Fin avril 2009, des cas de grippe causés par un nouveau virus grippal A/H1N1 d’origine porcine sont confirmés au Mexique et aux Etats-Unis. Rapidement, le virus est détecté aux quatre coins du globe causant la première pandémie du XXIème siècle. Les différents travaux présentés dans cette thèse retracent les moyens mis en œuvre pour obtenir des informations permettant d’estimer le taux d’attaque réel de ce nouveau virus et des informations sur les populations à risque. Durant les premiers mois, nous avons mis en place une plateforme de sérologies comprenant un laboratoire de réception et de traitement des échantillons pour l’exécution de notre technique d’IHA. Le traitement d’environ 40.000 sérums provenant de plusieurs endroits du globe : France, Bolivie, Djibouti, Mali, île de la Réunion et Laos a permis l’analyse de données sérologiques et leur comparaison. Nos études sérologiques de la grippe A(H1N1)pdm09 montrent que 10% à 40% des populations testées ont été infectées par ce nouveau virus après la première vague de 2009. Les plus forts taux d’attaque ont été observés chez les enfants et les jeunes adultes alors que les personnes âgées ont été relativement épargnées du fait qu’elles étaient déjà protégées contre des virus antigéniquements proches qui circulaient avant 1957 (virus pandémique et/ou saisonniers). L’analyse des données sérologiques ont également permis de tenter de définir les facteurs de risque à l’infection de A(H1N1)pdm09. / In late April 2009, news swine-origin A/H1N1 influenza virus cases were confirmed in Mexico and the United States. Quickly, it was spread worldwide causing the first flu pandemic of the 21st century. Different works presented in this thesis describe the means used to obtain information to estimate the actual attack rate of this new virus, and information on risk populations. During the first months, we have established a serology platform including a reception-processing samples laboratory for implementing our hémagglutination Inhibition technique (IHA). Processing of 40,000 sera from several parts of the world: France, Bolivia, Djibouti, Mali, Reunion and Laos, has allowed the analysis of serological data and their comparison. Our serological studies of influenza A(H1N1) pdm09 show that 10% to 40% of people tested were infected with this new virus after the first wave in 2009. The highest attack rates were observed in children and young adults, while the elderly were relatively spared because they were already protected again antigenic close viruses that circulated before 1957 (pandemic and / or seasonal). The analysis of serological data were also used to try to identify the risk factors for A(H1N1)pdm09 infection. It appears that infection with influenza A(H1N1)pdm09 was ubiquitous on the French territory, whatever the socio-demographic factors, and the Flu virus transmission can probably conditioned by the environmental and hygienic conditions in household.

Submitted by Heitor Rapela Medeiros (heitor.rapela@ufpe.br) on 2015-03-06T13:45:50Z No. of bitstreams: 2 TESE MARIA JOSE COUTO OLIVEIRA.pdf: 3444115 bytes, checksum: 23f3f8857155eb1b13b9abe031e11e3b (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-06T13:45:50Z (GMT). No. of bitstreams: 2 TESE MARIA JOSE COUTO OLIVEIRA.pdf: 3444115 bytes, checksum: 23f3f8857155eb1b13b9abe031e11e3b (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-02-29 / Durante a pandemia (2009-2010) com o vírus influenza A(H1N1)pdm09 foi recomendado o tratamento com o oseltamivir ou zanamivir. Com o aumento da