Dissertations / Theses on the topic 'H3N2 virus'

L'objectif de cette étude était double. Il s'agissait dans un premier temps de caractériser l'immunosuppression produite chez des porcs infectés par une souche de virus influenza H3N2. La deuxième partie de cette étude consistait à tester le potentiel adjuvant d'un dérivé de mannose (l'Acemannan) aux propriétés immunostimulantes. Lors de la première partie de cette étude, les effets d'une infection par le virus influenza furent évalués chez le porc. Il fut démontré par différents essais que la prolifération mitogène-dépendante ainsi que la sous-population de lymphocytes T auxilliaires sont affectées de façon négative lors de ce type d'infection. L'activité des cellules N. K est grandement influencée par le stress des animaux. Dans la deuxième partie de cette étude, les effets de l'Acemannan furent déterminés sur la réponse immunitaire du porc en réponse à un vaccin anti-virus influenza. Ceci a été effectué dans le but de vérifier le potentiel de l'Acemannan comme adjuvant couplé à des vaccins. Ces expériences ont permis de démontrer que l'Acemannan possède des effets activateurs ou inhibiteurs selon le type cellulaire en cause. Des effets activateurs ont été mis en évidence au niveau de la production d'anticorps et de la réponse proliférative des lymphocytes mononucles du sang périphérique du porc (PBML) tandis qu'un effet inhibiteur a été rapporté au niveau de l'activité des cellules N. K. [Résumé abrégé par UMI].

L'empaquetage des huit segments du génome des virus influenza de type A est une des étapes clef du cycle viral. Il intervient également dans l'apparition de virus réassortants, les virus pandémiques par exemple, ce qui en fait un enjeu fondamental de la recherche actuelle.Nous avons étudié ce mécanisme au cours de deux études, la première portant sur les vaccins antigrippaux (réassortiment), la seconde visant à construire un virus vecteur (incorporation d'un segment hétérologue). Les semences vaccinales sont obtenues par co-infection d'oeufs de poule embryonnés avec deux souches virales une donneuse (souche circulante de référence) et une accepteuse (A/Puerto Rico/8/34 (H1N1) (PR8)). L'analyse de la composition génétique de treize semences vaccinales H3N2 montre que le segment PB1 de la souche donneuse est présent dans plus de 50 % des semences analysées et qu'une grande variété de réassortants,allant de 6:2 à 2:6 (PR8:H3N2), peut résulter de ces coinfections. Des expériences de compétition d'encapsidation de segments à l'aide de la génétique inverse révèlent que l'encapsidation sélective du segment PB1 dépend de son environnement génétique notamment l'origine virale des segments HA et NA. La seconde partie de mon travail de thèse a été consacrée à la construction d'un vecteur réplicatif sur la base d'un virus influenza H3 naturel sans segment NA. Aucune des constructions contenant le transgène gfp n'a été incorporée dans les particules virales, contrairement à ce qui a été décrit dans la littérature. Bien que les mécanismes moléculaires régissant l'incorporation des segments des virus influenza A demeurent très complexes, le fond génétique semble être déterminant pour ce processus.

Les virus influenza sont des pathogènes respiratoires responsables d’épidémies saisonnières touchant 10 à 20% de la population mondiale chaque année, constituant donc un problème majeur de santé publique. La vaccination annuelle réduit l’impact des épidémies grippales; cependant, un mésappariement entre les souches vaccinales et circulantes peut parfois survenir et résulter en un échec de protection de la population. Dans ces cas, il est important d’avoir un traitement adéquat afin de traiter l’infection virale. Les inhibiteurs de la neuraminidase (INAs) constituent la principale classe d’antiviraux recommandée pour la prévention et le traitement des infections grippales. Les INAs lient de façon compétitive le site actif de la neuraminidase (NA), ce qui bloque la libération des virions des cellules hôtes inhibant de la sorte la dissémination du virus dans le tractus respiratoire. L’émergence sporadique de virus résistants à l’oseltamivir et/ou au zanamivir avec de faibles taux de transmission a été identifiée lors de traitements des souches saisonnières de l’influenza. Le développement de nouveaux antiviraux devient donc un sujet important d’investigation. Le peramivir, un nouvel INA disponible depuis peu en Amérique du Nord, exerce une activité sur des virus influenza A et B et son efficacité contre des mutants résistants à l’oseltamivir ou au zanamivir n’a pas encore été complètement caractérisée. À cause des différences dans la liaison des INAs avec l’enzyme cible, la nature des mutations de résistance peut varier d’un INA à l’autre bien que certaines mutations pourraient engendrer une résistance croisée à plusieurs INAs. Nous avons démontré que le peramivir s’avère très actif contre les différents sous-types de grippe saisonnière, quoique certains variants aient présentés des phénotypes de multi-résistance à l’oseltamivir, au zanamivir ainsi qu’au peramivir. À cet égard, un nouveau mécanisme de résistance d’un variant menant à la résistance croisée aux INAs a été décrit (I427T/Q313R) dans le cadre de ce mémoire et a permis de comprendre comment des substitutions retrouvées hors du site actif de la NA peuvent affecter la capacité de réplication du virus et sa résistance aux antiviraux.
