1. Variability and evolution of entEroviruses (R. Crainic, F. Delpeyroux, S Guillot, J Balanant)
Human Enteroviruses (picornaviridae, 64 serotypes) include in polioviruses coxsackieviruses, echoviruses, parechoviruses, enteroviruses 68-71. Their transmission is digestive, but the infection of the enteric tract is often sub-clinical. However, they may cause a great variety of severe diseases as meningitis, poliomyelitis, respiratory illnesses, acute myocardiopathy sometimes leading to chronic cardiovascular disease, diabetes, acute hemorrhagic conjunctivitis, congenital malformations and sometimes fatal neonatal diseases. Poliovirus, the agent of poliomyelitis, is a neurotropic enterovirus of 3 serotypes. (1, 2 and 3). Two vaccines are currently used to protect against poliomyelitis: injectable, with inactivated virus (IPV) and the oral, with attenuated virus (OVP). Only the OPV elicits a long-lasting digestive immunity, capable to stop the wild poliovirus transmission.
The genetic variability of RNA viruses (mutation and recombination) is the basis of viral diversity and evolution. By multiplication in the gut of the vaccinees, the attenuated OPV strains frequently revert to neuropathogenicity and, sometimes induce sporadic vaccine associated paralytic poliomyelitis (VAPP) to vaccinees and their contacts. Recently, a vaccine-derived poliovirus (VDPV) which reverted to neuropathogenicity and transmissibility, both wild poliovirus characters, induced a poliomyelitis outbreak in Dominican Republic and Haiti. together with our colleagues in CDC (Atlanta), we determined that, before it induced the outbreak, VDPV circulated for about 3 years and had a recombinant genome with another enterovirus (poliovirus?) of yet unknown origin.
Natural genetic recombination of poliovirus (S. Guillot, N. Cuervo, V. Caro, F. Delpeyroux, R. Crainic)
The genetic recombination between the OPV polioviruses is favored by simultaneous multiplication of the three strains of different serotypes (implicitly genotypes) in the gut of the vaccinees. We found that that more than 3/4 strains isolated from VAPP cases have an intertypic recombinant genome. In some cases, recombination with wild polioviruses and with other enteroviruses, was also found..
We are currently investigating the mechanism of recombination. The poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes. The repercussion of the recombination events on the phenotypic changes of OPV strains and in particular the acquiring of pathogenic characters is still to be determined.
Molecular taxonomy of enteroviruses (V. Caro, S. Guillot, R. Crainic)
To check whether there is a correlation between the antigenicity, the base of the classification of enterovirus serotypes, and their genomic structure, we initiated in 1996 and coordinated a research project in international collaboration on enterovirus molecular taxonomy. We recently discovered (Valérie Caro, PhD theses) that that the gene coding for the protein of capsid VP1, carrying the structure of an important viral neutralization antigenic site, displayed nucleotide sequences in a perfect correlation with enterovirus antigenic properties.
With a single couple of primers that we designed it was possible to obtain an RT-PCR amplicon, from practically all reference strains and from some 100 field isolates The alignment and the treatment of amplicons nucleotide sequences allowed the molecular classification of enteroviruses perfectly overlapping the serological classification. This open the way to identify enteroviruses serotypes by the simple sequencing of a segment of their genome. This diagnosis method will replace the presently in use serological method, which is more and more difficult to practice because of the exhaustion of type-specific neutralizing sera.
Phylogeny of epidemic Enteroviruses (A. Szendroi, S. Chevaliez, J. Balanant, F. Delpeyroux).
In order to understand the circulation and intra-epidemic evolution of enteroviruses, we analyzed genetically several Echovirus 11 (E11) strains implicated in a severe hemorrhagic syndrome outbreak in Hungary. The Hungarian strains were closely related to each others at the genetic level (99 % nucleotide identity) indicating that they do have a recent common ancestor strain. Comparison with previously or contemporarily circulating E11 isolates from sporadic cases and from environment in Finland, Romania, Netherlands, Russia end Japan have shown that Hungarian strains were closely related to most European and to the Japanese strains. A very close relationship was found with Romanian and a Finnish strain isolated from a sporadic hemiparesis case and from environmental waters, respectively. These results prepare the way for the detailed analysis of the viral determinants which differentiate the epidemic E11 from its neighboring strains isolated in non-epidemic period.
2. Physiopathologie of hepatitis C Virus (HCV) infection(A. Budkowska, P. Maillard, M. Sidorkiewicz, A. Poujol)
Hepatitis C virus infection is a major problem for public health in France and world-wide. The frequent evolution of the HCV infection in chronic disease (80% of cases) often leads to the development of liver cirrhosis and primary liver carcinoma. There is currently no way of preventing and treating HCV-related liver disease effectively. Our knowledge of the properties and functions of the structural HCV proteins relies on the molecular data and the expression models in vitro. The structure and biological properties of viral particles occurring in the serum of HCV infected individuals remain undetermined.
We showed that viral particles with physicochemical, antigenic and morphological properties of non-enveloped HCV nucleocapsids are present in the serum of HCV-infected individuals. These viral particles have a density of 1,32-1.34 g/ml, 38-43 or 54-62 nm in diameter and express on their surface specific epitopes localized in the immunodominant region of HCV core protein (amino acids 24-68). Using monoclonal antibodies produced by immunization with serumderived HCV nucleocapsids we showed the presence of HCV core antigen in the cytoplasm of hepatocytes of experimentally infected chimpanzees by immunofluorescence and confocal microscopy (coll. with CDC, Atlanta, USA).
HCV nuceocapsid is the only HCV particles isolated from serum and characterized. The production of non-enveloped HCV nucleocapsids and their release from serum seems to be a particular property of HCV morphogenesis. The presence of these particles in serum and accumulation of the core protein in hepatocytes during infection might contribute to the induction of HCV persistence and multiple immunopathological effects in the infected host.
3. RESEARCH AND PUBLIC HEALTH SUPPORT ACTIVITY (S. Guillot, J. Balanant, P. Maillard, A. Budkowska, F. Delpeyroux, R. Crainic)
This activity is formalized by the following structures functioning in our laboratory:
- WHO Collaborative Center for Reference and Research on Poliomyelitis;
- WHO Collaborative Center for Standardization of Viral Vaccines
- the "Study Group for Enteroviruses" (F. Delpeyroux) based on viral laboratories of International Network of Pasteur Institutes involved mainly on research on poliomyelitis
and on molecular identification of enteroviruses.
- Regional Reference Laboratory (Europe) and Specialized Laboratory (global, together with CDC Atlanta, KTL Helsinki, RIVM Bilthoven, NIBSC London and NIID Tokyo) of the WHO Global Polio Laboratory Network for poliomyelitis eradication
- "Inco-Copernicus" International Collaborative Study Groups (coordination of) on poliovirus (Radu Crainic) et sur le HCV (Agata Budkowska) financed by the EU.
Part of the research presented in the previous chapters was the result of the international collaboration as presented in this chapter