Urgent Response to Biological Threats  

  HEADDr Jean-Claude Manuguerra / jmanugu@pasteur.fr
  MEMBERSARNOUX Yolande, AYTAC Marie-Dominique, BATEJAT Christophe, BURGUIERE Ana Maria, CORALIE Gilberte, DUBLINEAU Amélie, Dr. DUBOIS Philippe, FICHENICK Frédéric, Dr. FOUQUE Florence, Dr. LECLERCQ India, LE FLECHE Anne, DR LE GOUIL Meriadeg, LOMPREZ Fabienne, PARIENTE Kevin, ROUSSEAUX Claudine, Dr. SAUVAGE Virginie, SHIGEMATSU Sayuri, VANHOMWEGEN Jessica

  Annual Report

Creation-history-core missions

The Laboratory, called in French Cellule d’Intervention Biologique d’Urgence(CIBU), was created at the end of 2002, under the impulse of the Directorate-General of Health of the Ministry in charge of Health and the Director General of the Pasteur Institute, to respond to "specialized biological urgencies". These urgencies are dealt with in the event of epidemics, accidents, or bioterrorist attacks using biological weapons which can endanger public health.

Area of intervention

CIBU, which is divided into 2 major poles (Bacteriology –PIB- and Virology –PIV) intervenes 24/7 in the detection and identification of a wide array of pathogens in particular the following microbiological agents: 1/ Bacteriology: agents of botulism, anthrax, plague, and tularaemia; 2/ Virology: detection of class 4 agents by molecular biology without virus amplification in cell culture (this is made to avoid working on class 4 agents without knowing so): Ebola and Marburg, Crimea Congo, Lassa and Nipah viruses. Detection of influenza A(H5N1) viruses, SARS (Severe Acute Respiratory Syndrome) coronavirus and orthopoxviruses. An array of other microbial agents are also detected and identified by CIBU in no emergency situations. Besides intervention and microbiological identification activities, CIBU develops research oriented activities and research projects in direct connection with its core missions.

Research oriented activities and research projects

RIVERS (2007-2010):This project called ‘Resistance of Influenza Viruses in Environmental Reservoirs and Systems’ with the acronym of RIVERS is coordinated by CIBU with a strong involvement of the whole team. RIVERS (http://www.rivers-project.eu) is sharply focused and is a research and technological development project designed to gain new knowledge to improve or develop new products and processes. The surge of the global avian influenza epizootic caused by the genotype Z high pathogenic avian influenza virus (HPAIV) has posed numerous questions, in particular to risk managers and policy makers. Scientific knowledge was very thin on many aspects of the ecology and environmental properties of HPAIVs, in particular H5N1. Virus survival, a key element in control strategies, was an illustration of this paucity of knowledge. More specific objectives are: 1/ to understand the basis of virus survival from a virological viewpoint; 2/ to understand the impact of physical and chemical elements on virus survival; 3/ to evaluate the role of environmental reservoirs; 4/ to propose standardised protocols for the concentration and detection of AIVs in waters, including waste waters, and in different matrices including food; 5/ to provide a database together with analytical tools to allow the generation of evidence based guidelines for the prevention and control of influenza outbreaks in animal and human populations, especially at times of restocking.

Detection of a wide array of pathogens by massively parallel technologies (2002-Present)

This area of research is approached by many complementing projects including the following ones: PEVASE/NEVADA(Prévention des Emergences Virales en Asie du Sud-Est/New Emerging Viral Agents Discovery and Analysis: 2004-present), Pathogen ID(2005-2008), DEVA (2008-2011) and the set of projects under the umbrella of Pathodisk (2009-present).

Some, such as PEVASE and PathogenID, are based on very high density resequencing DNA microarrays, others such as those depending on Pathodisk are based on high throughput technologies (HTS) also sometimes called New Generation Sequencing (NGS). In addition, we have been working on stages upstream of sequencing, in particular on nucleic acid extraction and purification and on isothermal nucleic acid amplification.

Identification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We worked on an approach based on whole-genome amplification and resequencing microarrays for “unbiased” pathogen detection. This 10 h process identified a broad spectrum of bacterial and viral species and predicts antibiotic resistance,pathogenicity and virulence profiles. Using the first generation of PathogenID (v1.0), we successfully identified a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identified organisms by hybridization of their DNA in as little as 1–4 h. Using this method, we identified Monkeypox virus and drug-resistant Staphylococcus aureusin a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner. Using the second generation PathogenID v2.0, we rapidly identified a novel influenza A virus strain of swine origin in the context of the emergence of the H1N1 pandemic virus (H1N1pdm) in Mexico in 2009. This novel virus resulted from a reassortment between the triple reassortant swine influenza virus that had caused considerable problems for pig farmers for several years and a Eurasian swine lineage. From the start of the outbreak, our laboratory, together with the National Influenza Centre for Northern France here at Institut Pasteur, faced the urgent need to investigate several suspected cases of human infection by the novel virus. We showed that such an approach could prove useful for public health authorities attempting to detect and identify reassortment events in future outbreaks.

Recently, together with other laboratories in various projects, we engaged in new kinds of technologies: high throughput sequencing technologies. Today, they are not compatible with the timeframe given to a laboratory like ours to output identification results as yet. However when the bottlenecks of sequencing itself but more importantly bioinformatic analysis are overcome or if we face a totally new agent such as the virus causing SARS in 2003, this kind of technologies can prove very useful for a prompt Public Health Response.

Keywords: Emergency, response, virus, bacteria, outbreak, epidemic, threat, pandemic influenza, diagnostic, identification


N. Berthet, I. Leclercq, A. Dublineau, S. Shigematsu, A. M. Burguière, Cl. Filippone, A. Gessain and J. C. Manuguerra, 2010 “High density Resequencing DNA Microarrays Can Prove Useful in Public Health Emergencies : application to the influenza A(H1N1) pandemic” Nature Biotechnology, 28(1):25-7

I. Leclercq, N. Berthet, C. Batéjat, C. Rousseaux, P. Dickinson, I. G Old, K. Kong, G. C Kennedy, S.T. Cole and J.C. Manuguerra2010 “Use of consensus sequences for the design of high density resequencing microarrays: the influenza virus paradigm” BMC Genomics, 11:586

Leclercq A, Clermont D, Bizet C, Grimont PA, Le Flèche-Matéos A, Roche SM, Buchrieser C, Cadet-Daniel V, Le Monnier A, Lecuit M ; Allerberger F. 2009 « Listeria rocourtiae sp.nov. » Int J Syst Evol Microbiol. 13.

N.Berthet , A. K. Reinhardt , I. Leclercq, S. van Ooyen , C. Batejat, P. Dickinson , R. Stamboliyska, I. G Old , K A Kong , L Dacheux , H Bourhy , G C Kennedy , C Korfhage S T Cole and J. C. Manuguerra2008 “Phi29polymerase based random amplification of viralRNA as an alternative to random RT-PCR” BMC Molecular Biology, 9:77

N. Berthet, P. Dickinson, I. Filliol, A.K. Reinhardt, C. Batejat, T. Vallaeys, .K.A. Kong, C. Davies, W. Lee, S. Zhang, Y. Turpaz, B. Heym, G. Coralie, L. Dacheux, A.M. Burguière, H. Bourhy, I.G. Old, J.C. Manuguerra, S.T. Cole, G.C. Kennedy 2008 “Massively parallel pathogen identification using high-density microarrays” Microbial Biotechnology 1: 78-86

Activity Reports 2010 - Institut Pasteur
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