Influenza A viruses are important pathogens of humans, other mammals and birds. Swine are considered to be the ‘mixing vessel’ for influenza viruses because of their susceptibility to infection with not only swine influenza viruses but also human and avian influenza viruses. After infection of pigs with different influenza viruses, reassortment events between genomic RNA segments and point mutations can take place which can result in novel influenza virus strains capable of causing human pandemics. To combat infections, vaccination is available in many countries for humans, but not typically used in pigs. However, anti-influenza drugs have been used to treat livestock, and mutations conferring drug resistance occur in circulating strains. The mechanisms responsible for the emergence and spread of drug resistant mutations against amantadine and oseltamivir have been studied previously but often gave conflicting results. Therefore, this PhD thesis focused on resolving the mechanisms responsible for this rapid drug resistance spread. In chapter one I examine the extent of reassortment events in swine influenza A viruses by analysing within subtype reassortment and extrapolating the results for the between subtype reassortment. Reassortment is one of the mechanisms that can be responsible for mutations, conferring resistance to drugs, to spread between strains, and thus spread in the host population. The findings of this chapter show that the genomic segments most prone to reassortment code for a polymerase (PB1) and both glycoproteins, within all three subtypes studied. Since particular mutations in the matrix protein (MP) segment cause resistance to amantadine, my study focused on MP compared to other segments and revealed moderate level of reassortment. MP reassorts well with polymerases, both within and between subtype, while nonstructural (NS) is least likely to reassort. Chapter two of this thesis aimed at resolving the origin and spread of the most common drug resistance conferring mutation in swine influenza viruses which causes amantadine resistance. I show first that this mutation occurred in swine influenza viruses and was therefore not transmitted from the recently ancestral avian influenza strains, and second that the prevalence of resistance in swine influenza viruses is due to functional linkage of mutations at other sites and not by direct drug pressure. In chapter three I examine the mechanisms responsible for the rapid rise and spread of oseltamivir resistance in human influenza H1N1 viruses which arose in the absence of drug use. The primary mutation lies in the neuraminidase glycoprotein but because of the close functional interaction I focus on changes in haemagglutinin that occurred in association with resistance. The results showed several mutations in haemagglutinin were associated with resistance suggesting selection acting on haemagglutinin in order to balance the activity of both glycoproteins. Overall these results show the importance of functional linkage between segments as a mechanism for the occurrence of drug resistance conferring mutations, and reassortment as a means of spreading these mutations into newly emerging strains.

Les profils de viabilité sur surface, de virus non enveloppés : murine minute virus (MVM), coxsackievirus B4 (CVB4), virus simien 40 (SV40), et de virus enveloppés : virus de grippe A (H1N1), et virus herpès simplex de type 1 (HSV-1), ainsi que la résistance à la chaleur et aux ultraviolets C (UVc) de ces virus ont été étudiés.Pour déterminer la viabilité de MVM, CVB4, H1N1 et HSV-1 sur surface, 50 µL de suspension virale ont été déposées sur des couvercles de boites de Pétri et séchés sous un flux d’air avant d’être récupérés et titrés sur des lignées cellulaires appropriées. Les virus enveloppés ont persisté moins de 5 jours alors que CVB4 et MVM restent infectieux pendant plusieurs semaines. Cependant, les cycles répétés de séchage et de remise en suspension ont eu un effet plus virucide sur CVB4 que sur H1N1 et HSV-1. Aucun effet des répétitions de ces cycles n’a été observé sur le titre infectieux du MVM. Quand il est exposé au séchage, les concentrations initiales d’albumine de sérum bovin, de sérum de veau fœtal et de chlorure de sodium, ont un impact sur la survie de CVB4. Dans un milieu riche en protéines, CVB4 a été plus