Influenza viruses are respiratory pathogens responsible for seasonal epidemics affecting 10 to 20% of the world's population every year, thus constituting a major public health impact. Annual vaccination reduces the impact of influenza epidemics; however, a mismatch between the vaccine strain and the circulating strain can sometimes occur and result in an unsuccessful attempt in protecting the population. In such cases, it is important to have adequate treatment to treat influenza infections. Neuraminidase inhibitors (NAIs) are the primary class of antiviral agents recommended for the prevention and treatment of influenza infections. NAIs competitively bind the neuraminidase (NA) active site, blocking the release of virions from host cells and thereby inhibiting the spread of the virus into the respiratory tract. The sporadic emergence of oseltamivir- and/or zanamivir-resistant viruses with low transmission rates was identified in seasonal influenza strains. The development of new antivirals thus became an important subject of investigation. Peramivir, a new NAI recently available in North America, exerts its activity against influenza A and B viruses, but its effectiveness against mutations conferring resistance to oseltamivir or zanamivir has not yet been fully characterized. Due to differences in the binding of NAIs to the target enzyme, the nature of the resistance mutations may vary from one NAI to another, although some mutations could induce global NAI cross-resistance. We have demonstrated that peramivir is highly active against the different seasonal influenza subtypes, although some variants have shown multi-resistance phenotypes to oseltamivir, zanamivir as well as peramivir. In this regard, a new resistance mechanism by which a NA variant leads to NAI cross-resistance (I427T/Q313R) has been described in this thesis and has helped to understand how substitutions found outside the NA active site can affect the replication kinetics of the virus and its resistance to antivirals.

Durant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une des causes des surinfections bactériennes post-grippales étant l’altération et/ou la perte de l’intégrité de l’épithélium pulmonaire, nous nous sommes proposés d’étudier le rôle potentiel de cette cytokine dans un modèle expérimental de surinfection bactérienne à S. pneumoniae. Nous avons ainsi pu montrer que si cette cytokine ne joue pas un rôle majeur dans la réponse anti-virale de l’hôte, l’IL-22 participe au contrôle de l’inflammation au cours de l’infection grippale et joue un rôle protecteur dans la surinfection bactérienne.Par ailleurs, l’utilisation de souris dépourvues en cellules iNKT (Jα18-/-) a permis de montrer que les cellules iNKT limitent la susceptibilité aux surinfections et réduisent le synergisme létal de la coinfection virus/bactérie. Au moment de l’infection bactérienne, les cellules iNKT des souris grippées sont incapables de produire de l’IFN-γ, cytokine dont nous avons montré le rôle essentiel dans les mécanismes de défense antibactérienne. Le défaut d’activation des cellules iNKT chez les souris surinfectées est lié à l’interleukine-10 (IL-10), cytokine immunosuppressive induite par l’infection virale, plutôt qu’à un défaut intrinsèque des cellules iNKT. L’IL-10 inhibe l’activation des cellules iNKT en réponse au pneumocoque en inhibant la production d’IL-12 par les cellules dendritiques dérivées de monocytes (MoDCs). La neutralisation de l’IL-10 restaure l’activation des cellules iNKT et augmente la résistance à la surinfection. Ainsi, les cellules iNKT ont un rôle bénéfique (en amont de la colonisation bactérienne) dans le contrôle de la surinfection bactérienne de la grippe et représentent une cible de l’immunosuppression.Nous avons par la suite étudié la possibilité que le superagoniste des cellules iNKT, l’ α-galactosylceramide (α-GalCer) puisse limiter la surinfection bactérienne. Pour cela, les souris ont été traitées par voie intranasale avec de l’α-GalCer à différents temps post-influenza, juste avant l’infection par le pneumocoque. Le traitement à jour 3, au pic de la réplication virale, limite fortement la surinfection. Cependant, l’inoculation d’α-GalCer pendant la phase aiguë du virus (jour 7) ne permet pas d’activer les cellules iNKT pulmonaires et n’a pas d’effet sur la surinfection. L’absence d’activation des cellules iNKT n’est pas intrinsèque et est associée à une disparition complète des cellules dendritiques CD103+ respiratoires (cDCs), lesquelles sont cruciales dans l’activation des cellules iNKTs. À des temps plus tardifs (jour 14), les cDCs repeuplent le poumon et l’α-GalCer promeut l’activité antibactérienne des cellules iNKT.Pris dans son ensemble, cette étude souligne le rôle des cellules iNKT dans la surinfection bactérienne de la grippe et ouvre de nouvelles voies thérapeutiques afin de limiter les surinfections bactériennes post-influenza
XDurant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une desDuring the infection by the virus Influenza A ( IAV), the physical and immunological changes of the lung predispose the host to the bacterial secondary infections. The invariant cells(units) T Natural Killer iNKT ) are lymphocytes T innate being able to have beneficial or noxious roles during the infection. Our objectives aimed at i) to study the natural role of cells(units) iNKT and ii) to look for the effect of an exogenous activation of cells(units) iNKT in the bacterial secondary infection post-influenza. During my arrival, the laboratory had just described, for the first time in infectious context, that cells(units) iNKT were capable of producing of IL-22 during the flu-like infection. This cytokine plays a major role in the processes of preservation and repair of epitheliums [...]