detecção de vírus de Influenza A (H1N1) sazonal resistente ao oseltamivir houve a preocupação de que o mesmo ocorresse com o novo vírus pandêmico. Nesta pandemia, muitos pacientes com suspeita de infecção pelo vírus A(H1N1)pdm09 tiveram o teste negativo para influenza A, ficando sem uma definição do agente etiológico. Testes moleculares podem detectar a presença de mutações relacionadas à resistência ao oseltamivir, à virulência e antigenicidade do vírus, assim como podem definir o diagnóstico etiológico por vírus respiratórios. Para esclarecer essas questões dois estudos foram realizados. O primeiro foi a “Caracterização genética dos vírus influenza A (H1N1)pdm09 detectados no Estado de Pernambuco, Brasil, no período de maio de 2009 a maio de 2010”, com o objetivo de verificar a resistência desse vírus ao oseltamivir e também avaliar a diversidade genética dos vírus circulantes. Foram analisadas 118 amostras do vírus A(H1N1)pdm09 através de pirosequenciamento, precedida da transcrição reversa e reação em cadeia da polimerase em tempo real (rRT-PCR) para amplificação do H1N1pdm-N1 fragmento C e posterior detecção da mutação H274Y, utilizando o equipamento PyroMark Q-96 ID no modo SNP (single nucleotide polymorphism). Foram sequenciados os genes da hemaglutinina de 31 amostras, pela técnica de Sanger, de acordo com o Protocolo do CDC para Influenza. Foi utilizado o kit “Big Dye® terminator Cycle Sequencing” (Applied Biosystem) e o produto submetido ao método de precipitação X-terminator. A mutação H274Y não foi observada, indicativo de que os vírus sequenciados eram sensíveis ao oseltamivir. As 31 amostras sequenciadas mostraram-se intimamente relacionadas com a cepa de referência A/California/7/2009(H1N1), entretanto, foram detectados 14 tipos de mutações, porém sem implicação no aumento da virulência. O segundo estudo realizado: ”Aspectos epidemiológicos e virológicos da infecção por Influenza A(H1N1)pdm09 e frequência de outros vírus respiratórios no Estado de Pernambuco, Brasil: 2009 – 2010” teve como objetivo analisar a pandemia de influenza no estado e identificar os vírus respiratórios responsáveis pelo quadro clínico que levou à hipótese diagnóstica da influenza pandêmica. Foram analisados espécimes de 705 casos para detecção do vírus da influenza A, utilizando-se a PCR em tempo real, sistema TaqMan, de acordo com o Centers for Disease Control and Prevention / Atlanta, das quais, 26,3% (186/705) foram positivas para o vírus A(H1N1)pdm09 e 2,3% (16/705) positivas para influenza A sazonal. Para detecção de outros vírus respiratórios foram analisadas 146 amostras negativas para o vírus A (H1N1)pdm09 por RT-PCR multiplex, com o kit “FTD Respiratory21 PLUS”. Entre as amostras negativas para o vírus A(H1N1)pdm09, 36,5% (53/146) foram positivas para outros vírus respiratórios, com três casos de infecção viral múltipla. Foram detectados: rhinovírus (41%), coronavírus 43 (14,3%), metapneumovírus humano (14,3%), bocavírus (7,1%), vírus respiratório sincicial (5,3%), influenza B (3,6%), parainfluenza 2 (3,6%), parainfluenza 3 (3,6%), adenovírus (1,8%), coronavírus HKU (1,8%), enterovírus (1,8%) e parainfluenza 1 (1,8%). Estes resultados mostram a circulação, além da Influenza A(H1N1)pdm09, de outros vírus respiratórios no estado em 2009-2010; evidenciam a necessidade da análise laboratorial dos casos suspeitos de influenza e a importância do monitoramento laboratorial das infecções respiratórias, uma vez que o diagnóstico etiológico baseado apenas em critérios clínicos nem sempre é acurado.

