Research on infectious diseases: a global challenge

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1 Research on infectious diseases: a global challenge La recherche sur les maladies infectieuses : un défi planétaire June 26th and 27th, 2008 / 26 et 27 juin 2008 Institut Pasteur, Paris, France ORGANISING COMMITTEE : Roberto BRUZZONE, Hong Kong University - Pasteur Research Centre Jean-Marc CAVAILLON, Infection and Epidemiology Dpt, Institut Pasteur, Paris Suzanne CHANTEAU, Institut Pasteur in New Caledonia Yves CHARPAK, International Affairs Director, Institut Pasteur, Paris Eliane COËFFIER, International Affairs Department, Institut Pasteur, Paris Antoine DANCHIN, Genomes and Genetics Dpt, Institut Pasteur, Paris Vincent DEUBEL, Institut Pasteur of Shanghai Antoine GESSAIN, Institut Pasteur, Paris Marcel HOMMEL, Institut Pasteur, Paris Marc JOUAN, International Affairs Department, Institut Pasteur, Paris Jacques LOUIS, Parasitology and Mycology Dpt, Institut Pasteur, Paris Jean-Louis SARTHOU, Institut Pasteur in Cambodia

2 TABLE OF CONTENTS Theme of the conference...1 Scientific program...2 Oral communications...6 Sessions: Session 1: Virology / Epidemiology...7 Session 2: Bacteriology...24 Session 3: Parasitology...30 Posters...38 Authors index...128

3 THEME DE LA CONFERENCE The symposium deals with field work as well as fundamental mechanisms of the diseases, but also with the new advances in disease control. The selected oral communications are those which: - the best illustrate collaborations and synergies between the institutes of the RIIP and various French or international teams - show the impact of the research activities on the development of the local public health capacities - show the articulation between basic and operational research - describe the impact of technological developments on research in the institutes of the network, as well as the original and new approaches to local problems. The symposium begins with an invited conference on the research in the field of the infectious diseases in the 21st century; it will focus on the need to match the geographical distribution of the challenges, the research priorities and the research locations. The symposium ends by an invited forward-looking presentation of the research in the RIIP, its multidisciplinary, international connections and partnerships and the interactions with major interventions in developing countries. Le colloque aborde la recherche sur le terrain, la compréhension des mécanismes des maladies, mais aussi les avancées dans le contrôle des maladies. Les communications orales sélectionnées sont en priorité celles qui : - illustrent les collaborations et synergies entre les instituts du RIIP et les équipes de l'institut Pasteur, voire d'autres équipes française ou étrangères - montrent l'impact des activités de recherche sur le développement des compétences de santé publique locales - montrent l'articulation entre la recherche fondamentale et la recherche opérationnelle, - décrivent l'impact des développements technologiques sur la recherche dans les instituts du Réseau, ainsi que des approches originales et nouvelles des problèmes locaux. Par ailleurs, le colloque s ouvre par une conférence invitée sur la recherche dans le domaine des maladies infectieuses au 21 ème siècle et la nécessaire articulation de la géographie des enjeux et des priorités de recherche avec les lieux d'exercice de la recherche. Le colloque se terminera par une conférence invitée sur la prospective de la recherche dans le RIIP, la nécessaire multidisciplinarité des recherches, les perspectives de connexions et partenariats internationaux de cette recherche dans des pays en développement. 1

4 SCIENTIFIC PROGRAM Thursday June 26th, :00 am Registration of participants 8:45 am-9:00 am Welcome address Alice DAUTRY, Directrice Générale de l Institut Pasteur Yves CHARPAK, Directeur des Affaires Internationales, Institut Pasteur, Paris 9:00 am-9:30 am Opening Lecture David HEYMANN, WHO, Geneva 9:30 am-4:00 pm Session 1 Virology / Epidemiology Chairs: Félix REY / Guillaume DIGHIERO 9:30 am-9:45 am S1-1: Sirenda VONG, Institut Pasteur du Cambodge Rabies in Cambodia: a vaccine preventable death 9:45 am-10:00 am S1-2: Hervé BOURHY, Institut Pasteur, Paris The Origin and Phylogeography of Dog Rabies Virus 10:00 am-10:15 am S1-3: Monique LAFON, Institut Pasteur, Paris Rabies virus has more than one trick up its sleeve to manipulate the host defences 10:15 am-10:30 am S1-4: Laurent DACHEUX, Institut Pasteur, Paris Du diagnostic intra-vitam de la rage humaine à l analyse du transcriptome cérébral chez les patients infectés 10:30 am-10:45 am S1-5: Philippe CAVAILLER, Institut Pasteur du Cambodge Frequency of Poultry to Human H5N1 transmission and evidence of Transmission via contaminated Environment in Cambodia 10:45 am-11:00 am S1-6: John NICHOLLS, Hong Kong University Pasteur Research Centre Determination of the haemagglutinin sialic acid receptor interaction of H5N1 and other influenza viruses at the atomic level 11:00 am-11:30 am Coffee break and posters 11:30 am-12:00 am Keynote speaker Malik PEIRIS, Hong Kong University Pasteur Research Centre 12:00 am-12:15 am S1-7: Julien POTHLICHET, Institut Pasteur, Paris Innate immune response triggered by influenza A virus is negatively regulated by suppressor of cytokine signalling (SOCS)1 and SOCS3 through a RIG-I/IFNAR1 2

