Par le passé les virus influenza A ont montré leur capacité d’émergence dans la population humaine. En 2009, l’apparition d’un nouveau variant réassortant a provoqué une pandémie. Ces travaux avaient pour objectifs d’étudier la propagation de la pandémie en France et Bolivie. Premièrement en Franve avec l’étude sérologique d’une population hospitalière et la surveillance hebdomadaire mise en place grâce au programme SéroGrippeHebdo. La séroprévalence prépandémique était de plus de 20% (au1/80) chez les plus de 60ans et de moins de 10% dans les autres groupes. Il a été possible de calculer un taux d’attaque d’environ 12% au sein de la population française métropolitaine. Mais aussi d’observer la perte d’anticorps rapide après le pic d’infection. Les taux de séroconversions les plus importants étaient chez les 0-24 ans (23.4%). La deuxième partie de ce travail s’est déroulée à Santa Cruz de la Sierra, en Bolivie. Le programme CoPanFlu international en association avec le CENETROP a permis l’étude de la sérologie sur une cohorte de foyers en 2010. Par ailleurs, nous avons caractérisé les pathogènes respiratoires de 2010 à 2012 à Santa Cruz. La grippe représente entre 40 et 58% des cas chaque années, suivie des rhinovirus, des coronavirus et des VRS. L’épidémiologie moléculaire des virus influenza a mis au jour un cluster de circulation sud américain pour les virus H1N1(2009).Les sérologies pré-pandémiques montrent une séroprévalence de 23% pour les ≥60ans. Après la pandémie la distribution par tranche d’âge est différente entre les villes de haute altitude et les autres.Enfin une estimation de la séroconversion montre que les jeunes adultes entre 20-39 sont les plus touchés. / Influenza viruses A have shown their ability to emerge in the human population and in 2009, the appearance of a new variant has caused a pandemia. The objective of this work was to study the pandemia’s spread in France and in Bolivia.In France, first, with the serological study of a hospital population and the weekly supervision established with SeroGrippeHebdo. The prepandemic seroprevalence was more than 20%(au1/80) for the >sixty years, and less than 10% in the others groups. It was possible to reckon an attack rate for about 12% in the metropolitan French population, and to observe a quick loss of antibody after the infection rate. Most important seroconversion rates concerned the 0-24 years(23.4%).The second part of this work took place in Santa Cruz de la Sierra, in Bolivia, developing country in the tropical zone of South America. The international CoPanFlu in partnership with the CENETROP, has enabled to study serology among a cohort of families in 2010. Moreover, we have characterized etiologies of respiratory pathogenesis from 2010 to 2012, in Santa Cruz. Flu represented about 40 to 58% of the cases each year, followed by rhinoviruses, coronaviruses and SRV. The molecular epidemiology of influenza viruses has shown a South American circulation cluster for the H1N1 viruses(2009). The prepandemic serologies in Santa Cruz show a seroprevalence of 23% for the ≥60. After the pandemia, the distribution by age is different for the towns at high altitude and others. Finally, an estimation of the sero-conversion showed that young adults between 20 and 39 were more affected in Bolivia.