O vírus influenza A (IAV) é um agente zoonótico de grande relevância tanto para saúde humana como animal. A influenza suína teve seu primeiro reconhecimento clínico em 1918, em suínos do Meio Oeste dos EUA, coincidindo com a pandemia de influenza em humanos. Desde então, o IAV permanece como um importante patógeno para a indústria suinícola em todo o mundo. A grande variabilidade genética destes vírus é causada por dois principais mecanismos genéticos: mutações pontuais e recombinações genéticas. A influenza é endêmica em muitos países e a emergências de recombinantes tem desafiado o controle e o diagnóstico desta enfermidade. No Brasil, a infecção pelo IAV em suínos (swIAV) não está bem caracterizada; poucos relatos evidenciam a prevalência deste agente antes do ano de 2009, especialmente no Estado do Rio Grande do Sul, que alberga um dos maiores rebanhos de suínos do Brasil. Em vista disso, este trabalho teve como objetivo investigar ocorrência de swIAV em alguns rebanhos suínos comerciais do Estado do Rio Grande do Sul, Brasil, no período de 2013-2014, e determinar os tipos e subtipos de vírus circulantes naquelas propriedades. O primeiro capítulo deste estudo reporta os aspectos clínicos, patológicos e virológicos da ocorrência de influenza suína e co-infecções identificadas em seis propriedades suinícolas selecionadas na região do Vale do Taquari. Neste estudo foram analisados suabes nasais coletados de 66 animais e 6 amostras de tecido pulmonar de suínos com sinais de infecção respiratória. A detecção viral foi feita através de uma PCR de triagem e confirmada através do isolamento viral em células MDCK. A identificação dos subtipos virais foi feita através de uma PCR em Tempo Real (rRT-PCR) para o subtipo A(H1N1)pdm09 ou através de uma PCR multiplex (RT-PCR) para outros subtipos de swIAV. A detecção de agentes bacterianos foi realizada apenas nas amostras de tecido pulmonar, através da pesquisa de genomas bacterianos por PCR. O subtipo A(H1N1)pdm09 foi identificado em 4/6 granjas e o subtipo H1N2 em 2/6 granjas. Além disso, agentes envolvidos no complexo respiratório dos suínos foram identificados em todas as granjas; Pasteurella multocida foi identificada em 5/6 granjas e Mycoplasma hyopneumoniae em 3/6 granjas. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) e PCV2 (1/6) também foram detectados. O segundo capítulo deste estudo teve como objetivo o sequenciamento do genoma completo de um novo recombinante H1N2 de origem humana, detectado em suínos. O genoma completo foi gerado através de uma RT-PCR. Os produtos foram purificados e submetidos ao sequenciamento utilizando a plataforma MiSeq (illumina). A análise filogenética revelou que as sequencias dos genes HA e NA correspondem a genes de IAV de origem humana, enquanto que as sequencias dos genes que codificam as proteínas internas do vírus (PB1, PB2, PA, NP, M e NS) correspondem a genes de amostras do vírus A(H1N1)pdm09. O terceiro capítulo reporta o sequenciamento completo dos genomas de 8 amostras de vírus influenza identificados nas populações de suínos amostradas. Foram identificados dois subtipos virais de origem humana (H1N2 e H3N2), além do vírus A(H1N1)pdm09. Os subtipos de origem humana possuem os genes HA e NA similares a vírus sazonais de humanos e os genes internos são estreitamente relacionados com o vírus A(H1N1)pdm09. / Influenza A virus (IAV) is a zoonotic agent of great relevance to human and animal health. Swine influenza was first recognized clinically in pigs in the Midwestern U.S., in 1918, coinciding with the human influenza pandemic. Since that time swine influenza has remained of importance to the swine industry throughout the world. The great genetic variability of influenza viruses is caused by two main genetic mechanisms: point mutations (antigenic drift) and gene reassortment (antigenic shift). Influenza is endemic in pigs in many countries and the emergence of new viruses has been challenging its control and diagnostics. Influenza virus (swIAV) infection in Brazilian swine population is not well characterized, and little evidence existed of swIAV circulation before 2009, especially in Rio Grande do Sul State, which hosts one of the largest swine populations in Brazil. Thus, this study aimed to investigate the occurrence of IAV in commercial swine herds in the state of Rio Grande do Sul, Brazil, between 2013-2014 and to know the types and subtypes of swine influenza viruses that are circulating in these herd. The first chapter of this study reports the clinical, pathological and virological aspects of the occurrence of swine