5 SCIENTIFIC PROGRAM Thursday June 26th, :15 am-12:30 am S1-8: Veronika Von MESSLING, INRS-Armand Frappier, Canada Innate and Adaptive Immune Responses to Influenza in Ferrets 12:30 am-12:45 am S1-9: Philippe BUCHY, Institut Pasteur du Cambodge Towards a better understanding of the circulation and the evolution of H5N1 viruses in Cambodia and within the regionusing bioinformatics tools 12:45 am-1:00 pm S1-10: Béatrice NAL, Hong Kong University Pasteur Research Centre Development of molecular tools for SARS-CoV single-virus tracking 1:00 pm-1:15 pm S1-11: Yongjin WANG, Institut Pasteur de Shanghai Human coronavirus OC43 Heptad Repeat 2 (HR2) domain induces neutralizing antibodies against SARS coronavirus and other human coronaviruses 1:15 pm-2:30 pm Lunch at Institut Pasteur 2:30 pm-4:00 pm Session 1 Virology Epidemiology Chairs: Antoine GESSAIN / Vincent DEUBEL 2:30 pm-2:45 pm S1-12: Juliet BRYANT, NCLE, Laos Multiplex detection of viral respiratory pathogens by luminextechnology in the Lao People s Democratic Republic 2:45 pm-3:00 pm S1-13: Vincent LACOSTE, Institut Pasteur de la Guyane Dengue infection in neotropical forest mammals 3:00 pm-3:15 pm S1-14: Emmanuel NAKOUNE, Institut Pasteur de Bangui Ebola virus RNA sequences in mammals from Central African Republic 3:15 pm-3:30 pm S1-15: Sabine PLANCOULAINE, Institut Pasteur, Paris A major susceptibility locus for HTLV-1 infection in childhood Maps to chromosome 6q27 3:30 pm-3:45 pm S1-16: Farzin ROOHVAND, Institut Pasteur d Iran French-Iranian collaborative studies on HCV vaccines (CD8- polytopic DNA constructs and protein based immunogens astherapeutic and prophylactic vaccine candidates) 3:45 pm-4:00 pm Discussion 4:00 pm-4:30 pm Coffee break and posters 3

6 SCIENTIFIC PROGRAM Thursday June 26th, :30 pm-6:30 pm Session 2 Bacteriology Chairs: Agnès LABIGNE / Mireille DOSSO 4:30 pm-5:00 pm Keynote speaker Laurent ABEL, Inserm 5:00 pm-5:15 pm S2-1: Sara EYANGOH, Centre Pasteur du Cameroun A multidisciplinary approach to decipher transmission mode(s) of Buruli ulcer : preliminary results 5:15 pm-5:30 pm S2-2: Albert KO, Fondation Oswaldo Cruz, Brazil Vaccine Development for Leptospirosis: From the Favelas tothe Candidate Protein 5:30 pm-5:45 pm S2-3: Sébastien BREUREC, Institut Pasteur de Dakar Etude de la diversité génétique de souches d Helicobacter pylori originaires d Afrique, d Asie, d Europe et d Océanie 5:45 pm-6:00 pm S2-4: Voahangy ANDRIANAIVOARIMANANA, Institut Pasteur de Madagascar Kinetics of IgG production and memory T cell proliferation in confirmed plague patients 6:00 pm-6:15 pm S2-5: Laurent DEBARBIEUX, Institut Pasteur, Paris Phage therapy of an acute lung infection in mice 6:15 pm-6:30 pm Discussion 6:30 pm-8:30 pm Posters session and Wine and Cheese 4

