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  Gmvr


  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, hepatitis C virus). 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 of the genetic variability of the viral genomes. One group addresses the functional analysis of neurological deficits and cognitive disorders associated with infectious pathologies, in particular HIV infection.



  report

cale

I. "MOLECULAR GENETCS 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 MARASCESCU)

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. We have thus shown that the nature of nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA were important determinants of type specificity and were also involved in the efficiency with which recognition by the polymerase complex occurred depending on the virus strain from which the polymerase complex was derived.

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. Furthermore, we observed that the level of proteolysis induced by the PA subunit of the polymerase complex was variable depending on the origin of PA and that the two activities associated with PA (i.e. proteolysis and transcription/replication) were not strictly correlated.

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

Genetic immunization with recombinant replicons in the form of naked RNA

Replicons derived from the genomes of Semliki Forest Virus (SFV), poliovirus (PV), or Mengo virus (MV) that express the influenza nucleoprotein (NP) were obtained. Upon injection in the form of naked RNA into mice, the recombinant replicons derived from SFV or MV were found to elicit a protective immunity towards a challenge infection with homologous influenza virus. The level of protection was comparable to that elicited by genetic immunization with plasmid DNA expressing the NP. Furthermore, we showed that replicons derived from the poliovirus genome could direct expression of a glycosylated protein such as the influenza hemagglutinin (HA), provided its expression was uncoupled from that of the rest of the poliovirus polyprotein by means of insertion of an IRES in a dicistronic construct.

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 according to two different strategies. The first one which relies on the insertion of the BiP IRES sequence renders the segment dicistronic for translation of the corresponding mRNAs. The second one 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 establish the superiority of the second approach and to show that the size of the insert negatively affected the viability of the transfectant viruses.

c) Molecular epidemiology of influenza viruses

Specificity of interaction of the hemagglutinin (HA) of influenza A(H3N2) viruses with the receptor, sialic acid (Rita MEDEIROS, Nadia NAFFAKH, Nicolas ESCRIOU, Jean-Claude MANUGUERRA)

Based on the 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. In parallel, we pursue the study of a substitution at residue 193, that is conserved for viruses from a given host and seems to be involved in the specificity of the HA towards a2,3Gal-linked sialic acids.

Molecular epidemiology of animal influenza viruses (Jean-Claude MANUGUERRA, Claudine ROUSSEAUX)

Phylogenetic analysis of the genes (PB1, PB2, PA) of equine A(H3N8) influenza viruses isolated in France between 1993 and 1999 suggests a linear evolutionary pathway and shows that they belong to the same lineage as strain A/Eq/London/1416/73(H7N7), in agreement with the hypothesis of the occurence of a reassortment event between A(H7N7) and A(H3N8) viruses that resulted in the substitution of the internal genes. Furthermore, evolution of the PA genes was found to be more rapid than that of the PB1 and PB2 genes.

Specimens were collected from wild birds and avian influenza viruses were isolated. Their characterization is ongoing.

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, Valérie LORIN, Claudine ROUSSEAUX, Valérie SEFFER)

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 european level by electronic exchange of data within the European Influenza Surveillance Schemle (EISS) and by piloting the EUROGROG network. Isolation, identification and antigenic characterization of respiratory viruses from specimens from cases of influenza like illness are carried out. During the 2000/2001 season the influenza epidemic was weak. Influenza A(H1N1) viruses predominated and were found to be antigenically related to the A/New Caledonia/20/99(H1N1) reference strain which was included in the vaccine composition. Influenza B viruses, that circulated later during the season, were found to be generally close but antigenically distinct from the vaccine strain B/Yamanashi/166/99.

II. "VIRUS des HÉPATITES"

directed by Annette MARTIN

Towards the use of chimeric GB-B viruses bearing hepatitis C virus determinants in tamarins as a surrogate animal model for hepatitis C. (Annette MARTIN, Lisette COHEN, Danièle BÉNICHOU, David GHIBAUDO)

In an effort to circumvent the lack of an in vitro cell culture system able to support efficient HCV replication and the fact that chimpanzees constitute the only experimental animal model, we are using GB-B virus (GBV-B), which is phylogenetically the most closely related virus to HCV among the Flaviviridae family. In addition, GBV-B is also hepatotropic and it replicates in small primates (tamarin species). Several hybrid GBV-B/HCV molecular clones were obtained by substitution of sequences encoding part or all of the structural proteins. These are designed to help identifying the molecular determinants of host range specificity for HCV and GBV-B, and to provide tools to study selected features of HCV replication and pathogenesis in tamarins. We showed that proteolytic processing of the corresponding hybrid polyproteins was accurate. Recombinant baculoviruses are currently being produced and used to determine whether structural polypeptides generated from hybrid structural precursors can assemble into virus-like particles. Other chimeric molecular clones have been generated by exchanging sequences involved in viral replication, such as sequences of the major viral proteinase or the IRES sequence responsible for internal initiation of translation. Beside their potential use in HCV replication studies in vivo, they should provide a mean to evaluate candidate antivirals in small primates (coll. S.M. Lemon, UTMB, Galveston, TX, USA). The infectivity of these chimeric genomes is currently under investigation after intrahepatic inoculation to tamarins.

III. "Cognitive deficits in various neuropathological diseases : NeuroAIDS, Cerebral Malaria and Schizophrenia"

directed by Catherine VIDAL

Functional analysis of animal models of infectious neuropathologies : anatomopathology, brain imaging (MRI) and in vivo spectroscopy, behavior.

  • Model of HIV encephalitis and dementia in the rat
  • Cerebral malaria in a murine model

Clinical studies: functional MRI and neuropsychological tests in human mental disorders:

  • HIV associated cognitive disorders
  • Schizophrenia


  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

NAUBRON Christine, secretary

COHEN Lisette, University Paris 11

ESCRIOU, Nicolas, Institut Pasteur

MARTIN Annette, Institut Pasteur

MANUGUERRA Jean-Claude, Institut Pasteur

NAFFAKH Nadia, CNRS

VIDAL, Catherine, Institut Pasteur

van der WERF Sylvie,Université Paris 7 & Institut Pasteur

GHIBAUDO David, PhD student

MASSIN Pascale, PhD student

MEDEIROS Rita, PhD student

VIEIRA MACHADO Alexandre, PhD student

VIGNUZZI Marco, PhD student

BÉNICHOU Danièle, CNRS, Engineer
CRESCENZO-CHAIGNE Bernadette, Institut Pasteur, Engineer

GERBAUD Sylvie, Institut Pasteur, Engineer

TARDY-PANIT Maryse, Institut Pasteur, Engineer

BATEJAT Christophe, , Institut Pasteur, Technician

LORIN Valérie, Institut Pasteur, Technician

MARASCESCU Monika, Institut Pasteur, Technician

ROUSSEAUX Claudine, Institut Pasteur, Technician

SEFFER Valérie, Institut Pasteur, Technician

ANSELME-VATIN Alex, Institut Pasteur

BLIN Josiane, Institut Pasteur

CLAVEL Sandrine, Institut Pasteur


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