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  Director : DEUBEL Vincent (vdeubel@cervi-lyon.inserm.fr)


  abstract

 

The Mérieux-Pasteur Research Centre in Lyon is a new association between Mérieux Foundation and Pasteur Institute established for the development of researches on emerging diseases caused by highly pathogenic agents. With the support of the BSL-4 laboratory, the first suite-lab in Europe, Pasteur Institute and its international network provide to the international community a scientific support to help populations suffering of haemorrhagic diseases. The objectives of the Centre are oriented to a surveillance of emerging diseases and to a better understanding of the pathogenesis of haemorrhagic fevers for the development of potent prophylaxis and therapy.



  report

cale

Several episodes of haemorrhagic fevers and of viral encephalitis have alerted the organisms of public health and collaborating reference centres this last decade: Ebola, Marburg, dengue haemorrhagic fever, yellow fever, Rift Valley fever, Crimean-Congo haemorrhagic fever, West Nile… There is a crucial need of treatments and vaccines against these viruses that are highly pathogenic for humans. One of the difficulties to reach this task is the high risk to manipulate these viruses that require BSL-3 or BSL-4 containments. The only BSL-4 in Europe has been opened in Lyon.in December 2000. This laboratory built up under the initiative of Dr Charles Mérieux belongs to Mérieux Foundation that maintains permanent logistic and biosecurity supports. A newly created association between Mérieux Foundation and Pasteur Institute, named Mérieux-Pasteur Research Centre in Lyon (CRMPL), has been he starter of new research topics dedicated to molecular diagnostics for the identification of P4 agents and to study of the biology of these agents. These researches will help to the development of potent therapy and prophylaxis.

The scientific team that belong to Pasteur Institute is gathered in the newly created Unit of Biology of Viral Emerging Diseases (UBIVE) that includes the National Reference Center (NRC) and the WHO Collaborating Center (WHOCC) for Arbovirus and Haemorrhagic fevers. UBIVE is part of the Department of Virology in Pasteur Institute in Paris and part of IFR74 "Center of Study and Research in Virology and Immunology" Directed by INSERM.

Over its academic research on emerging pathogens, the CRMPL represents a model of delocalisation of a Pasteur Institute research unit in Lyon. This Centre profits of the highest scientific environment provided by the IFR74, by the Ecole Normale Supérieure, by the Institute of Chemistry and Biology of Proteins, by the University Claude Bernard Lyon I…

UBIVE has privileged collaborations with the International Network of Pasteur Institutes, the European Network of Viral Imported Diseases (ENIVD) and the WHO bureau, Department of Transmissible Diseases, Surveillance and Action in Lyon. One of its objectives is to confirm its French and European expertise on Emerging Viral Diseases and zoonoses

Finally, formation and teaching of professionals and information of a larger public about emerging viral diseases and their pathogens are part of the missions of the research and biosecurity teams of the CRMPL.

I. The BSL-4 in Lyon

1. Diagnostic activities (M-C. Georges, H. Zeller, P. Marianneau, I. Marendat, S. Lacote)

One of the tasks of the CRMPL, in connection with the NRC for Arboviruses and Haemorrhagic Fevers, is to provide rapid diagnosis for haemorrhagic fevers carried out on samples from patients living in countries with epidemics of VHF and from imported cases. Lassa, Ebola, Marburg, Crimean-Congo haemorrhagic fever and Nipah virus genome amplification by RT-PCR has been established. Molecular tools are being developed to avoid the use of the BLS-4 laboratory for the preparation of antigens and for the diagnostics. Recombinant virus envelope and nucleocapsid proteins are produced using the baculovirus system. Reagents will then be available for laboratories of the International Network of Pasteur Institutes, and for other collaborating laboratories located in endemic and epidemic areas, and for laboratories of ENIVD.

With the support of a grant from the European Community, a new diagnostic test has been developed which is based on the detection of viral genomes by RT-PCR after virus capture on polymers coated on magnetic beads. This test is simple and reliable, and can be used in the field. The polymer-coated beads have been developed by the Unité mixte CNRS-BioMérieux UMR503 in Lyon. RT-PCR has been developed for the diagnosis of yellow fever, Lassa, Ebola, Marburg, Crimean-Congo haemorrhagic fever, and Nipah. Results have been compared to those obtained by classical RT-PCR and virus titration techniques and the different steps of the protocols have been optimized for field use. The new technique has been tested in Guinea, Ivory Coast and Gabon where epidemics of yellow fever, Lassa and Ebola were notified.

In order to develop a safe ELISA test to detect IgG and IgM in sera from patients infected with Lassa virus, recombinant viral nucleoprotein NP and glycoprotein GP have been produced in the baculovirus expression system. The antigenic reactivity of the recombinant proteins with immunized animal and patient sera is under trial.