Les probiotiques font partie du microbiote commensal. Ils ont le potentiel de stimuler la réponse immunitaire intestinale et systémique. Le virus de la grippe est à l’origine de morbidité importante. De plus, il favorise les infections bactériennes secondaires telles que Neisseria meningitidis. Nous nous sommes intéressés à l’étude des mécanismes de protections conférées par la souche Lactobacillus paracasei CNCM I-1518 sur une infection par le virus de la grippe dans un modèle de souris. Nos résultats ont montré que le gavage des souris avec la souche L. paracasei pendant 7 jours avant l’infection grippale a permis une activation des cytokines pro-inflammatoires et un recrutement massif des cellules immunitaires dans les poumons. Cette activation du système immunitaire est responsable après infection grippale d’une meilleure clairance du virus de la grippe et d’une réponse inflammatoire moins importante comparée aux souris témoins. L’administration orale à des souris du peptidoglycane de la souche L. paracasei a permis de retrouver partiellement le phénotype protecteur observé avec la bactérie entière. Les effets protecteurs induits par L. paracasei CNCM I-1518 lui sont spécifiques car l’utilisation de 2 souches L. rhamnosus CNCM I-3690 et L. paracasei CNCM I-3689 n’ont pas conférées aux souris une protection contre la grippe. L’étude de l’impact de la souche L. paracasei CNCM I-1518 sur une infection secondaire par N. meningitidis après infection grippale a montré que le gavage des souris par la souche L. paracasei permet un meilleur état clinique associé à une modulation de la réponse immunitaire et une clairance de l’infection bactérienne secondaire plus efficace
Probiotics are part of the commensal microbiota. They play a potential role in stimulating the intestinal and systemic immune response. Several clinical studies addressed beneficial effect of probiotics against respiratory infections in particular on influenza infections. These infections are responsible for significant morbidity. The burden of flu is also worsened by secondary bacterial infections such as Neisseria meningitidis. In this work, we investigated the mechanisms of protection against influenza infection conferred by Lactobacillus paracasei CNCM I-1518 strain in mice. Our results showed that, L. paracasei consumption allow an early activation of pro-inflammatory cytokines and a massive recruitment of immune cells in the lungs prior to influenza infection. This activation of immune system was associated with a faster clearance of influenza virus after infection. We able to show that feeding mice with purified peptidoglycan from L. paracasei reproduced partially the above mentioned effects observed with L. paracasei bacteria feeding.The protective effects induced by L. paracasei CNCM I-1518 against the flu infection are strain specific as L. rhamnosus CNCM I-3690 and L. paracasei CNCM I-3689, tested under the same conditions did not confer to mice protection against influenza infection. Subsequently, we investigated the impact of L. paracasei CNCM I-1518 on secondary bacterial infection with N. meningitidis following influenza infection. Our results showed that consumption of L. paracasei CNCM I-1518 strain was associated with a better clinical status and a modulation of the immune response with a better clearance of secondary bacterial infection

Thesis (M.S.)--Florida State University, 2007.
Advisor: David Swofford, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Oct. 9, 2007). Document formatted into pages; contains viii, 48, [24] pages. Includes bibliographical references.

