus making the segment dicistronic for transcription/replication as it directs the synthesis of genomic as well as subgenomic vRNA, cRNA and mRNA. Analysis of the expression of various reporter genes such as CAT or GFP, allowed us to show that the size of the insert or the nature of certain sequences negatively affects the viability of the transfectant viruses. We undertook to generalize this approach to the expression of other heterologous sequences (HBs antigen, NP of LCMV) as well as to other genomic segments. Finally, having shown that the conservation of certain coding sequences from the NA segment is crucial for the viability of the viruses harboring a dicistronic segment, we initiated the analysis of the role of these sequences during the virus life cycle.

c) Molecular epidemiology and evolution of influenza viruses

Evolution of the specificity of influenza surface glycoproteins, the hemagglutinin (HA) and neuraminidase (NA) of recent influenza A(H3N2) isolates with sialic acids (Rita MEDEIROS, Nadia NAFFAKH, Nicolas ESCRIOU, Jean-Claude MANUGUERRA)

Based on the initial observation that recent A(H3N2) influenza virus isolates fail to agglutinate chicken red blood cells, we showed that this property is in particular determined by the nature of residue 226 of the HA. Sequential variations at this position are observed since the introduction of A(H3N2) virus in humans at the origin of the 1968 pandemic. These variations are not correlated with a change of specificity towards a2,6Gal-linked sialic acids, but rather could alter the affinity of the HA for these receptors. We also showed that residue 193, that is conserved for viruses from a given host, is involved in the specificity of the HA towards N-acetyl or N-glycolyl sialic acids in a2,3Gal linkage, but that this specificity also depends on the general context of the HA sequence.

In parallel, we initiated the analysis of the evolution of the NA of recent A(H3N2) isolates for which variations in their specificity and/or affinity towards sialic acids has been demonstrated.

d) National Influenza Center (Northern-France) and WHO Collaborating Center for Reference and Research on influenza viruses and other respiratory viruses (Sylvie van der WERF, Jean-Claude MANUGUERRA, Maryse TARDY-PANIT, Saliha AZEBI, Valérie LORIN, Claudine ROUSSEAUX)

As a Reference Center, the unit contributes to the surveillance of influenza and other respiratory viruses at the national level through the RENAL network of hospital laboratories and the GROG network of sentinel general practitioners and pediatricians, as well as on the international, particularly european, level by electronic exchange of data within the European Influenza Surveillance Scheme (EISS) and by piloting the EUROGROG network.The Center has also been invited by the WHO and NGOs such as MSF to participate in the investigation of epidemics associated with a high mortality rate in african countries. Isolation, identification and antigenic characterization of respiratory viruses from specimens from cases of influenza-like illness are carried out. During the 2001/2002 season the influenza epidemic was moderate with co-circulation of influenza A and B viruses. Influenza A(H3N2) viruses were largely predominant and were found to be antigenically related to the A/Panama/2007/99(H3N2) reference strain which was included in the vaccine composition. Influenza viruses of the H1 sub-type were antigenically close to the vaccine strain A/New Caledonia/20/99(H1N1). However, the striking event of the 2001/2002 season was the emergence of the new sub-type A(H1N2) viruses, that resulted from a reassortment event through which A(H3N2) viruses exchanged their HA segment with that of A(H1N1) viruses. Concerning influenza B viruses, they were found either to be close but antigenically distinct from the vaccine strain B/Yamanashi/166/99, or to be related to viruses from the B/Victoria/2/87 lineage, to which also belongs strain B/Hong Kong/330/2001, that was included in the vaccine composition of the 2002/2003 season.

II. "VIRUS des HÉPATITES" directed by Annette MARTIN

Study of in vitro and in vivo replication of hepatitis C and GB-B viruses. Development of a surrogate animal model to study hepatitis C pathobiology. (Annette MARTIN, Lisette COHEN, David GHIBAUDO)

In an effort to circumvent the lack of in vitro cell culture systems able to support efficient replication of hepatitis C virus (HCV) and the lack of an alternative experimental animal model to chimpanzees, we are using GB virus B (GBV-B), which is phylogenetically the most closely related virus to HCV among the Flaviviridae family. GBV-B causes hepatitis in small New World primates such as tamarins and marmosets, and replicates efficiently in primary hepatocytes of these species. Assessment of the infectivity of chimeric GBV-B molecular clones containing various segments of HCV 5' noncoding region (5'NC) substituted for equivalent sequences of GBV-B allowed us to characterize RNA replication signals within the 5'NC region of the viral genome. In addition, a chimeric GBV-B genome containing the functional domain of the HCV 5'NC region that determines internal entry of ribosomes (IRES) replicates efficiently in tamarins and provides a good mean to evaluate candidate antivirals targeting HCV translation in small primates (Coll. S.M. Lemon, U.T.M.B., Galveston, TX, USA & R.E Lanford, S.F.B.R., San Antonio, TX, USA).

Other studies aim at characterizing GBV-B proteins, notably the structural proteins. Using recombinant baculoviruses, we showed that chimeric GBV-B/HCV structural precursors are accurately processed by cellular signalases and that heterologous capsid and envelope proteins can assemble into virus-like particles (Coll. M-C. Prévost, Plateforme Microscopie Electronique). Reverse genetics experiments using HCV replicons help us study the molecular requirements for polyprotein processing and RNA replication in a hepatocellular cell line. These studies are also a prerequisite to the design of chimeric GBV-B/HCV molecular clones containing substitutions of part or all of either the structural-protein or replication-enzyme encoding sequences. Analysis of the in vivo infectivity of these chimeric genomes upon intrahepatic inoculation is aimed at identifying the molecular determinants of host range specificity for these hepaciviruses and at providing tools to study selected features of HCV infection in small primates.

Keywords: influenza, hepatitis C virus, picornavirus, replication, vaccine, vector, virology, molecular epidemiology, animal model



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  NAUBRON Christine, secretary, cnaubron@ pasteur.fr COHEN Lisette, University Paris 11,lisemc@pasteur.fr

ESCRIOU, Nicolas, Institut Pasteur,escriou@pasteur.fr

LECLERCQ, India, University Paris 7,ileclerc@pasteur.fr

MARTIN Annette, Institut Pasteur, annettem@pasteur.fr

MANUGUERRA Jean-Claude, Institut Pasteur,jmanugu@pasteur.fr

NAFFAKH Nadia, CNRS,nnaffakh@pasteur.fr

van der WERF Sylvie,Université Paris 7& Institut Pasteur,svdwerf@pasteur.fr

GHIBAUDO David, University Paris 7, PhD student

MASSIN Pascale, University Paris 6, PhD student

MEDEIROS Rita, University Paris 7, PhD student

VIEIRA MACHADO Alexandre, University Paris 7, PhD student

CRESCENZO-CHAIGNE Bernadette, Institut Pasteur, Engineer

GERBAUD Sylvie, Institut Pasteur, Engineer

TARDY-PANIT Maryse, Institut Pasteur, Engineer

AZEBI Saliha, Institut Pasteur, Technician

BATEJAT Christophe, , Institut Pasteur, Technician

LORIN Valérie, Institut Pasteur, Technician

MARASESCU Monika, Institut Pasteur, Technician

ROUSSEAUX Claudine, Institut Pasteur, Technician

ANSELME-VATIN Alex, Institut Pasteur

BLIN Josiane, Institut Pasteur

FILLODEAU Anne-Marie, Institut Pasteur


Activity Reports 2002 - Institut Pasteur
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