(Collaborations : B. Mandrant, C. Akoua-Koffi, L. Koivogui, E. Leroy, A. Sall)

2. Research activities on Lassa virus

Research programmes in the BSL-4 are oriented to the study of the pathophysiology of Lassa fever, an haemorrhagic disease caused by Lassa virus, genus arenavirus of the Arenaviridae family. Like all arenaviruses, the virus is maintained in nature by transmission of a persistently-infected. rodent of Mastomys sp to another rodent. Humans are infected by contact or inhalation of secretions of infected rodents (urine, feces, saliva) and can be the source of inter-human contamination. The endemic zone of Lassa virus is located in western Africa with an estimated of 300,000 cases with 5000 deaths. Few days after contamination, the fever increases and flue-like symptoms appear. Severe disease is accompanied with diarrhoea, vomiting, facial and cervical oedema, conjunctiva haemorrhages and sometimes bleedings. Patients generally die of hypovolemic shock and respiratory distress. There is no vaccine and Ribavirin is an efficient therapeutic if provided within the first days after appearance of the symptoms.

A- Study of the immune response to Lassa virus infection

(S. Baize, I. Grosjean, M-C. Georges)

The immune responses induced during Lassa fever are not known in detail but are presumed to be involved in the outcome of the patients. Dendritic cells and macrophages play a crucial role in the initiation and the regulation of the immune responses, and the later are known to be target cells of Lassa virus infection. We are studying Lassa virus interactions with dendritic cells obtained from human blood monocytes cultured in the presence of GM-CSF and IL-4, and with macrophages differentiated in the presence of M-CSF.

Preliminary results of immunofluorescence, flow cytometry and virus titration show that dendritic cells and macrophages are sensitive to Lassa virus infection and are activated during virus infection. Pro-inflammatory and anti-inflammatory cytokines produced by the infected cells are being studied. In addition, interactions between dendritic cells and macrophages with T-cells will be investigated.

This study will provide information on the consequences of the viral tropism for antigen-presenting cells in the induction of the immune response and to on a possible role of immune cells in the pathophysiology of Lassa fever.

B - Study of endothelial cell responses to Lassa virus infection (P. Marianneau, P. Loth)

This project focuses on the study of the origins of the increase of the vascular permeability during Lassa virus infection. The study of human endothelial cell responses either directly to Lassa virus infection or to soluble factors released by Lassa virus-infected macrophages is investigated.

Guinea pigs showing respiratory distress after Lassa virus infection would provide an interesting model to study vascular permeability in vivo and to test new treatment in preventing pleural effusion.

C- Study of antivirals against Lassa virus (F. Martineau, I. Grosjean)

The objective of this study is to identify new therapeutics of arenavirus infection. Ribavirin, an analogous of guanosine, is efficient to treat Hantavirus and Lassa virus diseases. However, it is efficient if it is provided during the first days after the onset of the disease. It is toxic when injected to high doses and it cannot reach the central nervous system. Therefore, it is not efficient for treatment of encephalitis encountered in Lassa fever. It would be relevant to identify new potent drugs against Lassa virus.

Our first approach is to screen new molecules analogous to nucleosides using Ippy virus, a P2 arenavirus that infects neuronal cells in vitro. Any potent molecules will then be tested in the BSL-4 against Lassa virus infection of human dendritic cells, macrophages and endothelial cells. Molecules with a virostatic effect will then be tested in a guinea pig model of Lassa virus infection.

3 - Research activities on Nipah virus (I. Grosjean, P. Loth, M-C. Georges)

Nipah virus has be identified for the first time in Malaysia in 1998 and 1999. Humans have been infected by direct contact with pigs that had primarily amplified the virus. Acute encephalitis in 265 pig farmers has led to 105 deaths. Pigs have shown a respiratory distress with a case fatality rate of 5%. Nipah virus has been isolated from the urine of flying foxes that are presumed to be the reservoir of the virus.

The structures of the genome, a negative single-stranded RNA and of the pleomorphic enveloped virus of 100-500 nm in diameter, have allowed to classify Nipah virus in the Paramyxoviridae family. This virus can be distinguished from the other genus of the family, (rubulavirus, paramyxovirus, morbillivirus) by a nucleotide sequence divergence of more than 50%, by the absence of serological cross-reactivity, by a genome larger of about 3000 nucleotides, and by the absence of neuraminidase and hemagglutinin activities of the envelope glycoproteins. The virus is pleiotropic but causes necrosis of endothelial cells and is largely neurotropic.

In order to identify more clearly the pathophysiology of the disease in humans, studies of virus infection have been initiated in vitro and in animal models. These studies would allow the development of a therapy that is needed against this emerging disease. The tools that are being developed for this study would be useful for a surveillance of the virus circulation in southeast Asia and for the identification of new potential reservoirs.