influenza and related co-infections in six pig properties of the Taquari Valley region. In this study were analyzed nasal swabs collected from 66 animals and six lung tissue samples from pigs showing clinical signs of respiratory disease. IAV detection was performed by PCR screening and confirmed by virus isolation in MDCK cells and hemagglutination (HA). Influenza A subtyping was performed by real-time reverse transcription PCR (rRT-PCR) to detect the 2009 H1N1pandemic A(H1N1)pdm09; other swIAV subtypes were identifieded by multiplex RT-PCR. Bacterial infections were identified through detection of bacterial genomes by PCR, only in lung samples. Influenza A was detected by screening PCR in 46/66 swab samples and from 5/6 lungs. Virus was recovered from pigs of the six herds. Subtype A(H1N1)pdm09 was detected in 4/6 herds and H1N2 in the other 2/6 herds. In lung tissues, further agents involved in porcine respiratory disease complex were detected in all cases; Pasteurella multocida was identified in 5/6 samples and Mycoplasma hyopneumoniae in 3/6. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. The aim of the second chapter was to sequence the whole-genome of a novel human-like H1N2 swine influenza virus. Wholegenome sequences were generated by RT-PCR. Amplicons were purified followed by sequencing in the MiSeq sequencing platform (Illumina). Phylogenetic analyses revealed that the HA and NA genes clustered with influenza viruses of human lineage, whereas the internal genes (PB1, PB2, PA, NP, M and NS) clustered with the A(H1N1)pdm09. The third chapter reports the genetic sequencing of the full genomes of eight swine influenza viruses circulating in the sampled pig population. Two swine human-like subtypes (H1N2 and H3N2) and the A(H1N1)pdm09 virus were identified. The human-like subtypes have the HA and NA genes similar to the human seasonal strains and the internal genes are closely related to the virus A(H1N1)pdm09.

Despite the benefits of applying methods of geographic information science (GIScience), the use of such methods in health service planning and provision remains greatly underutilized. Spread of epidemic diseases is a constant threat to mankind and the globalization of the world increases the risk for global attacks from multi-resistant bacteria or deadly virus strains. Therefore, research is needed to better understand how GIScience could be used in epidemiologic analyses and other health applications. This thesis is divided into two parts; one for epidemiologic analyses and one for neighbourhood studies. The overall objective of the epidemiologic part of this research is to understand more about the spatial spread of past pandemics and to find out if there are any common patterns. This overall objective is divided into four specific research objectives; 1) to describe the spatial spread of the Russian Influenza in Sweden, 2) to create models of propagation of the Black Death in Sweden, 3) to establish spatiotemporal characteristics common to past pandemics in Sweden and 4) to visualize the spatiotemporal occurrence of salmonella among animal herds in Sweden. This thesis also discusses some other aspects of health related to place. Are differences in neighbourhood deprivation related to the amount of presence of goods and services? Is the way cities are planned affecting the behaviour within the local population regarding spontaneous walking and physical activity? The specific research objectives for this part are to define how deprivation is related to presence of goods and services in Sweden and to create walkability indices over the city of Stockholm including a quality test of these indices. Case data reported by physicians were used for the epidemiologic studies. The pandemics discussed covered the entire world, but our data is from Sweden only and as regards the Black Death there was no case data at all. The data for the goods and services analyses are from all of Sweden, whereas the walkability indices are based on data from the city of Stockholm. Various methods have been used to clean, structure and geocode the data, including hand written reports on case data, maps of poor geometric quality, information from databases on climate, demography, diseases, goods and services, income data and more, to make this data feasible for spatial analysis, modeling and visualization. Network analysis was used to model food transports in the 14th century as well as walking in the city of Stockholm today. Proximity analysis was used to assess the spatio-temporal spread of the Russian Influenza. The impact