碩士
國立交通大學
分子醫學與生物工程研究所
99
Influenza A virus causes significant morbidity and mortality in humans. H1N1 is one of the current circulating influenza A subtypes in human. The H1N1 pandemic occurred in the early 20th century and resulted in approximately 20 million deaths in the world. Recently, the emerged swine-origin H1N1 virus has infected human population and cause the 2009 influenza pandemic. Hemagglutinin (HA), which is an antigenic glycoprotein on the surface of influenza virus, is neutralized by antibodies during infection or vaccination. Accumulation of mutations on HA can lead to antigenic drift. The emergence and spread of antigenic variants often requires a new vaccine strain to be selected before coming epidemic. Most of studies on HA focused on the H3N2 subtype. However, the genetic evolution and antigenic evolution of the HA is poor understood for subtype A (H1N1). To study the genetic and antigenic evolution of subtype A (H1N1) is an emergent issue for public health and vaccine development. In this thesis, we performed the genetic and antigenic analysis to the HA of A (H1N1) viruses. In the sequence level, we collected 1525 HA sequences and used Shannon entropy to quantify the genetic diversity of each amino acid. In the vaccine efficacy level, we collected 202 pairs of HI assays from weekly epidemiological record (WER) and publications in last 40 years. Based on the collected Hemagglutination Inhibition (HI) assays, we applied a statistical index to quantify the antigenic score of each amino acid on HA. Finally, a decision tree tool (C4.5) was used to build a model for predicting the antigenic variants of H1N1 virus. We select 30 critical positions of H1N1 hemagglutinin by the genetic and antigenic analysis. There are 26 positions on the surface of the HA and 9 positions on the H1N1 epitopes. Based on the genetic and antigenic analysis on HA, we found that there are two sites with both high genetic diversity and antigenic score in A (H1N1) virus. These two sites include one site around the receptor binding site and the other antigenic site about 45 Å distant from receptor binding site. In contrast, there is only one site, which is around the receptor binding site, have high genetic diversity and high antigenic in A (H3N2) virus. By comparing the HA of two subtypes of influenza A virus, we found that some amino acid positions locating on the antigenic sites of influenza A (H3N2) virus are potential epitope residues for influenza A (H1N1) virus. In addition, the accuracy of our model for predicting antigenic variants was 85% by using HA sequences as input. We believe that our methods are useful for the vaccine development and understanding the genetic and antigenic evolution of influenza A (H1N1) virus.

碩士
國立臺灣大學
醫事技術學研究所
93
Influenza virus is a member of Orthomyxoviridae, and infection of influenza virus can cause severe morbidity and mortality in the elderly and children. Influenza A viruses are enveloped and have two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Both of HA and NA undergo antigenic shift and antigenic drift. Antibody to HA is the most important determinant of immunity because it can neutralize the infectivity of influenza virus. Although anti-NA antibodies do not neutralize virus infectivity, they appear to modify the disease and reduce both pulmonary virus titer and the extent of lung lesions. Therefore, antigenic variability of the NA protein should also be considered when analyzing the epidemic impact of influenza virus and predicting newly emerging viruses. However, limited information is available concerning the molecular change of the influenza NA genes. Analysis of NA gene is particularly important since the use of influenza NA inhibitors that target the highly conserved catalytic site of the enzyme. In order to understand the variation of NA gene of influenza A (H3N2) virus in northern Taiwan, 43 strains of clinical isolates in Taipei during 2000-2004 were collected for this study. The result indicated that the amino acid variation rate of NA was about 0.5% per year. As compared with the A/Moscow/10/99 vaccine strain, amino acid changes within at least one of the seven NA antigenic determinants (I-VII) were found in approximate half of the isolates (20/43) and the most common changes were at position 332, 401, 431 and 432. Only one amino acid change (D151G) was observed in the catalytic site of NA. All isolates contained the seven conserved asparagine-linked glycosylation sites found in the NA of the progenitor A/Hong Kong/8/68 strain. In addition, most strains (38/43) had the new glycosylation sites at positions 93 and 329. To understand whether there is gene reassortment recently, we also analyzed the evolutionary relationship of these isolates. It appears that no HA/NA reassortment was found. Variation of plaque size was observed in the plaque assay. After purification of virus, a small-plaque virus strain (NA-) was obtained and a 586 nucleotide deletion (303-888nt) of NA gene was found. Although the deficiency of NA enzyme activity, it still can grow in MDCK cell. However, the virus yield was 45-fold less than wild-type when low MOI (10-5) was used. The receptor-binding ability of the defective virus was low but no compensatory substitutions in the HA gene were found. TamifluTM, a kind of anti-influenza drug, did not influence on NA- virus in vitro. Thus, our results suggest that NA activity may not be essential for influenza A virus growth.