7 SCIENTIFIC PROGRAM Friday June 27th, morning 9:00 am-12:15 am Session 3 Parasitology Chairs: Jacques LOUIS / André SPIEGEL 9:00 am-9:30 am Keynote speaker Jacques LOUIS 9:30 am-9:45 am S3-1: Alejandro BUSCHIAZZO, Institut Pasteur de Montevideo Structure/functional studies of trypanosomal neuraminidases 9:45 am-10:00 am S3-2: Maman Laminou IBRAHIM, CERMES, Niger Analyse de 34 SNPs associées à la résistance de P.falciparum aux antipaludiques par les puces à ADN au Niger 10:00 am-10:15 am S3-3: Ronan JAMBOU, Sydney University Allergic immune response and histamine pathway in malaria: new perspective to improve the treatment of cerebral malaria R 10:15 am-10:30 am S3-4: Elisabeth RAVAOARISOA, Institut Pasteur de Madagascar Recombinant Fab fragments specific for the histidine-rich protein2 and heat-shock protein 72 of Plasmodium falciparum : implications for malaria diagnosis 10:30 am-11:15 am Coffee break / Posters 11:15 am-11:30 am S3-5: Robert MENARD, Institut Pasteur, Paris In vivo imaging of Plasmodium pre-erythrocytic stages in a rodent model 11:30 am-11:45 am S3-6: Hechmi LOUZIR, Institut Pasteur de Tunis Approaches for the identification of vaccines against human leishmaniasis 11:45 am-12:00 am S3-7: Wilson SAVINO, Fondation Oswaldo Fiocruz, Brazil Abnormal T cell traffic in experimental Chagas disease 12:00 am-12:15 am Discussion 12:15 am-12:45 am Closing lecture Michèle BARZACH, Ancienne Ministre de la Santé, Présidente de l'association Les Amis du Fonds Mondial Europe, Paris 12:45 am-1:00 pm Conclusions Alice DAUTRY, Directrice Générale de l Institut Pasteur Yves CHARPAK, Directeur des Affaires Internationales, Institut Pasteur, Paris 5

8 LECTURE ABSTRACTS 6

9 SESSION 1 Virology / Epidemiology 7

10 S1-1 RABIES IN CAMBODIA: A VACCINE PREVENTABLE DEATH. S. Ly 1, P. Buchy 1, S. Ong 1, NY Heng 1, JL Sarthou 1, H. Bourhy 2, S. Vong 1 1. Institut Pasteur du Cambodge 2. Institut Pasteur de Paris Rabies, a fatal but preventable zoonosis, is a major public health problem in developing countries. However in Cambodia the disease burden is largely underestimated because patients with encephalitis following dog bites are rarely hospitalized and die at home. Since 1998 the Institut Pasteur (IPC), Phnom Penh (PP) has been the only source of free post exposure treatment (PET) and post mortem diagnosis. Methods: The data compiled by IPC was analyzed to identify all treated patients, confirmed human rabies cases and results of human and animal testing. From bites' characteristics, we defined a suspected rabies injury as a unprovoked bite from a dog that died spontaneously, or was reported sick. We applied a deterministic probability model to estimate 2007 rabies mortality in Cambodia from the clinical characteristics of biting dogs, the incidence of rabies suspected dog bites injuries, the distribution of bite wounds and the probability of PET access. Results: Over 120,000 patients received PET at IPC during (average: 12,470 and range: ,475). In 2007, 14,475 patients were treated (101/100,000 for Cambodia) including 8,606 (59.5%) residing in PP (615/100,000) and 5,305 (37%) in 5 neighboring provinces. Sixty suspected human cases of rabies were reported and tested at IPC in pre or post-mortem by PCR or IFD during , and 73% tested positive. Of the positive patients, all were bitten by dogs and all died. From 1998 to 2007, IPC tested 1,255 animal brain samples, 1,214 (97%) were from dogs and 610 (49%) were positive. Of 596 positive dogs, 88% lived in PP or neighboring provinces. Assuming IPC reports reflected true incidence of dog bites in Phnom Penh, the predictive model estimated 117 human rabies deaths would occur in 2007 (95%CI = ) despite PET; an incidence of 0.8 (95%CI = ) per 100,000. Conclusions: Rabies is endemic in all parts of Cambodia including PP. Access to PET is only available for PP since IPC is the only PET center in Cambodia. In 2007, the estimated rabies related mortality is similar to that of dengue reported in A national rabies control program is needed to improve surveillance and access to PET, and initiate a vaccination campaign in dogs. Furthermore, because data are still lacking regarding the burden of rabies in endemic countries, this type of approach could be proposed with minimal adjustment to the Réseau International des Instituts Pasteur where PET centers and diagnostic capacity exist. 8