of climatological factors on the propagation of the Asian Influenza was analyzed and geographically weighted mean (GWM) calculations were used to discover common characteristics in the spatio-temporal spread of three past pandemics. Among the results generated in the epidemiologic study the following should be noted in particular; the local peaking periods of the Asian Influenza were preceded by falling temperature, the total peaking period for the three pandemics (Russian, Asian and A(H1N1)pdm09) was approximately 10 weeks and their weekly GWM followed a path from southwest to northeast (opposite direction for the A(H1N1)pdm09). From the neighborhood studies one can note that compared to the results measured and reported by tested individuals there is a positive (small but significant) association between neighborhood walkability and physical activity outcomes. The main contribution of this work is that it gives epidemiologists and public health specialists new ideas, not only on how to formulate, model, analyze and visualize different health related research questions but also ideas on how new procedures could be implemented in their daily work. Once the data reporting is organized in a suitable manner there is a multitude of options on how to present important and critical information to officials and policy makers. / <p>QC 20140313</p>

O vírus influenza A (IAV) é um agente zoonótico de grande relevância tanto para saúde humana como animal. A influenza suína teve seu primeiro reconhecimento clínico em 1918, em suínos do Meio Oeste dos EUA, coincidindo com a pandemia de influenza em humanos. Desde então, o IAV permanece como um importante patógeno para a indústria suinícola em todo o mundo. A grande variabilidade genética destes vírus é causada por dois principais mecanismos genéticos: mutações pontuais e recombinações genéticas. A influenza é endêmica em muitos países e a emergências de recombinantes tem desafiado o controle e o diagnóstico desta enfermidade. No Brasil, a infecção pelo IAV em suínos (swIAV) não está bem caracterizada; poucos relatos evidenciam a prevalência deste agente antes do ano de 2009, especialmente no Estado do Rio Grande do Sul, que alberga um dos maiores rebanhos de suínos do Brasil. Em vista disso, este trabalho teve como objetivo investigar ocorrência de swIAV em alguns rebanhos suínos comerciais do Estado do Rio Grande do Sul, Brasil, no período de 2013-2014, e determinar os tipos e subtipos de vírus circulantes naquelas propriedades. O primeiro capítulo deste estudo reporta os aspectos clínicos, patológicos e virológicos da ocorrência de influenza suína e co-infecções identificadas em seis propriedades suinícolas selecionadas na região do Vale do Taquari. Neste estudo foram analisados suabes nasais coletados de 66 animais e 6 amostras de tecido pulmonar de suínos com sinais de infecção respiratória. A detecção viral foi feita através de uma PCR de triagem e confirmada através do isolamento viral em células MDCK. A identificação dos subtipos virais foi feita através de uma PCR em Tempo Real (rRT-PCR) para o subtipo A(H1N1)pdm09 ou através de uma PCR multiplex (RT-PCR) para outros subtipos de swIAV. A detecção de agentes bacterianos foi realizada apenas nas amostras de tecido pulmonar, através da pesquisa de genomas bacterianos por PCR. O subtipo A(H1N1)pdm09 foi identificado em 4/6 granjas e o subtipo H1N2 em 2/6 granjas. Além disso, agentes envolvidos no complexo respiratório dos suínos foram identificados em todas as granjas; Pasteurella multocida foi identificada em 5/6 granjas e Mycoplasma hyopneumoniae em 3/6 granjas. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) e PCV2 (1/6) também foram detectados. O segundo capítulo deste estudo teve como objetivo o sequenciamento do genoma completo de um novo recombinante H1N2 de origem humana, detectado em suínos. O genoma completo foi gerado através de uma RT-PCR. Os produtos foram purificados e submetidos ao sequenciamento utilizando a plataforma MiSeq (illumina). A análise filogenética revelou que as sequencias dos genes HA e NA correspondem a genes de IAV de origem humana, enquanto que as sequencias dos genes que codificam as proteínas internas do vírus (PB1, PB2, PA, NP, M e NS) correspondem a genes de amostras do vírus A(H1N1)pdm09. O terceiro capítulo reporta o sequenciamento completo dos genomas de 8 amostras de vírus influenza identificados nas populações de suínos amostradas. Foram identificados dois subtipos virais de origem humana (H1N2 e H3N2), além do vírus A(H1N1)pdm09. Os subtipos de origem humana possuem os genes