碩士
國立成功大學
醫學檢驗生物技術學系碩博士班
100
Influenza A virus is an important pathogen with worldwide prevalence. The major circulating subtypes are H3N2 and H1N1. Influenza A virus contains 8 negative stranded RNA segments that encode 11 or 12 viral proteins. Among them, NS (non-structural) gene encodes NS1 protein which can increase viral pathogenicity and virulence by regulating viral translation and antagonizing host defense immune responses, especially type I interferon (IFN-α/β) production and antiviral activities of IFN-induced proteins. Our phylogenetic analysis of NS genes of H3N2 viruses from 1999 to 2011 showed that most NS genes fall into one group, H3 group, except that NS genes of 2002 isolates (H3 variants, H3v) were separated into another group, H3v group. Two amino acid changes, E71G in the RNA binding domain and V82A in the effector domain, were identified in NS1 proteins of these 2002 isolates. To test whether these amino acid mutations contribute to IFN response, we investigated IFN-β protein production, IFN-β promoter activity and IFN-α susceptibility of isolates in H3 group and H3v group. The result showed that the H3 variants induced higher IFN-β protein production and IFN-β promoter activity. In addition, the H3 variants showed comparable reduction to other isolates in virus productions when pretreated A549 cell with IFN-α. The result indicated that the two substitutions V82A and E71G in NS1 protein of H3 variants can affect the IFN-β protein level and promoter activation. We further constructed recombinant NS1 protein to elucidate the effect of E71G and V82A mutations on IFN-β and ISRE promoter activity. Recombinant NS1 proteins with E71G or double mutation of E71G/V82A substitution showed higher induction of IFN-β promoter than NS1 protein from H3 group. Interestingly, recombinant NS1 proteins with either substitution exhibited stronger inhibitory effects on ISRE promoter activity when compared with H3 NS1. We further investigated the effect of recombinant virus with difference in NS gene sequence on replication curve. It showed that the replication ability of recombinant virus with different NS gene were similar. Our results indicated that NS1 with E71G and V82A mutations may affect type I IFN response in the IFN-β protein secretion, promoter activation and the downstream pathway of IFN-β which may be the disadvantage to virus infection and lead to disappearance in the later season. The substitutions in NS1 can be applied to vaccine strain development. Our study will be helpful to understand the contribution of NS1 genetic variation to the pathogenesis of influenza viruses.

碩士
國立中興大學
微生物暨公共衛生學研究所
100
Introduction: Pigs play an important role in gene reassortment of the eight RNA segments of influenza viruses since they are susceptible to the viruses with both avian and human origin. Because of the great change of antigenicity, the reassorted viruses have the potential to cause global pandemic such as 2009 pandemic H1N1. As the reports of the increase of human infection by the newly-reassorted swine influenza viruses subtype H3N2 carrying genes from 2009 pandemic H1N1 were observed, it is crucial to understand the antibody cross-reactivity between swine and human-origin H3N2 as well as the protection provided by the seasonal influenza vaccination (TIV). Besides, this study would evaluate different vaccination strategies as well as the changes of vaccine component on the sustainability of antibody against. In this study, we used hemagglutination inhibition (HI) assay to test the antibody distribution. Results: (1) The swine-origin H3N2 viruses used in this study included A/Swine/Taiwan ex USA/28-9/2010, A/Swine/Taichung/50-1/2004, A/Swine/Yunlin/113-3/2010, and A/Swine/Obihiro/10/1985 from Japan. The HA lineage of A/Swine/Taiwan ex USA/28-9/2010 was from 2005-like human influenza and others were from 1980s-like human influenza. Human sera were collected from school-aged children as well as their family adults at Taichung and Nantou between 2008 and 2010 before and after seasonal influenza vaccination. The results showed that GMT and seroprotection rate against Taiwan swine influenza were gradually elevated with increasing age with GMT from 5 to 10.315 and the seroprotection rate from 0 to 23.59% of the young and old age group, respectively. However, the titer decreased through year in adults with GMT from 14.42 to 5.8 and the seroprotection rate from 38.83 to 2.68% during 2008 and 2010, respectively. (2) The results of receiving different vaccination strategies showed that the seroprotection rate of grade 4-6 with 2009 pandemic vaccine only decreased to 64.71 % before 2010 vaccination, while grade 1-3 were 82.76 %. Furthermore, the seroprotection rate of grade 4-6 was significant lower in group 1 (64.71 %) than in group 2 (94.30 %) before 2010 vaccination. (3) The only vaccine component changed was H3N2 during 2010-11 season. Although receiving TIV from both seasons (2009-10 and 2010-11) had significant higher GMT and seroprotection rate in H1N1 (2009 and 2010 vaccine strains were the same), antibody response was significant lower in H3N2 (2009 and 2010 vaccine strains were different), compared to the group receiving only TIV during 2010-11 season. Conclusion: Our data demonstrated that both children and adults are susceptible to the infection by swine influenza virus and suggested the necessity of specific vaccine against newly-reassorted pandemic virus from pigs because of the low cross-reactivity between seasonal TIV and swine influenza viruses. Furthermore, boosting of the same vaccine strain would enhance immune response, while the change of vaccine strain might reduce the ability of enhancing antibody response among school-aged children.