Dans le contexte de la menace pandémique liée au virus influenza A(H5N1), un projet «GRIPPE AVIAIRE ET GRIPPE PANDÉMIQUE » a émergé au sein de LyonBioPôle avec comme objectif le développement d’outils de caractérisation des virus influenza pour la production de vaccins. Pour étudier le réassortiment génétique entre virus influenza, nous avons développé 3 systèmes de génétique inverse : virus humain A(H3N2) et aviaires A(H5N2) et A(H5N1) et produit des virus réassortants de composition déterminée. Leurs capacités réplicatives ont été évaluées par cinétiques de croissance virale sur MDCK avec quantification de la production virale par qRT-PCR temps réel. L’émergence du virus influenza A(H1N1)2009 pose deux questions sur l’acquisition par réassortiment génétique, d’une résistance à l’oseltamivir d’une part ou de facteurs de virulence d’autre part. Nous avons donc développé un protocole de co-infection virale de cellules MDCK pour étudier les constellations de gènes des réassortants entre différents virus: A(H1N1)2009-A(H1N1) H275Y et A(H1N1)2009-A(H5N1). Nous montrons par deux approches différentes, génétique inverse et co-infections virales, que le réassortiment génétique entre souches aviaires et humaines et surtout aviaires et porcines est possible, en privilégiant certaines constellations. Nous rapportons que le virus pandémique peut acquérir la NA H275Y des virus A(H1N1) Brisbane-like résistants à l’oseltamivir sans que ses capacités de réplication ne soient altérées. De même nous montrons que son réassortiment avec un virus hautement pathogène A(H5N1) est possible. Ces observations renforcent la nécessité de promouvoir la vaccination afin de limiter les risques de co-infection virale chez un même individu. / In the context of A(H5N1) pandemics threat, an « avian flu and flu pandemics » project was proposed by LyonBioPole to develop influenza viruses characterization tools for vaccine production. To study genetic reassortment between influenza viruses, 3 reverse genetic systems of A(H3N2) human virus and A(H5N2) and A(H5N1) avian viruses were developed and reassortant viruses were produced. Their replicative capacities were evaluated using growth kinetics on MDCK cells with viral production quantification by real-time qRT-PCR. The A(H1N1)2009 emergence raises two questions about the acquisition by genetic reassortment of oseltamivir resistance and/or pathogenicity determinants. A co-infection protocol on MDCK cells was developed to study gene constellations of reassortant viruses like A(H1N1)2009-A(H1N1) H275Y and A(H1N1)2009-A(H5N1). We report here that genetic reassortment is possible between avian, human and swine strains using reverse genetic and viral co-infection and that some specific constellations emerged. We also report, that pandemic A(H1N1)2009 can acquire the H275Y mutated NA from seasonal oseltamivir resistant A(H1N1) viruses without any modifications on replicative capacities. This genetic reassortment is also possible with A(H5N1) viruses. These observations strenght the importance of vaccination against all these influenza strains to reduce the risk of one-individual viral co-infection.

An influenza is an illness annually affecting 5-15 percent of the world population. During the influenza pandemy 40-50 percent of world population can be affected and millions of people can die.The measures resulting from the pandemic plans help to limit the influenza virus spreading, to reduce morbidity and mortality. In April 2009 the first cases of the flue pandemic caused by Pandemic A (H1N1) 2009 virus occurred on the American continent, in the Czech Republic there was the first case registered in May, in the Pilsen region in July. Based on these facts I decided to assess the pandemic plans at the level of the Pilsen region and to analyse the course of the pandemy in the Pilsen region too. Having studied the Pandemic plan of the Pilsen region and the Pandemic plan of the Regional Hygiene Station of the Pilsen Region I got to the conclusion that in case of the pandemy caused by the highly virulent tribe of the influenza it would not be possible to use up the pandemic plans efficiently. The disadvantages concern mainly the way of the distribution of the pandemic vaccine and antivirotics. The other disadvantage is the absence of a parenteral form of antivirotics. A bad awarness of the inhabitants also came out effecting mass rejection of vaccination by the pandemic vaccine and preventative taking antivirotics. The analyse of the course of the pandemy in the Pilsen region proved that at many patients with the flue pandemic there was present a risky factor of more serious course of the influenza in the anamnesis. If these patients had been vaccinated by the pandemic vaccine they had been entitled for, they could have been protected against this illness, for some of them the vaccination might have meant life-saving. It was also proved that originally the pandemic tribe of Pandemic A (H1N1) 2009 virus became the causer of the common seasonal influenza in the season of 2010-2011.

In the thesis there are chronologically processed data about incidence of influenza Pandemic (H1N1) 2009 from its first incidence in spring 2009 in Mexico until the official end of the 6th phase of pandemic announced by the WHO in august 2010. These data were collected by means of secondary analysis. The thesis is focused on the Region of South Bohemia from the first proved incidence of Pandemic influenza (H1N1) 2009 here. The data necessary to meet the objectives of the work and to answer the research questions were collected in cooperation with the Regional Hygienic Station of the South Bohemia in České Budějovice. 3 deaths were analysed in the context of incidence of Pandemic influenza (H1N1) 2009 in the Region of South Bohemia and anti-epidemic measures were assessed. Differences in 121 people with Pandemic influenza (H1N1) 2009 in the Region of South Bohemia from the point of view of age and sex were described. In 52 people with the flu from the Region of South Bohemia ?traveller? history was recorded (these people were infected during their stays abroad) and most of them had stayed in Germany. Indicators of morbidity (ARI) in the Region of South Bohemia and in the whole Czech Republic were also processed and then graphically compared.