HA e NA similares a vírus sazonais de humanos e os genes internos são estreitamente relacionados com o vírus A(H1N1)pdm09. / Influenza A virus (IAV) is a zoonotic agent of great relevance to human and animal health. Swine influenza was first recognized clinically in pigs in the Midwestern U.S., in 1918, coinciding with the human influenza pandemic. Since that time swine influenza has remained of importance to the swine industry throughout the world. The great genetic variability of influenza viruses is caused by two main genetic mechanisms: point mutations (antigenic drift) and gene reassortment (antigenic shift). Influenza is endemic in pigs in many countries and the emergence of new viruses has been challenging its control and diagnostics. Influenza virus (swIAV) infection in Brazilian swine population is not well characterized, and little evidence existed of swIAV circulation before 2009, especially in Rio Grande do Sul State, which hosts one of the largest swine populations in Brazil. Thus, this study aimed to investigate the occurrence of IAV in commercial swine herds in the state of Rio Grande do Sul, Brazil, between 2013-2014 and to know the types and subtypes of swine influenza viruses that are circulating in these herd. The first chapter of this study reports the clinical, pathological and virological aspects of the occurrence of swine influenza and related co-infections in six pig properties of the Taquari Valley region. In this study were analyzed nasal swabs collected from 66 animals and six lung tissue samples from pigs showing clinical signs of respiratory disease. IAV detection was performed by PCR screening and confirmed by virus isolation in MDCK cells and hemagglutination (HA). Influenza A subtyping was performed by real-time reverse transcription PCR (rRT-PCR) to detect the 2009 H1N1pandemic A(H1N1)pdm09; other swIAV subtypes were identifieded by multiplex RT-PCR. Bacterial infections were identified through detection of bacterial genomes by PCR, only in lung samples. Influenza A was detected by screening PCR in 46/66 swab samples and from 5/6 lungs. Virus was recovered from pigs of the six herds. Subtype A(H1N1)pdm09 was detected in 4/6 herds and H1N2 in the other 2/6 herds. In lung tissues, further agents involved in porcine respiratory disease complex were detected in all cases; Pasteurella multocida was identified in 5/6 samples and Mycoplasma hyopneumoniae in 3/6. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. The aim of the second chapter was to sequence the whole-genome of a novel human-like H1N2 swine influenza virus. Wholegenome sequences were generated by RT-PCR. Amplicons were purified followed by sequencing in the MiSeq sequencing platform (Illumina). Phylogenetic analyses revealed that the HA and NA genes clustered with influenza viruses of human lineage, whereas the internal genes (PB1, PB2, PA, NP, M and NS) clustered with the A(H1N1)pdm09. The third chapter reports the genetic sequencing of the full genomes of eight swine influenza viruses circulating in the sampled pig population. Two swine human-like subtypes (H1N2 and H3N2) and the A(H1N1)pdm09 virus were identified. The human-like subtypes have the HA and NA genes similar to the human seasonal strains and the internal genes are closely related to the virus A(H1N1)pdm09.

O vírus influenza A (IAV) é um agente zoonótico de grande relevância tanto para saúde humana como animal. A influenza suína teve seu primeiro reconhecimento clínico em 1918, em suínos do Meio Oeste dos EUA, coincidindo com a pandemia de influenza em humanos. Desde então, o IAV permanece como um importante patógeno para a indústria suinícola em todo o mundo. A grande variabilidade genética destes vírus é causada por dois principais mecanismos genéticos: mutações pontuais e recombinações genéticas. A influenza é endêmica em muitos países e a emergências de recombinantes tem desafiado o controle e o diagnóstico desta enfermidade. No Brasil, a infecção pelo IAV em suínos (swIAV) não está bem caracterizada; poucos relatos evidenciam a prevalência deste agente antes do ano de 2009, especialmente no Estado do Rio Grande do Sul, que alberga um dos maiores rebanhos de suínos do Brasil. Em vista disso, este trabalho teve como objetivo investigar ocorrência de swIAV em alguns rebanhos suínos comerciais do Estado do Rio Grande do Sul, Brasil, no período de 2013-2014, e determinar os tipos e subtipos de vírus circulantes naquelas propriedades. O primeiro capítulo deste estudo reporta os aspectos clínicos, patológicos e virológicos da ocorrência de influenza suína e