11 S1-2 THE ORIGIN AND PHYLOGEOGRAPHY OF DOG RABIES VIRUS. Hervé Bourhy 1, Jean-Marc Reynes 2*, Eleca J. Dunham 3, Laurent Dacheux 1, Florence Larrous 1, Vu Thi Que Huong 4, Gelin Xu 5, Jiaxin Yan 5, Mary Elizabeth G. Miranda 6, Edward C Holmes 3,7 1. Institut Pasteur, UPRE Lyssavirus Dynamics and Host Adaptation, France 2. Institut Pasteur du Cambodge, Phnom Penh, Cambodge 3. Center for Infectious Disease Dynamics, The Pennsylvania State University, Mueller Laboratory, University Park. USA 4. Institut Pasteur of Ho Chi Minh City, Ho Chi Minh City, Vietnam 5. Wuhan Institute of Biological Products, Wuhan, Hubei Province, China 6. Veterinary Research Department, Research Institute for Tropical Medicine, Metro Manila, Philippines 7. Fogarty International Center, National Institutes of Health, Bethesda, USA. Rabies is a progressively fatal and incurable viral encephalitis caused by a lyssavirus infection. Almost all of the 55,000 annual rabies deaths in humans result from infection with dog rabies viruses (RABV). Despite the importance of rabies for human health, little is known about the origin and spread of RABV in dog populations, and patterns of biodiversity have only been studied in limited geographical space. To address these questions on a global scale we sequenced 62 new isolates and performed the largest comparative analysis of RABV gene sequence data undertaken to date, representing 192 isolates sampled from 55 countries. From this, we identified six clades of RABV adapted to non-flying mammals, each of which has a distinct geographic distribution, most likely reflecting major physical barriers to gene flow. Indeed, a detailed analysis of phylogeographic structure revealed only limited viral movement among geographical localities. Using Bayesian coalescent methods we further reveal that the sampled lineages of canid RABV derive from a common ancestor that originated within the last 1500 years. Additionally, we found no evidence for either positive selection or widespread population bottlenecks during the expansion of canid RABV. In sum, our study reveals that the stochastic processes of genetic drift and population subdivision are the most important factors shaping the global phylogeography of canid RABV. * present address: Institut Pasteur de Madagascar, Tannanarive, Madagascar. 9

12 S1-3 RABIES VIRUS HAS MORE THAN ONE TRICK UP ITS SLEEVE TO MANIPULATE THE HOST DEFENCES. Monique Lafon 1, Heinz Wiendl 2 and Thiravat Hemachudha 3 1. Institut Pasteur, Paris 2. University of Würzburg, Germany 3. Chulalongkorn University hospital and University Hospital Bangkok,Thailand Rabies virus is a pathogen well-adapted to the mammalian nervous system where it infects the neurons. It causes rabies- an acute myelo-encephalitis- fatal in most mammalian species, and humans in particular. Rabies virus is transmitted by saliva of an infected animal through bites or scratches, by unfortunate transplantation of organs originated from unsuspected rabid donors and more rarely by aerosols. Rabies virus enters the nervous system via a motor neuron through the neuromuscular junction, or via a sensory nerve through nerve spindles. It then travels from one neuron to the next, along the spinal cord to the brain. Then, rabies virus infection reaches the salivary glands and virus particles are excreted in the saliva.. Intriguingly, once the rabies virus has entered the CNS, its progression is interrupted neither by destruction of the infected neuron nor by the immune response, two classical strategies developed by the host to usually battle viral infection. Successful invasion of the nervous system by rabies virus seems to be the result of rabies virus capacity to escape the host mechanisms of defence.we showed that rabies virus neuroinvasiveness results of the selection of multiple factors : not only neuronotropic rabies virus avoids to induce neuron cell death, but also protective T cells that migrate into the infected nervous system are exhausted or killed by apoptosis, as a result of the overexpression by the infected neurons of at least three immunosubversive molecules: Fas-L, HLA-G and B7- H1. We also observed that fast killing virus strains limit local inflammation of the infected nervous tissues. Preservation of the integrity of neurons and neuronal network can be understood as a perquisite for the long journey of the virus through the nervous system from the site of entry up to the salivary glands. One would expect that the host s natural capacity to fight such a well adapted virus is greatly limited, explaining why in the absence of post-exposure vaccination, rabies is one of the very few human infections with a near 100 % mortality rate. Implications of these findings for new rabies treatment will be discussed. 10