In the United States and other developed countries, despite great efforts in time and funding for the prevention of foodborne and airborne diseases, there is still an unacceptable level of common pathogens spread via food, water, and air. To this end, lab-on-a-chip (LOC) technologies were developed for field-deployable assays and point of care diagnostics. These devices have potential applications in hospitals, agricultural farms, processing plants, and even on fields of battle. Two successful types of assays in the recent years towards point of care diagnostics are immunoassays and nucleic acid detection assays. In the Appendix A, we demonstrated a complete, field-deployable particle immunoassay encased within a microfluidic chip that detects small quantities of Salmonella Typhimurium in poultry fluid samples. Because the necessary reagents are pre-loaded and the test and negative control channels are fed by a single sample inlet, single pipetting of sample is possible. This assay demonstrated a 10 CFU/mL limit of detection, which is considerably lower than PCR and enzyme-linked immunosorbent assay (ELISA). Total assay time, including sample reading in an integrated handheld device, was 10 minutes, which was much lower than conventional methods. Because of the simplified protocol and assay time, this biosensor has potential in clinical and field diagnostic applications. In Appendix B, we fit the particle immunoassay to test for Influenza A H1N1/2009 virus and included aerosol sampling from a scaled-down mock classroom. To make the assay field deployable, we used an iPhone for signal detection. The detection limit of the assay was 1 pg/mL (10 pg/mL using the iPhone), which is well below the limit of detection for RT-PCR. This protocol demonstrated that immunoassays can be effective in the presence of interfering dust particles and that viruses can be collected from aerosol with minimal sample preparation. In Appendix C, we demonstrated that paper microfluidics, a newer vision of microfluidics, is a cheap and easy method to extract nucleic acid from S. Typhimurium in a variety of samples, including poultry packaging liquid, whole blood, and feces. Fluorescent detection with an iPhone allows for field and clinical testing. This protocol interfaces with rapid PCR and is a true diagnostic tool.

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Dans le contexte de la menace pandémique liée au virus influenza A(H5N1), un projet "GRIPPE AVIAIRE ET GRIPPE PANDÉMIQUE " a émergé au sein de LyonBioPôle avec comme objectif le développement d'outils de caractérisation des virus influenza pour la production de vaccins. Pour étudier le réassortiment génétique entre virus influenza, nous avons développé 3 systèmes de génétique inverse : virus humain A(H3N2) et aviaires A(H5N2) et A(H5N1) et produit des virus réassortants de composition déterminée. Leurs capacités réplicatives ont été évaluées par cinétiques de croissance virale sur MDCK avec quantification de la production virale par qRT-PCR temps réel. L'émergence du virus influenza A(H1N1)2009 pose deux questions sur l'acquisition par réassortiment génétique, d'une résistance à l'oseltamivir d'une part ou de facteurs de virulence d'autre part. Nous avons donc développé un protocole de co-infection virale de cellules MDCK pour étudier les constellations de gènes des réassortants entre différents virus: A(H1N1)2009-A(H1N1) H275Y et A(H1N1)2009-A(H5N1). Nous montrons par deux approches différentes, génétique inverse et co-infections virales, que le réassortiment génétique entre souches aviaires et humaines et surtout aviaires et porcines est possible, en privilégiant certaines constellations. Nous rapportons que le virus pandémique peut acquérir la NA H275Y des virus A(H1N1) Brisbane-like résistants à l'oseltamivir sans que ses capacités de réplication ne soient altérées. De même nous montrons que son réassortiment avec un virus hautement pathogène A(H5N1) est possible. Ces observations renforcent la nécessité de promouvoir la vaccination afin de limiter les risques de co-infection virale chez un même individu.