A ce jour, les données génétiques et moléculaires se rapportant aux virus influenza de type A (VIs) présents dans la population porcine au Québec sont relativement rares. Pourtant, ces informations sont essentielles pour la compréhension de de l'évolution des VIs à grande échelle de 2011 à 2015. Afin de remédier à ce manque de données, différents échantillons (pulmonaires, salivaires et nasaux) ont été prélevés à partir de 24 foyers dans lesquelles les animaux présentaient des signes cliniques. Ensuite, les souches virales ont été isolées en culture cellulaire (MDCK) ou sur oeufs embryonnés. Les 8 segments génomiques des VIs de 18 souches virales ont par la suite été séquencés et analysés intégralement. La résistance aux drogues antivirales telles que l’oseltamivir (GS4071) carboxylate, le zanamivir (GS167) et l’amantadine hydrochloride a également été évaluée par des tests d'inhibition de la neuraminidase (INAs) ainsi que par un test de réduction sur plaque. Deux sous-types viraux H3N2 et H1N1 ont été identifiés dans la population porcine au Québec. Douze souches des VIs de sous-type trH3N2 ont été génétiquement liées au Cluster IV, avec au moins 6 profils de réassortiment différents. D'autre part, 6 souches virales ont été trouvées génétiquement liées au virus pandémique A(H1N1)pdm09 avec au moins trois profils de réassortiment génétique différents. Le sous-type trH3N2 des VIs est le plus répandu dans la population porcine au Québec (66,7%). La cartographie d'épitope de la protéine HA de sous-type H3 a présenté la plus forte variabilité avec 21 substitutions d’acides aminés sur 5 sites antigéniques A (5), B (8), C (5), D (1), et E (2). Toutefois, la protéine HA du sous-type H1 avait seulement 5 substitutions d'aa sur les 3 sites antigéniques Sb (1), Ca1 (2) et Ca2 (2). Un isolat H1N1 (1/6 = 16,7%) et 1 autre trH3N2 (1/12 = 8,3%) ont été trouvés comme étant résistants à l'oseltamivir. En revanche, 2 isolats du H1N1 (2/6 = 33,3%) et 2 autres du trH3N2 (2/12 = 16,7%) ont révélé être résistants au zanamivir. Dans l'ensemble, le taux de résistance aux INAs et à l’amantadine était compris entre 33,3% et 100%. La présence des VIs résistants aux drogues antivirales chez les porcs ainsi que l'émergence possible de nouvelles souches virales constituent des préoccupations majeures en la santé publique et animale justifiant ainsi la surveillance continue des VIs dans la population porcine au Québec.
Data about genomic variability of swine influenza A viruses (SIV) in Quebec herds are scarce. Yet, this information is important for understanding virus evolution in Quebec from until 2015. Different clinical samples were obtained from 24 outbreaks of swine flu in which animals were experiencing respiratory disease. Samples including lung tissues, saliva and nasal swabs were collected and virus isolation was attempted in MDCK cells and embryonated eggs. All eight gene segments of the 18 isolated SIV strains were sequenced and analysed. Antiviral drugs resistance against oseltamivir carboxylate (GS4071), zanamivir (GS167) and amantadine hydrochloride was evaluated by neuraminidase inhibition assays (NAIs) and plaque reduction assay. Two subtypes of SIV, H3N2 and H1N1, were identified in Quebec pig herds. Twelve SIV strains were genetically related to trH3N2 Cluster IV and at least 6 different reassortment profiles were identified. On the other hand, 6 Quebec SIV strains were found to be genetically related to the pandemic virus A(H1N1)pdm09 and from which three reassortment profiles were identified. Overall, the trH3N2 was the most prevalent subtype (66.7%) found in Quebec swine herds. The epitope mapping of HA indicated that the H3 subtype was the most variable with a possibility of 21 amino acids (aa) substitutions within the 5 antigenic sites A(5), B(8), C(5), D(1) and E(2). However, the HA protein of the H1 subtype had only 5 aa substitutions within 3 antigenic sites Sb(1), Ca1(2) and Ca2(2). One H1N1 (1/6 = 16.7%) and one trH3N2 (1/12 = 8.3%) were identified as strains resistant against oseltamivir. In contrast, two H1N1 (2/6 = 33.3%) and two trH3N2 (2/12 = 16.7%) strains were found to be resistant against zanamivir. Overall, the SIV resistance against antiviral neuraminidase inhibitor drugs was (33.3%). All strains were resistant against the M2 inhibitor antiviral drug, amantadine. The presence of antiviral drug resistance in Quebec swine herds and the possible emergence of new SIVs strains are public health concerns supporting the surveillance of SIVs.