13 S1-4 DU DIAGNOSTIC INTRA-VITAM DE LA RAGE HUMAINE A L'ANALYSE DU TRANSCRIPTOME CEREBRAL CHEZ LES PATIENTS INFECTES. L. Dacheux 1, J.M. Reynes 2-3, Ph. Buchy 3, B. Regnault, 4 T. S. Leng 5, B. Diop 6, D. Rousset 2, C. Rathat 7, N. Jolly 8, J.B. Dufourcq 5, C. Nareth 5, R. Rajerisson 9, G. Soubigou 4, J.Y. Coppée 4, C. Sadorge 8, H. Bourhy 1 1. Institut Pasteur, UPRE Dynamique des Lyssavirus et Adaptation à l Hôte, France 2. Institut Pasteur de Madagascar, Madagascar 3. Institut Pasteur du Cambodge, Cambodge 4. Institut Pasteur, Plate-forme Puces à ADN-Pasteur Génopole Ile de France, France 5. Hopital Calmette, Phnom Penh, Cambodge, 6Hopital Fann, Dakar, Sénégal 7. Hopital de Soavinandriana (CENHOSOA), Antananarivo, Madagascar 8. Institut Pasteur, Centre de Recherche Vaccinale et Biomédicale, France 9. Centre Hospitalier Universitaire de Befelatanana, Antananarivo, Madagascar La rage est une infection virale toujours mortelle, dont l incidence mondiale est estimée à décès par an. Cependant, ce chiffre est largement sous-estimé. L un des facteurs responsables réside dans l'absence de diagnostic biologique facilement réalisable. Seul le diagnostic post-mortem sur prélèvement cérébral permet actuellement de porter un diagnostic de certitude, difficilement réalisable dans les zones d endémie. Un nouveau protocole standardisé de diagnostic biologique intra-vitam de la rage, basé sur la détection d'arn viraux par RT-PCR, a été évalué en collaboration avec les laboratoires référents pour la rage situés dans les Instituts Pasteur du Cambodge, de Madagascar et de Dakar au Sénégal. Au final 51 patients issus des hôpitaux des pays concernés ont été inclus dans cette étude et plus de 400 prélèvements biologiques ont été collectés et analysés. Cette étude a permis de définir un protocole simple basé sur des prélèvements peu ou pas invasifs et présentant une sensibilité de plus de 98%. Pour 34 de ces patients, une biopsie cérébrale terminale a pu être obtenue. Afin d'analyser les dysfonctionnements du système nerveux central lors de l infection rabique, une étude a été initiée en utilisant la technologie des puces à ADN (Affymetrix). Après analyse des résultats obtenus, l activation ou la répression de différentes voies métaboliques a pu être mise en évidence, notamment celles impliquées dans la transmission synaptique et l homéostasie neuronale. De plus, les gènes impliqués dans les principales voies de réponses immunitaires antivirales (de type innée ou adaptative) ont été retrouvés très faiblement exprimés, voire réprimés. Ce résultat reste surprenant comparé à différents modèles animaux, comme la souris chez laquelle une très forte réponse antivirale de type interféron a été observée. 11