facilement inactivé par le séchage, alors qu’en présence de chlorure de sodium le pouvoir virucide du séchage a été réduit. Ces résultats montrent que la résistance des virus vis-à-vis du séchage, n’est pas due à une hétérogénéité de populations virales, mais peut être influencée par la composition du milieu et la concentration des composants.Nous avons évalué la résistance thermique de MVM, CVB4, H1N1 et HSV-1 contenus dans des gouttelettes. Quatre microlitres de suspension virale ont été déposés sur une surface chauffée et exposés à des températures comprises entre 70 et 130°c pendant 0 à 90min, selon le virus, avant d’être titrés. Clairement, MVM a été plus résistant que H1N1, lui-même plus résistant que HSV-1 et CVB4. Pour la première fois, l’inactivation de particules virales contenues dans des gouttelettes exposées à des températures supérieures à 100°C a été étudiée. Il apparaît que le chauffage peut provoquer un effet plus rapidement virucide que décrit précédemment.La résistance aux UVc (254nm) de MVM, CVB4, H1N1, HSV-1 et SV40 contenus dans des gouttelettes a été évaluée. Les virus à ADN double brins (HSV-1 et SV40) restaient infectieux après une exposition à 60mJ/cm² d’UVc, tandis que les virus à ARN (H1N1 et CVB4) et un virus à ADN simple brin (MVM) ont été totalement inactivés par une exposition inférieure ou égale à 35mJ/cm² d’UVc. De plus l’effet des UVc combiné à la chaleur sur la viabilité de MVM a été déterminé. Le titre infectieux de MVM, contenu dans une gouttelette a été totalement inactivé après une exposition à 27mJ/cm² d’UVc. Le chauffage (20s à 100°C) a provoqué une réduction modérée du titre viral de MVM (-1.8 log10TCID50), alors que le chauffage suivi par une exposition à 17mJ/cm² d’UVc entraine une inactivation complète.En conclusion, nos études montrent que les virus peuvent persister pendant des jours voire des semaines sur une surface hydrophobe. Le profil de résistance des virus vis-à-vis du séchage, n’est pas dû à une hétérogénéité de populations virales, comme l’ont montré les résultats obtenus avec CVB4. De plus, dans la mesure où la composition du milieu joue un rôle dans la viabilité des virus exposés au séchage, la persistance des virus devrait être étudiée dans des milieux naturels plutôt que dans des milieux définis. L’impact de temps d’exposition courts à la chaleur sur les virus contenus dans de petits volumes de suspension a été déterminé. La résistance thermique de H1N1 jusqu’à 100°C, supérieure à celle d’HSV-1, un autre virus enveloppé, et à celle de CVB4 un virus non-enveloppé a été observée. Une inactivation virale efficace peut être obtenue en combinant une exposition aux UVc et à la chaleur comme le montrent les résultats obtenus avec MVM. / The pattern of viability of non-enveloped viruses, minute virus of mice (MVM), coxsackievirus B4 (CVB4), and simian virus 40 (SV40) and enveloped-viruses, influenza A virus (H1N1), and herpes simplex virus type 1 (HSV-1) onto surfaces and their resistance to heating and to ultraviolet C (UVc) exposure have been investigated. To determine the viability of MVM, CVB4, H1N1 and HSV1 on surface, fifty microliters of viral suspension were applied onto petri dish lids and dried under air flow of biosafety cabinet. The recovered viral preparations were titered on appropriate cell cultures. Enveloped viruses persisted for less than 5 days while CVB4 and MVM persisted for weeks. However, repetitive cycles of drying and resuspension had more virucidal effect on CVB4 than on H1N1 and HSV-1. No effect of these repetitive cycles on infectious titer of MVM was recorded. When exposed to drying, initial concentrations of bovine serum albumin, foetal calf and sodium chloride (NaCl) had an impact on the viability of CVB4. In a protein rich medium, CVB4 was more likely inactivated by drying whereas in presence of NaCl, the impact of drying was reduced. Thus, it appears that the resistance of viruses toward drying is not due to a heterogeneity of viral populations, but it can be influenced by media composition and components concentrations.Heat inactivation of viruses was reported, however, the thermal resistance of viruses in droplets has not been studied. We evaluated the pattern of heat resistance of MVM, CVB4, H1N1 and HSV1 contained in droplets. Four microliters droplets containing viruses were applied onto warmed