(Collaborations : Fabian Wild, Robin Buckland, Sai Kit Lam, Kow Bin Chua)

II. Activities of the National Reference Centre for Arboviruses

1- Outbreaks : detection and investigation (B. Murgue, S. Murri, D. Coudrier, I. Marendat, H. Zeller)

Following the West Nile outbreak in Southern France in 2000, different epidemiological surveys have been conducted including multiple partners (Ministry of Health, Agriculture, Environment and other agencies), in which the NRC has been strongly involved : serosurvey in horses and birds in 2000 and surveillance programme by sentinel birds in 2001.

The genomes of West Nile isolates (France 2000 and Tunisia 1997) have been fully sequenced.

In April 2001, few patients with fever and jaundice are detected in Côte d'Ivoire. The Institut Pasteur of Abidjan suspects Yellow Fever, this will be confirmed by the NRC. All samples were then sent to the CNR for confirmation. A mission was done in October 2001 (P. Marianneau — UBIVE et A. Sall — Pasteur Institut of Dakar) to establish the RT-PCR in Abidjan. Virus was then detected in a few samples.

All over the year 2001, the NRC has been involved in the follow up of the yellow fever epidemic in Guinea.

(Main collaborators: J. Hars, S. Zientara, C. Akoua-Koffi, L. Koivogui)

2- Clinical and epidemiological aspects of dengue : imported dengue and dengue in endemic countries (B. Murgue, H. Zeller)

A prospective study on imported dengue in France has been conducted in 2000, with the collaboration of Pasteur-CERBA. Dengue infection has been laboratory-confirmed for 103 among 3103 patients (3.3%). Patients were returning from Antigua/Guiana (33%), Asia (27%), Africa (21%) and Central and South America (17%). Among the dengue patients, one DHF case was reported, however 6 patients had severe neurological signs, 3 patients had severe haemorrhage and 5 patients an added infection. One patient with an homozygous drepanocytic anaemia presented a vaso-occlusive crises due to dengue virus with severe manifestations.

A project on early clinical and biological diagnosis of severe forms of dengue has been taken in place with the Research Centre for Emerging Viral Diseases, IRD-Mahidol University (Bangkok) and the physicians of provincial hospitals in Thailand. This project will begin in January 2002.

A project on imported dengue in Europe, in the scope of ENIVD has been taken in place in order to evaluate the yearly incidence of dengue in Europe, to standardise the diagnostic procedure and to determine the clinical, epidemiological and virological aspects of imported dengue. A first meeting was organised in Institut Pasteur in December 2001.

(Main collaborators: JD. Poveda, JP. Gonzalez, X. Deparis, J. Groen)

3- Prediction and prevention of haemorrhagic fever with renal syndrom (HFRS) (H. Zeller, D. Coudrier, B. Murgue)

In France, the diagnosis of HFRS is performed only at the NRC. This allows to have an exhaustive overview on the epidemiological situation of this disease. Clethrionomys glareolus is the specific rodent reservoir for Puumala virus. The great majority of HFRS are reported in the North-East. Epidemics of HFRS occur every three years, the last one was in 1999 (115 cases). A collaboration with the CIRE-East has been established in October 2001 in order to reinforce the surveillance system based on clinical and epidemiological data. This system will allow to increase physician awareness for the next epidemic in 2002.

From January 1st to December 15, 2001, HFRS has been confirmed for 83 patients. The monthly repartition of cases is quite homogeneous (5 to 8 cases) , except in January (11 cases) and October (14 cases). These data are surprising as the year 2001 is an inter-epidemic one with usually less than 40 cases per year. However in 2000, HFRS was confirmed for 68 patients suggesting some modifications in the transmission of Puumala virus.

In March 2001, the first reported case of imported Hantavirus Pulmonary Syndrome in Europe is diagnosed by the CNR

An evaluation of commercial kits for hantavirus diagnosis was conducted. Data analysis is under progress.

(Main collaborators: M. Artois, P. Rollin)



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

STUBLJAR Isabelle 33 4 37 28 24 40

DEUBEL Vincent, ScD

BAIZE Sylvain, PhD

GEORGES-COURBOT Marie-Claude, MD

MARIANNEAU Philippe, PhD

MURGUE Bernadette, MD, PhD

ZELLER Hervé, Pharmacist

MARENDAT Ingrid, technician

MURRI Séverine, technician

LOTH Philippe, Technician

 

GROSJEAN Isabelle, Associated Ingineer

MARTINEAU Francis, PhD, post-doc

FLETY Géraldine, fellow tehnician

LACOTE Sandra, post-graduate fellow


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