Le nouveau virus grippal A(H1N1)v Vincent ENOUF - NIC Paris

Chronologie des évènements OMS Passage niveau 6 2 cas A(H1N1)v chez deux enfants en Californie Rétrospectif : premier cas mexicain le 17 mars Kit de détection Pasteur - envoi au labo de première ligne 2 premiers cas en France Premier cas en dehors du continent américain (Espagne) 21 27 30 1er 15 11 Avril Mai Juin 24 28 30 8 30 Alerte internationale OMS - cas humains de grippe d’origine porcine avec transmission inter-humaine au Mexique et aux Etats-Unis passage au stade 4A du plan pandémique français passage au stade 5A du plan pandémique français arrêt surveillance cas par cas aux Etats-Unis et Mexique - surveillance de type grippe saisonnière 299 cas confirmés H1N1v en France Vincent ENOUF - NIC of PARIS

Cas confirmés - Monde 6094 décès, hors Europe Vincent ENOUF - NIC of PARIS

Cas confirmés - Europe 414 décès en Europe Vincent ENOUF - NIC of PARIS 4

Diffusion du virus en Europe 414 décès en Europe dont : - 154 Angleterre - 73 Espagne - 25 Italie - 30 France Vincent ENOUF - NIC of PARIS

Activité grippale - hors France (S43) Toujours en augmentation dans sept pays, Tx positivité dépassant les 50 % Angleterre, Belgique, Irlande du Nord, Norvège, Pays-Bas, République d’Irlande et Suède. Intensité moyenne Espagne et Italie En europe, 82 % des prélèvements positifs pour les virus Influenzae sont dus au virus A(H1N1)v. We studied the impact of changes at these positions using a system which allow the characterization of enzymatic properties of transiently expressed recombinant NA. Briefly NA coding sequences are cloned in mammalian expression vector. 293T cells are transiently transfected. NA expression is monitored by FACS or western blotting and enzymatic assay is performed on the NA expressed at the cell surface using the fluorigenic substrate MUNANA. Vincent ENOUF - NIC of PARIS 6

Activité grippale - Ukraine (S43) Premier décès 30/10/09 (grippe A(H1N1)v) 154 décès suspects supplémentaires, 20-50 ans Saturation des services de santé Problèmes d’accès aux antiviraux Difficultés de prise en charge des formes graves 700 femmes enceintes infectées : 7 sous ventilations, 9 décès au 6 novembre 2009 We studied the impact of changes at these positions using a system which allow the characterization of enzymatic properties of transiently expressed recombinant NA. Briefly NA coding sequences are cloned in mammalian expression vector. 293T cells are transiently transfected. NA expression is monitored by FACS or western blotting and enzymatic assay is performed on the NA expressed at the cell surface using the fluorigenic substrate MUNANA. Vincent ENOUF - NIC of PARIS 7

Aspects épidémiologiques - France Métropole InVS - bulletin épidémiologique 3 novembre 2009 Vincent ENOUF - NIC of PARIS

Aspects épidémiologiques - France Augmentation de la circulation virale We studied the impact of changes at these positions using a system which allow the characterization of enzymatic properties of transiently expressed recombinant NA. Briefly NA coding sequences are cloned in mammalian expression vector. 293T cells are transiently transfected. NA expression is monitored by FACS or western blotting and enzymatic assay is performed on the NA expressed at the cell surface using the fluorigenic substrate MUNANA. Vincent ENOUF - NIC of PARIS

Aspects cliniques Tableaux cliniques atteinte bénigne et non fébrile des voies respiratoires supérieures aux pneumonies sévères voire mortelles symptômes digestifs (nausées, vomissements et/ou diarrhés) We studied the impact of changes at these positions using a system which allow the characterization of enzymatic properties of transiently expressed recombinant NA. Briefly NA coding sequences are cloned in mammalian expression vector. 293T cells are transiently transfected. NA expression is monitored by FACS or western blotting and enzymatic assay is performed on the NA expressed at the cell surface using the fluorigenic substrate MUNANA. Vincent ENOUF - NIC of PARIS

Aspects épidémiologiques Profil d’attaque différent de celui de la grippe saisonnière Tranches d’âges les plus touchées : comprises entre 5 et 50 ans grande majorité des cas graves et des décès, < à 59 ans (75 %) Groupes à risques (x-- soins intensifs) femmes enceintes (2ème et 3ème trimestres de grossesse) (x10) asthmatiques (x3) obèses morbides (x6) immunodéprimés diabétiques pathologies lourdes chroniques (cardiaques, pulmonaires, neurologiques) We studied the impact of changes at these positions using a system which allow the characterization of enzymatic properties of transiently expressed recombinant NA. Briefly NA coding sequences are cloned in mammalian expression vector. 293T cells are transiently transfected. NA expression is monitored by FACS or western blotting and enzymatic assay is performed on the NA expressed at the cell surface using the fluorigenic substrate MUNANA. Vincent ENOUF - NIC of PARIS