surface obtained by using a self-made heating device. Viral suspensions were exposed to temperatures ranging from 70 to 130°C for 0 to 90 min depending on the virus, and then the recovered viral preparations were titered. Clearly, MVM was more resistant than H1N1 that was more resistant than HSV-1 and CVB4. For the first time, the inactivation of viral particles contained in drops exposed to temperatures higher than 100°C has been investigated. It appears that heating can have an unexpected faster virucidal effect than previously described. The resistance to ultraviolet C (UVc) (254nm) of MVM, CVB4, H1N1, HSV-1 and SV40 contained in droplets has been evaluated. Double-stranded DNA viruses (HSV-1 and SV40) were still infectious after exposure to 60 mJ/cm² UVc, while RNA viruses H1N1, CVB4 and single-stranded DNA virus MVM were fully inactivated when they were exposed to a dose equal to or lower than 35 mJ/cm² UVc. Moreover the effect of UVc (254 nm) combined with heating onto the viability of MVM was determined. The infectious level of MVM suspension droplets applied onto petri dish lids was fully inactivated when exposed to 27 mJ/cm² UVc. Heating (100°C for 20s) provoked a moderate reduction of infectious level (-1.8 log10TCID50) of MVM, whereas heating followed by UVc exposure (17 mJ/cm²) resulted in a full inactivation.In conclusion, our studies show that viruses can persist for days or even weeks on dry hydrophobic surfaces. The pattern of resistance of viruses toward drying is not due to a heterogeneity of viral population as shown by results obtained with CVB4. In so far as media composition play a role in the viability of viruses exposed to drying, the persistence of viruses in natural media (clinical or environmental), instead of defined media, should be investigated. The impact of short time exposure to heat onto the infectivity of viruses contained in a small volume of suspension has been determined. The thermal resistance of H1N1 up to 100°C, higher than the one of HSV1 another enveloped virus, and CVB4 a non-enveloped virus has been observed. An efficient viral inactivation can be obtained by combining UVc exposure and heating as shown by results obtained with MVM.

Influenza is a major concern across healthcare environments. Annual vaccination of healthcare workers (HCWs) remains essential for maintaining the health and availability of HCWs, as well as influenza prevention in healthcare environments. Yet, annual vaccination coverage among HCWs continues to be below recommended standards during pandemic (pH1N1) and non-pandemic (sINFLU) influenza seasons. The primary aim of this research is to inform the design and implementation of effective HCW targeted influenza vaccination campaigns via a 1) systematic review of the existing literature on HCW pH1N1 vaccination, 2) qualitative content analysis of motivators and barriers to HCW pH1N1 and sINFLU vaccination, as well as 3) quantitative regression analysis of modifiable factors predicting pH1N1 and sINFLU vaccination. The qualitative and quantitative analysis processes were applied to data collected from a large-scale multi-professional sample of HCWs. Findings from all analysis sections were found to be consistent. Most attitudes, beliefs, motivators, and barriers influencing HCW influenza vaccination were similar for pH1N1 and sINFLU vaccinations. Yet, a number of notable differences were also identified. HCWs were likely to accept vaccination if they perceived, 1) vaccination to be safe, 2) vaccination to be protective against influenza for self, loved ones, patients or communities, and 3) influenza to be a serious and severe infection to self and others. Additionally, encouragement from supervisors and colleagues, physicians, and loves ones also enhanced vaccine uptake. Most HCWs avoided vaccination because of 1) limited knowledge or misinformation about vaccination, 2) concern for vaccine induced side-effects and 3) assuming vaccination was not a requirement for healthy adults. With respect to pH1N1 vaccination, mass media communications, perceptions of novel vaccinations, and rapid vaccine development processes especially deterred HCW pH1N1 vaccination. Future vaccination programs targeting HCWs should look towards influencing HCWs’ vaccination attitudes and promoting pro-vaccination cultures in healthcare workplaces.

