Unit: Molecular Genetics of Respiratory Tract Viruses - URA 1966 CNRS
Director: van der WERF Sylvie
The activities of the unit are focused on molecular genetics of positive and negative strand RNA viruses (influenza viruses, SARS coronavirus, 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 of the evolution and genetic variability of the viral genomes, as well as the development of vaccines and diagnostic tools, for instance for SARS.
I. "MOLECULAR GENETICS OF RESPIRATORY VIRUSES"
directed by Sylvie van der WERF
a) Molecular genetics of influenza virus transcription/réplication complexes
(Nadia NAFFAKH, Bernadette CRESCENZO-CHAIGNE, Emmanuel DOS-SANTOS, Pierre DUARTE-RODRIGUES, Monika MARASESCU)
Having shown that the nature of nucleotides and the stability of the secondary structure at the extremities of the viral RNA are 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 towards these determinants. In order to evaluate their role during virus multiplication, analysis of the effect of mutations of these determinants when introduced by reverse genetics in the context of an infectious virus was undertaken.
To complete our analysis of the determinants of host specificity harbored by the polymerase complexes derived from avian or human viruses in the context of avian versus mammalian cells, we performed the molecular cloning of hte avian polI polymerase. This provides a means to develop reverse genetics for influenza viruses in avian cells and to study functional interactions of the polymerase complex with host proteins in avian cells. With the aim to identify cellular interactors of the complex, influenza viruses expressing a tagged PB2 (one of the subunits of the polymerase complex) were obtained . This also provides a means to follow the intracellular localization of the polymerase complexes in the course of virus multiplication.
b) Viral vectors and vaccinology
(Nicolas ESCRIOU, Benoît CALLENDRET, Bernadette CRESCENZO-CHAIGNE, Sylvie GERBAUD, India LECLERCQ, Valérie LORIN)
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. MV replicons that direct the expression of the influenza hemagglutinin were produced. We are currently extending these observations to evaluate the ability of recombinant MV replicons that express various heterologous sequences (HbsAg, LCMV nucleoprotein) to induce a cytotoxic T-cell response and/or humoral immunity that could eventually be protective.
Transfectant influenza viruses with a dicistronic segment
Transfectant influenza viruses harboring a dicistronic segment by means of an internal duplication of the 3' non-coding sequences were produced by reverse genetics. These were shown to be able to induce a humoral or T-cytotoxic immune response towards the heterologous antigens. In the course of this study it was found that conservation of sequences from the extremities spanning the coding region is crucial for the viability of the viruses harboring a dicistronic segment. A possible role for packaging is being investigated, We currently attempt to generalize this approach to the other viral segments as well as to the expression of other heterologous sequences.
Expression of structural proteins of SARS-CoV
Sequencing of the complete genome of the SARS-CoV has been achieved directly from a respiratory specimen from a patient from the french hospital in Hanoi, Vietnam. After molecular cloning of the corresponding sequences, the various structural proteins were expressed by means of different prokaryotic and eukaryotic vectors with the aim to develop vaccine and/or diagnostic tools based on the recombinant proteins.
c) 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, Ana-Maria BURGUIERE, Maryse TARDY-PANIT, Saliha AZEBI, Aurélien BRIONNE, Patricia JEANNIN, Valérie LORIN, Claudine ROUSSEAUX)
Epidemiology and evolution of influenza viruses
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 the Democratic Republic of Congo. Isolation, identification and antigenic characterization of respiratory viruses from specimens from cases of influenza-like illness are carried out. The unit also performs the antigenic and genetic characterization of viruses isolated from swine, horses or from wild birds. Large-scale sequencing should provide a spatio-temporal analysis of the evolution of human and animal (porcine, equine, avian) influenza viruses as well as information about the frequency of reassortments between various lineages. Along this line, phenotypic and phylogenetic analysis of influenza viruses of the A(H1N2) subtype that recently emerged allowed us to show that these originated from multiple independant reassortment events.
Contribution to the investigation of the epidemic of Severe Acute Respiratory Syndrom
At the request of the WHO, the unit has been part of the WHO laboratory network for SARS etiology and diagnostics. Thus, in addition to the investigation of the french probable and suspect cases with the help of the CIBU, the unit contributed to the identification of the new coronavirus as the etiologic agent of SARS, to the definition of the virus excretion profile and to the development and validation of RT-PCR diagnostic tests for the detection of the SARS-CoV.
II. "VIRUS des HÉPATITES", directed by Annette MARTIN
Study of in vitro and in vivo replication of hepatitis C and GB-B viruses.
(Annette MARTIN, Lisette COHEN, David GHIBAUDO, Christophe CHEVALIER, Lucile WARTER)
Studies of hepatitis C virus (HCV) have been hindered by the lack of in vitro cell culture systems able to support efficient replication of HCV and the increasing unavailability of chimpanzees, the only experimental animal model susceptible to HCV. We have focused our interest on GB virus B (GBV-B), which is phylogenetically the most closely related virus to HCV. GBV-B causes hepatitis in small New World primates such as tamarins and marmosets and replicates efficiently in primary hepatocytes of these species. GBV-B is thus an attractive surrogate model for HCV studies.
We characterized proteolytic processing of GBV-B polyprotein within the structural region. In the course of this work, we identified a new protein in GBV-B that shares partial homology with HCV p7 protein. HCV p7 has recently been shown to function as an ion channel but its role in the virus infectious cycle remains unknown. A functional study of this GBV-B new protein will shed light on the role of this type of proteins in the infectious cycle of these viruses.
We are developing and using HCV and GBV-B RNAs (replicons), that are capable of autonomous replication in cell culture, to investigate molecular requirements for polyprotein processing and RNA replication. These studies also provide a basis for the rational design of chimeric GBV-B/HCV molecular clones in which GBV-B functional sequences are substituted by HCV homologous sequences. The infectivity of such chimeric genomes is assayed in small primates following intrahepatic inoculation of RNA. A chimeric GBV-B RNA containing the functional domain of the HCV internal ribosome entry segment (IRES) was shown to be infectious. The corresponding chimeric virus replicates efficiently in tamarins and provides a good model 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). In addition, we characterized adaptive mutations within the genome of this chimeric virus, suggesting the existence of genomic molecular interactions that are likely to be important in GBV-B replication.
Keywords: influenza, hepatitis C virus, picornavirus, SARS coronavirus, replication, vaccine, vector, virology, molecular epidemiology, animal model