Influenza virus belongs to the Orthomyxovirus family of viruses that causes respiratory infection in humans, leading to morbidity and mortality. The mature influenza A virion has an envelope that contains two major surface glycoproteins proteins – hemagglutinin (HA) and neuraminidase (NA). HA is a highly antigenic molecules and is responsible binding to host cell surface receptors (Sialic acid), and membrane fusion between the viral membrane and the host endosomal membrane. Most of the antibody response generated against influenza virus either by vaccination or by natural infection is directed against HA. Influenza virus has segmented negative–sense RNA genome which gives the virus the ability to evade the host immune response by incorporating mutations (antigenic drift) and/or by reassotment with other subtypes of influenza A viruses (antigenic shift). Currently licensed vaccines which include an inactivated vaccine, a live attenuated vaccine, and recombinant subunit vaccine are beneficial for providing protection against seasonal influenza viruses that are closely related to the vaccine strain but fail to provide protection against drifted strains. This limits their breadth of protection and thus requires annual revaccination with reformulated vaccines. Also, because selection of a vaccine strain for the next season is purely based on surveillance and prediction, sometimes mismatches do happen between the selected vaccine strains and circulating viruses, resulting in a drastic decrease in vaccine efficacy and thus high morbidity and mortality. Furthermore, the production of these seasonal vaccines takes 6-8 months on an average, and does not guarantee protection against infection with novel reassortant viruses which can cause pandemics. To overcome the draw-backs of seasonal influenza virus vaccines and to enhance our pandemic preparedness, there is an increasing need for game-changing influenza virus vaccines that can confer robust, long-lasting protection against a broad spectrum of influenza virus isolates. Influenza hemagglutinin (HA) is highly immunogenic and thus a major target for vaccine design. HA is synthesized as a precursor polypeptide (HA0), assembles into a trimer, matures by proteolytic cleavage along the secretory pathway and is transported to the cell surface. Mature HA has a globular head domain, primarily composed of the HA1 subunit, which mediates receptor binding, while the stem domain, predominantly comprises of the HA2 subunit, and houses the fusion peptide. At neutral pH, the HA stem is trapped in a metastable state but undergoes an extensive conformational rearrangement at low pH in the late endosome (host-cell endosome) to trigger the fusion of virus and host membranes. Clusters of ‘antigenic sites’ have been identified in the head domain of HA, indicating that it harbors an almost continuous carpet of epitopes that are targeted by antibodies. However, these immunodominant sites constantly accumulate mutations to escape immune pressure, and thereby narrow the breadth of head-directed neutralizing antibodies (nAbs). In contrast to the highly-variable head domain, the membrane-proximal HA stem subdomain has much less sequence variability and, thus, is a desirable target for influenza vaccine development. In the recent past, several broadly neutralizing antibodies (bnAbs) targeting this subdomain with neutralizing activity against diverse influenza A virus subtypes have been isolated from infected people, further proving that this subdomain of HA can be targeted as a vaccine candidate. Steering the immune response towards this conserved, subimmunodominant stem subdomain in the presence of the variable immunodominant head domain of HA has been quite challenging. Alternatively, mimicking the epitome of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogenic capable of eliciting a broadly protective immune response has been difficult because of the metastable nature of HA. Addressing the aforementioned challenges, here we describe the design, stabilization and characterization of novel stem derived immunogens from HA of influenza A viruses using a protein minimization approach. Chapter 1 gives an overview of the influenza virus life cycle, nomenclature and classification of influenza virus; outlines the structural organization and functional properties of different viral proteins. An introduction to the kind of immune responses generated during vaccination or natural infection with the virus is discussed. The conventional vaccines that are currently used and their limitations, recent progress in the field of novel vaccine developmental approaches targeting the conserved epitopes on HA, is also described in this chapter. This chapter also gives a broad overview of bnAbs that have been isolated in the recent past, which target the novel antigenic signatures on HA. The design of a stem domain construct from an H3N2 virus (A/HK/68) is described in Chapter 2. In order to ensure that HA2 folds into the neutral pH conformation, regions of HA1 interacting with it were included in the design. Additionally, two Asp mutations were introduced in the B loop of HA2 to destabilize the low pH conformation and stabilize the desired native, neutral pH conformation. Studies using small peptides (57-98 of HA2) indicated that Asp mutations at positions 63 and 73 destabilized the low pH conformation. Studies on mutants with additional pairs of introduced Cys residues showed that the designed protein H3HA6 was folded into the neutral pH form. Immunization studies using mice