The H1N1/09 (sub-type A) virus was declared to be a pandemic influenza on June 11th, 2009. In response, Canadian public health agencies planned mass vaccination clinics to protect the public. However, little information existed to aid in the planning of mass vaccination clinics, as they had not been used in previous pandemic flu outbreaks. This was further complicated by fear of a limited vaccine supply and nurse shortages. Public health nurses (PHNs), as the largest group of public health professionals were pivotal in implementing the mass vaccination clinics. Yet, the available evidence indicated that PHNs involvement in H1N1/09 response planning was limited and their experiences on the frontline in the mass vaccination clinics were not well understood. The purpose of this study was to give PHNs’ a voice to describe their experiences in the H1N1/09 mass vaccination clinics. A framework based on Foucault’s concepts of knowledge, power, and resistance was developed as the theoretical lens to guide the research. Using an interpretive descriptive methodology, a purposeful sample of 23 PHNs (16 front-line immunizers, seven clinic supervisors) participated in semi-structured interviews. Four pandemic planning documents containing policies in place during the pandemic outbreak were also reviewed to provide context to participants’ experiences. Interpretive descriptive analysis was used to analyze the interviews and pandemic documents. Guba and Lincoln’s (1994) trustworthiness framework was implemented to evaluate the rigour of the study’s findings. Two overall core themes emerged to describe participants’ experiences. The core theme ‘the necessity of knowledge’, illustrated participants’ feelings of unpreparedness entering into the H1N1/09 clinics. Limited notice of the pandemic response, uncertainties regarding the clinics’ anticipated timeframe, and a lack of knowledge on vaccination and clinic management, contributed to a loss of power in the participants’ role. In the second core theme ‘essential supports in protecting the population’, many perceived a lack of agency support when they tried to exercise power in their clinical practice. Although participants did not refuse to immunize in, or supervise, the mass vaccination clinics, participants at times did display subtle resistance. Insights gained from participants’ experiences have implications in terms of public health nursing administration, practice, research, and education. A key recommendation is to involve PHNs in future pandemic planning to optimize mass vaccination clinics’ operations. If this cannot happen, PHNs should at least be informed of the disciplinary discourse utilized to guide clinical decisions. This will help nurses be supported in their own pandemic roles and contribute to the provision of quality population care.

Influenza A viruses (IAVs) cause occasional pandemics and seasonal epidemics, thus presenting continuous challenges to public health. Vaccination is the primary strategy for the prevention and control of influenza outbreaks. The antigenicity matched high-yield seed strain is critical for the success of influenza vaccine. Currently, there are several limitations for the influenza vaccine manufacture: 1) the conventional methods for generating such strains are time consuming; 2) egg-based vaccines, the predominant production platform, have several disadvantages including the emergence of viral antigenic variants that can be induced during egg passage; 3) vaccine seed viruses often do not grow efficiently in mammalian cell lines. Previous studies suggested that mutations in the receptor binding site (RBS) that locates at the globular head of the HA1 can change IAVs’ binding specificity, antigenicity, and yield and thus RBS would be an potential target for engineering vaccine seed strain. However, systematic analysis of the mutations on RBS affecting those viral phenotypes is lacking. Specifically, this dissertation has following aims: Firstly, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. The error-prone PCR- based reverse genetic system could also be applied to gain-ofunction studies for influenza virus and other pathogens; Secondly, in this dissertation, we identified an Y161F mutation in the hemagglutinin (HA) that enhanced the infectivity and thermostability of virus without changing its original antigenic properties which would prompted the development of cell-based vaccines; Thirdly, the molecular mechanisms underlying host adaption of equine-origin influenza A(H3N8) virus from horses to dogs are unknown. This dissertation identified that a substitution of W222L in the HA of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to sialyl Lewis X motifs, which were found abundantly in the submucosal glands of dog trachea but not in equine trachea. To summary, this dissertation investigated the role of RBS in IAVs biology and expanded the current knowledge toward IAV vaccine strain engineering, IAV host adaption and evolution.