14 S1-5 FREQUENCY OF POULTRY TO HUMAN H5N1 TRANSMISSION AND EVIDENCE OF TRANSMISSION VIA CONTAMINATED ENVIRONMENT IN CAMBODIA. Vong S 1, Cavailler P 1, Garcia JM 2, Chu S 1, Buchy P 1 1. Institut Pasteur in Cambodia 2. HKU-Pasteur Research Centre Background: In Cambodia, H5N1 poultry outbreaks have been widespread and seven fatal human H5N1 cases were detected since January Direct contact with infected poultry is thought to be the main source of transmission. However, despite intense exposure, H5N1 viruses may not be easily transmitted from birds to humans. As H5N1 viruses continue to circulate and evolve among poultry, poultry to human transmission of H5N1 viruses could increase. We report the results of 4 studies conducted in four villages with human H5N1 cases to monitor the extent of illnesses from poultry to human transmission and to explore potential risk factors for H5N1 virus infection. Methods: In response to notification of 4 confirmed human H5N1 cases that occurred in 4 villages during May 2005 June 2007, we launched a series of investigations in the 4 cases villages including (i) retrospective poultry mortality surveys to determine the magnitude of exposure to infected birds; (ii) environmental sampling collection to identify the extent of environmental contamination; (iii) a sero-prevalence survey and a case control study few weeks following the outbreaks to determine the frequency of asymptomatic cases and potential risk factors. We interviewed villagers living near the households of H5N1 patients about potential exposures and bled them for H5N1 serological testing by microneutralization assay. Of note, we introduced a new serology testing which used H5 pseudotype-based assay for detection of neutralizing antibodies developed by Pasteur Research Centre in Hong Kong and Institut Pasteur in Cambodia. Findings: High intensity contact with poultry likely infected by H5N1 virus within 3 months prior to notification of H5N1-infected patients. The 2005 study found no seropositive cases in 351 villagers potentially exposed to infected poultry. In the studies, of 1,382 participants who were interviewed and bled, 12 (1%) tested positive for H5N1 antibodies. Of these, 7 were males and the median age was 12 years (range 4-20 years), which was significantly younger than that of the seronegative participants. The first 7 seropositive cases were more likely than the 24 controls to report bathing or swimming in household ponds (71.4% vs. 20.8%, matched odds ratio 11.3, 95 % CI , Wald test p=0.03); the analysis of the remaining 5 cases is pending. There were no differences between cases and controls regarding basic hygiene, poultry preparation practices or contact with an H5N1 patient. Viral RNA was detected in 27 of 77 specimens in mud, pond water, water plants and soil swabs. Discussion: Our research protocol following an H5N1 outbreak detected 12 sub-clinical H5N1 virus infections, but avian-to-human transmission of H5N1 virus appeared low despite extensive poultry contact. Our results also suggest the potential risks for bird-tohuman transmission via contaminated environment and underscore the needs for regular disinfection of poultry areas. 12

15 S1-6 DETERMINATION OF THE HAEMAGGLUTININ SIALIC ACID RECEPTOR INTERACTION OF H5N1 AND OTHER INFLUENZA VIRUSES AT THE ATOMIC LEVEL. Jean-Michel Garcia ±1, John M Nicholls ±1, Mark von Itzstein 2, Thomas Hazelhorst 2, Jimmy Lai 1 and JS Malik Peiris 1 1. Department of Pathology, The University of Hong Kong, Hong Kong University-Pasteur Research Centre 2. Institute for Glycomics, Griffith University, QLD, Australia Research into the pathogenesis of emerging viral infections has been hampered by the hazardous nature of many of these viruses, necessitating their study in BSL3 or 4 laboratories. This is especially the case with highly pathogenic H5N1 influenza virus. We have developed virus like pseudoparticles (VLP) using a retroviral core and surface haemagglutinin (HA) expression. Electron microscopy studies have demonstrated particle formation and NMR studies have shown that these particles bind to similar glycans than native virions in particular we have demonstrated there is a specific affinity for the SAα2-3Galß1-4GlcNAc moiety. Recent data indicates that the virus receptor recognition involves more than these terminal sugars and so we are continuing the studies using tetra and pentasaccharides and comparing these with virus affinity on glycan arrays. Our approach has some obvious advantages compare to previously reported methods. First, the NMR studies are invaluable for determining the virus host binding at the atomic level and the strengths of these interactions, as this information is not available from glycan array or lectin affinity. Second, the pseudoparticle system allows us to study the HA alone (in the absence of NA) with a infectious-potent virus-like topology. We have used this methodology to investigate the effect of naturally occurring point mutations in the haemagglutinin resulting in a switch of binding from an α2-3 to α2-6 binding for H3N2 virus and from α2-6 to α2-3 in H5N1 viruses. Using these mutated HA together with VLPs with different neuraminidases we will also analyze for the first time the synergy between the viral HA and NA. ± JMN and JMG contributed equally to the project. 13

16 S1-7 INNATE IMMUNE RESPONSE TRIGGERED BY INFLUENZA A VIRUS IS NEGATIVELY REGULATED BY SUPPRESSOR OF CYTOKINE SIGNALING (SOCS)1 AND SOCS3 THROUGH A RIG-I/IFNAR1- DEPENDENT PATHWAY. Julien Pothlichet, Michel Chignard, and Mustapha Si-Tahar Unité Défense Innée et Inflammation, Inserm U874, Institut Pasteur Influenza A virus (IAV) triggers a contagious respiratory disease that produces considerable lethality. Although this lethality is likely due to an excessive host inflammatory response, the negative feedback mechanisms aimed to regulate such response are unknown. Here, we investigated the role of the eight suppressor of cytokine signaling (SOCS) regulatory proteins in IAV-triggered cytokine expression in human respiratory epithelial cells. SOCS1 to SOCS7, but not cytokine inducible src homology 2-containing protein (CIS), are constitutively expressed in these cells and only SOCS1 and SOCS3 expression is upregulated upon IAV challenge. Using distinct approaches affecting the expression and/or the function of the IFNα/ß receptor (IFNAR)1, the viral sensors TLR3 and RIG-I as well as MAVS (a RIG-I signaling intermediate), we demonstrated that SOCS1 and SOCS3 upregulation requires a TLR3-independent, RIG-I/MAVS/IFNAR1-dependent pathway. Importantly, using vectors overexpressing SOCS1 and SOCS3, we revealed that while both molecules inhibit antiviral responses, they differentially modulate inflammatory signaling pathways. 14