showed that the protein was highly immunogenic and provided complete protection against a lethal dose of a homologous virus. Two constructs H3HA6a and H3HA6b, designed from the stem region of drifted H3N2 viruses (A/Phil/2/82 and A/Bris/10/07) were tested for protection against HK/68 to determine the extent of cross-strain protection provided by HA6. While HA6a (from A/Phil/2/82) provided near complete protection against HK/68, HA6b could protect against challenge only partially, possibly because of lower titers of antibodies elicited by this antigen. Studies using FcRγ chain knockout mice indicated that majority of the protection mediated by anti-HA6 antibodies was because of antibody mediated effectors functions, although neutralization as a mechanism of protection was also likely to contribute. In all the 18 subtypes of HA, the B loop contains residues that form the hydrophobic core of the extended coiled coil of the low pH form. As in the case of H3HA6, we suggest that these residues could be mutated to Asp to destabilize the low pH conformation. Two circularly permuted stem domain constructs from an H1N1 virus (A/PR/8/34) and an H5N1 virus (A/Viet/1203/04) were made. The design and characterization of these proteins is described in Chapter 3. H1HA6, H1HA0HA6 and H5HA6 were purified from inclusion bodies and refolded. The proteins H1HA6 and H1HA0HA6 were highly immunogenic and provided protection against a lethal challenge with homologous PR/8/34 virus. Anti-H1HA6 sera had higher titres of antibodies against heterogonous HAs as compared to convalescent sera. Stem derived immunogens from drifted H1N1 viruses (A/NC/20/99 and A/Cal/7/09) have been made and tested for cross-protection with PR/8/34 challenge. While H5HA6 also elicited high titers of antibodies, it could only protect partially against PR/8/34 challenge probably because high enough titers of cross-reactive protective antibodies were not elicited by this protein. These stem immunogens conferred robust subtype specific and modest heterosubtypic protection in vivo against lethal virus challenge. However, the immunogens, especially H1HA6, a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. The strategy used to improve the biophysical and biochemical properties and thus the immunogenicity of these stem derived immunogens is discussed in Chapter 4. A random mutagenesis library of H1HA6 was constructed by error prone PCR using modified nucleotide analogues. The library was displayed on the yeast cell surface to isolate mutants showing better surface expression and improvement in binding to the broadly neutralizing antibody CR6261 compared to the wild-type protein. We isolated few clones, of which one mutant (H1HA6P2) dominated the enriched population. The other mutants differed slightly from H1HA6P2. This mutant differs from the wild-type by two mutations K314E and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody foot-print (epitope). This mutant showed improved binding to CR6261 and exhibited significant improvement in surface expression. Improvement was also observed in binding of this mutant to F16v3-ScFv (another broadly neutralizing antibody). Two cysteine mutations were also introduced to further stabilize the trimeric form of the protein. Chapter 5 describes the biophysical and biochemical characterization of the high affinity isolated mutant at the protein level. We expressed this affinity matured mutant gene in E.coli and purified the protein from inclusion bodies. The stabilized mutant protein showed remarkable improvement in biophysical and biochemical properties and was recognized by stem directed conformation sensitive broadly neutralizing antibodies CR6261, F10 and F16v3 with affinity comparable to the full-length HA ectodomain. These results clearly suggest that this mutant protein is properly folded in its native pre-fusion conformation and thus can be an excellent candidate for eliciting stem directed broadly neutralizing antibodies. All these stabilized versions of stem derived immunogens will be tested for immunogenicity and cross-protection with different viral challenges. Chapter 6 describes the development of a method for mapping antibody epitopes (especially conformational epitopes) down to the residue level. Using a panel of single cysteine mutants, displayed on the yeast cell surface, this bypasses the need for laborious and time consuming protein purifications steps used in conventional methods for epitope mapping. We made a panel of single cysteine mutants, covering the entire surface of the antigen (CcdB, a bacterial toxin protein), displayed each mutant individually as well as in a pool, representing all mutants together on the yeast cell surface, and covalently labeled the cysteine with biotin-PEG2-maleimide to mask the area. The effect on antibody binding was monitored to identify the residues and relative positions important for antibody interactions with the displayed antigen by flow cytometry. By using this method we were able to map the conformational as well as linear epitopes of a panel of monoclonal antibodies down to the residue level with ease, and also identify the regions on the antigen which contribute to the antigen city during immunization in different animals. Since, this method is quite easy, rapid and gives in-depth information about antigenic epitopes, it can be useful in rational design of epitomes specific vaccines and other antibody therapeutics. It can easily be extended to other display systems and is a general approach to probe macromolecular interfaces.