The purpose of this thesis was to identify causes of disparities in affliction (infection) and mortality for minority populations (Blacks, Hispanics, Asians, and American Indian/Alaskan Natives) during the Swine Flu (H1N1) and COVID-19 (Sars-Cov-2) Pandemics. A literature review was conducted gathering peer-reviewed journal articles related to racial and socioeconomic disparities in affliction and mortality during both pandemics. The model of Blumenshine et al. (2008) was used as a guide for the analysis of this thesis, and measures of exposure, susceptibility, and treatment were hypothesized as causes for the disparities experienced by the minority populations during the two pandemics. Ultimately, it was established that the causes of the disparities noted were found to be differences in social determinants of health experienced by minority populations including poverty, education, occupation, and housing location. Differences in each of these social determinants of health then led to disparities in exposure, susceptibility, and treatment. All of these disparities combined together caused disproportionate affliction and mortality for minority populations during both pandemics. Organizing disparities in terms of social determinants of health and identifying possible explanations for disparities is important for future pandemic planning, and the model of Blumenshine et al. (2008) is a structured way to hypothesize certain causes of disparities during a pandemic based on social determinants of health. Emphasis needs to be placed on developing a pandemic vulnerability index based on the measures hypothesized so that future pandemic planning can direct resources to those most vulnerable.

Våren 2009 spreds en ny subtyp av influensavirus A(H1N1) mycket snabbt över världen. Media hårdbevakade pandemins framfart och förmedlade bilden av en allvarlig smitta med högre dödlighet i yngre åldrar. Hotet om påverkade samhällsfunktioner och en hårt belastad sjukvård ledde till beslutet att låta massvaccinera hela svenska folket. I frontlinjen stod primärvårdssjuksköterskorna som hade till uppgift att bemöta en orolig allmänhet. Syftet med studien var att undersöka primärvårdssjuksköterskors upplevelser av svininfluensapandemin A/H1N1. Hur upplevde de allmänhetens reaktion, sin egen arbetssituation och hur hanterade de situationen? Studien är empirisk och grundar sig på kvalitativa intervjuer med sex sjuksköterskor på tre olika vårdcentraler i Malmö. Analysmetoden var kvalitativ innehållsanalys såsom den beskrivs av Lundman och Hällgren Graneheim (2008). Resultatet redovisas i ett antal kategorier under tre teman. Temat för allmänhetens reaktioner var rädsla; människor uppgavs kontakta vårdcentralerna i långt större utsträckning än vanligt, vara vaksamma och ivriga att få vaccin samtidigt som de var misstänksamma mot vaccinets säkerhet. Det andra temat var hanterbart kaos, vilket representerar upplevelsen av arbetssituationen, som präglades av hög belastning, bristande beredskap, komplicerad rådgivning och etiska dilemman. Situationen hanterades genom målmedvetenhet som var det tredje framträdande temat, med försök att lugna, metodisk handläggning och kollegialt stöd som representativa kategorier. / In the spring of 2009 a new subtype of the influenza virus A(H1N1) spread very rapidly over the world. The media coverage of the pandemics’ rampage was intense and media conveyed the image of a severe infection with higher mortality at younger ages. The threat of interruptions to public services and a congested medical care led to the decision to undertake mass vaccination of the entire Swedish population. In the front line were the primary care nurses with the task to counter anxious citizens. The aim of this study was to investigate primary healthcare nurses’ experiences of the pandemic influenza A(H1N1). How did they experience the public’s reaction, their work situation and how did they handle the situation? This is an empirical study based on qualitative interviews with six nurses at three healthcare centers in Malmö. The method of the analysis was a content analysis, as described by Lundman and Hällgren Granheim (2008). The results are presented in a number of categories in three themes. The theme of the public reaction was fear; the informants stated that people contacted the health care centers in far greater extent than usual, they were vigilant and eager to receive the vaccine whilst they were suspicious of the vaccine’s safety. The second theme was manageable chaos, which represents the experience of the work situation, which was characterized by high stress, lack of preparedness, difficult counseling and ethical dilemmas. The situation was handled by determination, which is the third theme; attempts to calm, methodical management and collegially support are representative categories in this theme.