Virus grippaux Types et sous-types viraux Type B Type C Type A Virus pandémique HxNy H1N1 H2N2 H3N2 H5N1 – H9N2 – H7N7 Vincent ENOUF - NIC of PARIS

Virus grippaux de type A From Hatta et al., 2002 NA HA M2 M1 ARNv NP PA PB1 PB2 PB1-F2 NS1 NEP Type A, sous-type sérologique : HA : H1-H16 NA : N1-N9 Vincent ENOUF - NIC of PARIS

Cycle viral Vincent ENOUF - NIC of PARIS

Origine des virus porcins Transmission inter-espèce Adaptation des virus grippaux H1N1 H3N2 H1N2 H1 à H16 N1 à N9 Réservoir de virus humains Vincent ENOUF - NIC of PARIS

Réassortiment de virus grippaux Avian virus Human virus SA-2,3-Gal SA-2,6-Gal SA-2,3-Gal + SA-2,6-Gal Avian/Human reassortant virus Vincent ENOUF - NIC of PARIS

Emergence des nouveaux virus grippaux H2N2 Asian Flu 1957 1968 H3N2 Hong Kong Flu 1968 H1N1 Spanish Flu 1918 1931 1957 1977 H1N1 Russian Flu 2009 Swine Flu - Fort Dix 230 cases - 1 death 1976 H3N2 H2N2 H3N2 Classical swine H1N1 There was a trend in increase of antiviral resistance over the surveillance period (from ≈20% before the beginning, to ≈ 45% at the peak and 58% at the end of the epidemic). This increase was observed in all regions, whereas the impact of influenza was equivalent. 97-98 2003-06 H1N1 H1N1 H3N2 H1N2 Vincent et al. Adv Vs Res 2008:721 Vincent ENOUF - NIC of PARIS

Mise en évidence de virus grippaux triple-réassortants Cas sporadiques humains d’infection par des virus grippaux porcins aux Etats-Unis (2005-2009) Mise en évidence de virus grippaux triple-réassortants Clinical information collected at the onset of the disease from patients presenting with influenza infection was analyzed for 314 cases have including 130 patients with a resistant virus. The sex ratio of both groups of patients (sensitive vs resistant) were not statistically different (1.3 vs 1.2), as well as the age-range (0-81 vs 0-70) or the mean and median age (19 vs 22 and 12 vs 21,respectively). Clinical signs and symptoms of the two groups of patients were indistinguishable; there was no difference in the clinical presentation, nor in the severity. There was no difference in the rate of detection of sensitive vs resistant viruses in the hospitalized patients as compared to the community setting. Shinde et al. NEJM 2009:361 Vincent ENOUF - NIC of PARIS

Composition génomique du nouveau variant A(H1N1)v It was previously established that H274Y mutation altered the enzymatic properties of the N1 leading to decreased affinity for the substrate and decreased activity. In ferrets and mouse models it was shown that seasonnal H1N1 viruses with H274 Y mutation exhibits decreased infectivity and transmissibility. Thus Oseltamivir resistant H1N1 were supposed to be too attenuated to circulate in the human population in the absence of selective drug pressure. Since these viruses were able to circulate the question is : What are the 2007-08 H1N1 viruses particular features that lead to the circulation of naturally resistant viruses ? Garten et al. Science 2009 Vincent ENOUF - NIC of PARIS

Phylogénie HA Virus porcin classique, lignage Nord Américain But then we compared the enzymatic properties of the N1 of the recent H1N1 viruses with those of the N1 of previously circulating viruses. We found that the N1 recent viruses (jaune-orange) exhibited a significantly reduced Km and a significantly increased Vmax as compared with that N1 of the previously circulating viruses (bleu). The N1 of the recent viruses exhibit higher catalytic efficiency as compared with those of the previous ones. (In this conditions the catalytic efficiency of the H274Y mutant N1 (rose) remains higher as comapred to this of the previous N1. ) Virus porcin, lignage Eurasien Virus aviaire, lignage Nord Américain Virus humain saisonnier Vincent ENOUF - NIC of PARIS

Charactéristiques antigéniques Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Vincent ENOUF - NIC of PARIS

Technique de détection - RT-PCR RT-PCR en temps réel : M : virus grippal de type A H1v et N1v : virus grippal de type A(H1N1)v GAPDH : gène de ménage afin de vérifier la qualité du prélèvement Exclusion des cas saisonniers : H1N1 et H3N2 : virus grippaux saisonniers Seuil de sensibilité similaire entre PCR M et H1N1v : Cp H1v : 18,32 - 35,88 CP M : 18,7 - 37,74 Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Vincent ENOUF - NIC of PARIS