17 S1-8 INNATE AND ADAPTIVE IMMUNE RESPONSES TO INFLUENZA IN FERRETS. S. Pillet 1, I. Meunier 1, É. Somo-Youmbi 1, K. Obojes 1, G. Kobinger 2, M. Gray 2, V. von Messling 1 1. INRS-Institut Armand-Frappier, University of Quebec, Laval, Canada 2. Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada Ferrets are considered one of the best influenza animal models because they are naturally susceptible to human strains, and the course and signs of disease caused reproduce those in humans. However, the lack of suitable immunological reagents has limited their use for the assessment of the anti-viral immune response. To characterize the host aspect of influenza pathogenesis in this model in more detail, we adapted our real-time PCR assays for the cytokine mrna quantification in nasal wash cells and developed an ELISpot assay for interferon γ to assess cytotoxic T cell responses. We observed that strains causing a mild disease were associated with a strong and rapid innate response and upregulation of IL-8, while severe infections were characterized by a lesser induction of type I and II interferons and strong IL-6 upregulation. Regardless of the strains virulence, virus-specific serum IgM and nasal IgA were detected as early as three days after infection, followed by a strong sustained IgG response beginning at day seven. Interferon γ production in peripheral blood mononuclear cells upon exposure to peptides covering parts of the HA, NA, or NP proteins was first observed after ten days and was strongest against the HA- and NP-derived peptides. Both, humoral and cellular responses started to decline four weeks after infection. However, the antibody titer stabilized at protective levels, while the cellular response in most cases declined below background after two months. Taken together our study suggests that virulent strains interfere more efficiently with the innate response, but do not prevent a strong adaptive response. This study provides new insights in the host response to influenza and will contribute to the development of novel vaccine and treatment approaches exist. 15

18 S1-9 TOWARDS A BETTER UNDERSTANDING OF THE CIRCULATION AND THE EVOLUTION OF H5N1 VIRUSES IN CAMBODIA AND WITHIN THE REGION USING BIOINFORMATICS TOOLS. Philippe Buchy 1, Mardy Sek 1, Mathieu Fourment 1, Sorn San 3, Holl Davun 3, Ly Sovann 4, Sok Touch 4, Malik Peiris 5, Sylvie van der Werf 6, Sirenda Vong 2 1. Virology unit 2. Epidemiology unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodge. 3. Ministry of Agriculture, Cambodia 4. Ministry of Health, Cambodia 5. Department of microbiology, The University of Hong Kong and HKU-Pasteur Research Center, Hong Kong SAR 6. Unité de génétique moléculaire des virus respiratoires, Institut Pasteur, Paris, France. From 2004 to 2007, Cambodia experienced 24 outbreaks in poultry and 7 human cases of H5N1 infection. Phylogenetic and Bayesian analyses were performed on sequences from clade 1, 1, 2.1, 2.2, 2.3 viruses including 225 complete segment sequences from 33 representative Cambodian isolates belonging to clade 1. Our data suggest that H5N1 was introduced in Cambodia several times, by successive waves, until Cambodian viruses seemed to evolve independently. This hypothesis is supported by phylogenic analysis, differences in substitution rates and epidemiological data (9 months of lack or low transmission in Vietnam in 2006). For the clade 1 HA sequences, the mean substitution rate (number of substitutions/site/year) is 5.1 and with strict and relaxed clock, respectively. It increases to 7.5 and 7.9 when only Cambodian sequences are considered. For the latter, a positively selected site at position 138, corresponding to an antigenic site of the HA was identified. Poultry movements probably play a crucial role in the introduction of new viruses from, and eventually also to, neighbouring countries and in a sometimes complex evolution of viruses within Cambodia. The lack of introduction of clade 2.2 or clade viruses into Cambodia is compatible with the role of poultry trade (rather than wild birds) being responsible for the continued endemicity of the virus in Cambodia. 16