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OBJECTIFS : A l'automne 2009, le Québec a procédé à une vaccination de masse de sa population contre la grippe pandémique A(H1N1). Les objectifs de cette étude étaient de décrire le rehaussement du système de surveillance des manifestations cliniques indésirables (MCI) mis en place en vue de la campagne de vaccination contre 1'influenza pandémique et de comparer le profil des MCI rapportées dans le cadre de la surveillance faite durant la campagne de vaccination contre le virus pandémique H1N1 par rapport à ceux de la vaccination saisonnière des années précédentes. MÉTHODE : Pour le rehaussement de la surveillance, les données présentées ont été recueillies à partir des procès verbaux des rencontres des membres du groupe ESPRI depuis juin 2009, des conférences téléphoniques et des documents d'orientation pour le groupe. Pour la comparaison du profil des MCI rapportées dans le cadre de la surveillance faite durant la campagne de vaccination contre la grippe pandémique A(H1N1) par rapport à ceux de la vaccination saisonnière des six saisons précédentes (2003-2004, 2004-2005, 2005-2006, 2006-2007, 2007-2008 et 2008-2009), les données analysées proviennent des informations saisies au fichier ESPRI au 21 mai 2010. RÉSULTATS : Les principales modifications à la surveillance ont été la mise en place d'une surveillance spécifique du syndrome de Guillain Barré et la déclaration directe à la surveillance passive par les infirmières d'info-santé. Le vaccin pandémique a été associé à une fréquence trois plus élevée de MCI que le vaccin saisonnier soit 50,4 et 18,9 par 100 000 doses, respectivement. Les taux de chacune des MCI sont supérieurs avec le vaccin pandémique adjuvante que ceux des vaccins saisonniers. Cette augmentation s'est manifestée principalement pour les allergies (11.8 vs 4.1/100 000 doses), mais aussi pour des manifestations neurologiques de type anesthésie/paresthésie (2.5 vs 0.3/100 000 doses). CONCLUSION : Le Québec a opéré des modifications de sa surveillance des MCI lors de la campagne de vaccination massive de 2009. Bien qu'un taux plus élevé de MCI ait été rapporté, aucune MCI n'a été assez fréquente ou sévère pour mener à des modifications dans le déroulement du programme.

La sécurité des vaccins est évaluée par le biais du système de surveillance passive des manifestations cliniques indésirables (MCI). À cause du très grand nombre de personnes vaccinées lors des campagnes massives de vaccination contre l'influenza, si un effet secondaire modérément fréquent (ex. 1/1 000 vaccinés) survenait, le délai avant que la surveillance passive ait détecté un problème serait suffisamment long pour que des millions de personnes aient été vaccinés et que des milliers aient souffert de cet effet secondaire. Lors de la vaccination de masse contre l'influenza pandémique H1N1 en 2009, un projet pilote de surveillance électronique active de la sécurité du vaccin contre l'influenza pandémique chez plusieurs milliers de travailleurs de la santé a été mis sur pied. Cette surveillance visait à évaluer très rapidement la fréquence des problèmes de santé suffisamment sévères pour causer de l'absentéisme au travail ou une consultation médicale. La présente étude a évalué la qualité de cette surveillance par le biais de ses attributs comme définis dans le cadre de référence des Centers for Disease Control and Prevention (CDC) pour l'évaluation des systèmes de surveillance. Une méthodologie mixte (analyse quantitative et qualitative) a été utilisée. Les résultats montrent que la simplicité, la flexibilité, l'acceptabilité et la valeur prédictive positive (VPP) étaient des attributs plutôt acquis par le système de surveillance, mais que des déficiences étaient présentes dans le contrôle de la qualité des données, la sensibilité, la représentativité et la réactivité. Des recommandations ont été émises pour améliorer ce système, qui pourraient servir dans le cas d'une nouvelle pandémie ou s'intégrer au processus formel de surveillance de la vaccination saisonnière contre l'influenza.