Technique de détection - Tests rapides Faible sensibilité : 40 % Bonne spécificité : 100 % Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Vincent ENOUF - NIC of PARIS

Variations dans la poche catalytique N1/N4/N8 B Glu 119 Group 1 N1 type Glu 276 N2 Val 149 Asp 151 N3 N8 N1/N9 N5 N9 Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Glu 119 Group 2 N2 type Glu 276 N6 Ile 149 Asp 151 N1 N4 N7 Val 149 Russell et al. Nature 2006;443:45–9 Vincent ENOUF - NIC of PARIS

Résistance chez Influenza A R152K – Catalytic mutation E119V – Framework mutation D198N – Framework mutation H274Y – Framework mutation Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Asn294Ser – Framework mutation? R292K – Catalytic mutation Vincent ENOUF - NIC of PARIS

Analyse structurale : mutation H274Y / N1 A(H1N1) Ex : Mutation H275Y sur N1 de A(H5N1) Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Oseltamivir Zanamivir (Collins et al., Nature 2008) Vincent ENOUF - NIC of PARIS

Premier cas de résistance au Tamiflu Contexte médical homme sous traitement prophylactique (1/2 dose) 3 jours après le début du traitement, syndrôme grippal traitement curatif mis en place (dose curative) le patient est soigné Le virus grippal virus résistant au Tamiflu, mutation H274Y virus sensible au Relenza vitalité du virus réduite, incapable de diffuser actuellement dans la population Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Vincent ENOUF - NIC of PARIS

Acquisition de la résistance au Tamiflu Traitement par le Tamiflu 0,4 % adultes résistants 4 % des enfants resistants au Tamiflu Résistance Spontanée : exemple des virus saisonniers H1N1 résistants au Tamiflu Réassortiment avec un virus saisonnier cet hiver Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Vincent ENOUF - NIC of PARIS

Sérologie : cross-réactivité des vaccinés saisonniers contre le A(H1N1)v Etude sur les vaccinés des saisons suivantes : 2005-06,2006-07,2007-08, 2008-09 Test IHA ( globules de dinde) Sérum pré-vaccinal : enfants 0% 18-64 ans : 6-9 % > 60 ans : 33 % Sérum post-vaccinal enfants : RAS 18-64 ans : augmentation x2 > 60ans : RAS Microneutralisation Sérum pré-vaccinal : enfants 0% 18-64 ans : 9 % (≥160) > 60 ans : 33 % (≥160) Sérum post-vaccinal enfants : RAS 18-64 ans : 25 % (≥160) > 60 ans : 43 % (≥160)s Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Aucune cross-protection avec le vaccin saisonnier contre le A(H1N1)v Identité de H1 entre H1N1 saisonnier et A(H1N1)v = 72-73 % (97-98% entre les virus saisonniers de cette période) Vincent ENOUF - NIC of PARIS

Vaccin pandémique Souche vaccinale : virus réassortant (CBER-RG2) construit par génétique inverse à partir de la l’isolat A/California/04/2009. Production sur œufs embryonnés Combien de doses ? (Une ou deux) Vaccin pandémique adjuvanté et non adjuvanté Délai de production : délivrance avant la première vague !!! essais cliniques AMM Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Vincent ENOUF - NIC of PARIS

Vaccin pandémique - distribution Priorité Population Stratégie 1 Professionnels de santé, pompiers, gendarmes, police nationale Femmes enceintes à partir du début du T2 Vaccin sans adjuvant Entourage des nourrissons de <6mois Nourrissons de 6-24 mois avec facteurs de risques de complications graves 2 2-65 ans à risques de complications graves Nourrissons de 6-24 mois sans facteurs de risques 3 >65 ans avec facteurs de risques de complications graves 4 2-18 ans sans facteur de risque 5 19-64 ans sans facteur de risque 6 >65 ans sans facteur de risque Among these residues, 3 are of particular interest since they are located close to the NA catalytic site. Vincent ENOUF - NIC of PARIS

Merci de votre attention !! Phylogénie HA Merci de votre attention !! But then we compared the enzymatic properties of the N1 of the recent H1N1 viruses with those of the N1 of previously circulating viruses. We found that the N1 recent viruses (jaune-orange) exhibited a significantly reduced Km and a significantly increased Vmax as compared with that N1 of the previously circulating viruses (bleu). The N1 of the recent viruses exhibit higher catalytic efficiency as compared with those of the previous ones. (In this conditions the catalytic efficiency of the H274Y mutant N1 (rose) remains higher as comapred to this of the previous N1. ) Vincent ENOUF - NIC of PARIS