19 S1-10 DEVELOPMENT OF MOLECULAR TOOLS FOR SARS-COV SINGLE- VIRUS TRACKING. Kien Francois, Siu Lewis, Teoh KimTat, Millet Jean, Tse Jane, Altmeyer Ralf, Peiris Malik, Bruzzone Roberto and Nal Beatrice HKU-Pasteur Research Centre, Dexter HC Man Building, 8 Sassoon Road, Pokfulam, Hong Kong SAR, China Single-virus tracking using fluorescently labeled virus derivatives is a powerful approach which allows direct visualization of viral entry, trafficking and egress in live cells and analysis of dynamic interactions between viruses and host-cell factors. Severe acute respiratory syndrome coronavirus (SARS-CoV) is the aetiological agent of SARS which infected 8100 people worldwide, among which 774 people died in Imaging of life infected cells with a biosafety level 3 (BSL3) pathogen like SARS-CoV is undesirable due to safety concerns. The same safety concerns prompted us to generate viral genome-free SARS-CoV virus like particle (VLP) and replication-defective, SARS-CoV Spike pseudotyped lentiviral particle (SARS-CoVpp) as safe molecular tools to study SARS-CoV egress and/or entry. By co-expressing different combinations of the four SARS-CoV structural proteins in VeroE6 permissive cell line, we have developed an expression system that allows efficient secretion of SARS-CoV VLPs. Addition of fluorescent tags to viral proteins allowed us to visualize VLPs in producing cells. Pseudotyping replication defective lentiviral vectors with heterologous envelope or spike protein from enveloped viruses has been used to study viral entry and cell tropism. We and others have shown that SARS-CoV Spike can efficiently pseudotype lentiviral vector to generate SARS-CoVpp which faithfully mimic the SARS-CoV entry process. Incorporation of HIV Vpr accessory protein fused to GFP allowed us to track individual GFP-labeled SARS-CoVpp. We present here a detailed characterization of these molecular tools together with imaging studies, demonstrating their suitability for SARS-CoV single-virus tracking. Fluorescently labeled SARS-CoV VLPs and SARS-CoVpp are valuable tools used to probe dynamic interactions between viral structural proteins and cellular partners that we have previously identified by Yeast-Two-Hybrid and RNAi screens. Besides cultured cells, which are simplified model systems, virus-cell interactions will be also investigated using in vitro well-differentiated human airway epithelial cells and ex vivo cultures of human airway tissues. 17

20 S1-11 HUMAN CORONAVIRUS OC43 HEPTAD REPEAT 2 (HR2) DOMAIN INDUCES NEUTRALIZING ANTIBODIES AGAINST SARS CORONAVIRUS AND OTHER HUMAN CORONAVIRUSES. Yongjin Wang, Ying Gao and Vincent Deubel Institut Pasteur of Shanghai, Chinese Academy of Sciences, PR China Coronavirus (CoV) spike heptad repeat 2 (HR2) region is highly immunogenic and induces neutralizing antibodies. Monoclonal antibody against SARS CoV HR2 region inhibits HR1-HR2 postfusion core formation and entry of the virus into its susceptible cells. The HR2 region is relatively conserved for all CoV from the same or different serogroups. Our objective is to extend the understanding of the specificity of fusion process for all known human CoV (HcoV) by blocking their cell entry with antibodies directed against the HR2 region. In this study, we purified the HCoV OC43 HR2 protein fragment and produced the corresponding antibody in mice. Western-blot showed that this polyclonal antibody detected sspike protein of all known HCoV including HCoV OC43, 229E, NL63, HKU1 and SARS coronavirus. This antibody also recognized the native form of all five HCoV spike proteins by confocal experiment. To investigate the neutralization and cross-neutralization activities of this antibody, we performed neutralization assay against HCoV OC43, 229E and NL63 in cell culture by either plaque or TCID50 reduction test. Because the cellular receptor of HCoV HKU1 has not been identified, we constructed plasmids expressing chimeric gene of OC43 S1 and HKU S2 ligated by CoV cleavage residues and pseudotyped in lentiviral vector. SARS CoV spike protein was also pseudotyped with the same vector. Neutralization assay against SARS CoV and HCoV HKU1 HR1-HR2 postfusion core formation was performed on the pseudoviruses. The results showed that antibodies developed from HCoV OC43 HR2 region cross neutralized all known HCoV. This study brings new insights on the mechanisms and specificity of CoV neutralization and offers new tools for diagnosis of the virus infection and for immunotherapy strategies. 18

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