co-infecções identificadas em seis propriedades suinícolas selecionadas na região do Vale do Taquari. Neste estudo foram analisados suabes nasais coletados de 66 animais e 6 amostras de tecido pulmonar de suínos com sinais de infecção respiratória. A detecção viral foi feita através de uma PCR de triagem e confirmada através do isolamento viral em células MDCK. A identificação dos subtipos virais foi feita através de uma PCR em Tempo Real (rRT-PCR) para o subtipo A(H1N1)pdm09 ou através de uma PCR multiplex (RT-PCR) para outros subtipos de swIAV. A detecção de agentes bacterianos foi realizada apenas nas amostras de tecido pulmonar, através da pesquisa de genomas bacterianos por PCR. O subtipo A(H1N1)pdm09 foi identificado em 4/6 granjas e o subtipo H1N2 em 2/6 granjas. Além disso, agentes envolvidos no complexo respiratório dos suínos foram identificados em todas as granjas; Pasteurella multocida foi identificada em 5/6 granjas e Mycoplasma hyopneumoniae em 3/6 granjas. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) e PCV2 (1/6) também foram detectados. O segundo capítulo deste estudo teve como objetivo o sequenciamento do genoma completo de um novo recombinante H1N2 de origem humana, detectado em suínos. O genoma completo foi gerado através de uma RT-PCR. Os produtos foram purificados e submetidos ao sequenciamento utilizando a plataforma MiSeq (illumina). A análise filogenética revelou que as sequencias dos genes HA e NA correspondem a genes de IAV de origem humana, enquanto que as sequencias dos genes que codificam as proteínas internas do vírus (PB1, PB2, PA, NP, M e NS) correspondem a genes de amostras do vírus A(H1N1)pdm09. O terceiro capítulo reporta o sequenciamento completo dos genomas de 8 amostras de vírus influenza identificados nas populações de suínos amostradas. Foram identificados dois subtipos virais de origem humana (H1N2 e H3N2), além do vírus A(H1N1)pdm09. Os subtipos de origem humana possuem os genes HA e NA similares a vírus sazonais de humanos e os genes internos são estreitamente relacionados com o vírus A(H1N1)pdm09. / Influenza A virus (IAV) is a zoonotic agent of great relevance to human and animal health. Swine influenza was first recognized clinically in pigs in the Midwestern U.S., in 1918, coinciding with the human influenza pandemic. Since that time swine influenza has remained of importance to the swine industry throughout the world. The great genetic variability of influenza viruses is caused by two main genetic mechanisms: point mutations (antigenic drift) and gene reassortment (antigenic shift). Influenza is endemic in pigs in many countries and the emergence of new viruses has been challenging its control and diagnostics. Influenza virus (swIAV) infection in Brazilian swine population is not well characterized, and little evidence existed of swIAV circulation before 2009, especially in Rio Grande do Sul State, which hosts one of the largest swine populations in Brazil. Thus, this study aimed to investigate the occurrence of IAV in commercial swine herds in the state of Rio Grande do Sul, Brazil, between 2013-2014 and to know the types and subtypes of swine influenza viruses that are circulating in these herd. The first chapter of this study reports the clinical, pathological and virological aspects of the occurrence of swine influenza and related co-infections in six pig properties of the Taquari Valley region. In this study were analyzed nasal swabs collected from 66 animals and six lung tissue samples from pigs showing clinical signs of respiratory disease. IAV detection was performed by PCR screening and confirmed by virus isolation in MDCK cells and hemagglutination (HA). Influenza A subtyping was performed by real-time reverse transcription PCR (rRT-PCR) to detect the 2009 H1N1pandemic A(H1N1)pdm09; other swIAV subtypes were identifieded by multiplex RT-PCR. Bacterial infections were identified through detection of bacterial genomes by PCR, only in lung samples. Influenza A was detected by screening PCR in 46/66 swab samples and from 5/6 lungs. Virus was recovered from pigs of the six herds. Subtype A(H1N1)pdm09 was detected in 4/6 herds and H1N2 in the other 2/6 herds. In lung tissues, further agents involved in porcine respiratory disease complex were detected in all cases; Pasteurella multocida was identified in 5/6 samples and Mycoplasma hyopneumoniae in 3/6. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. The aim of the second chapter was to sequence the whole-genome of a novel human-like H1N2 swine influenza virus. Wholegenome sequences were generated by RT-PCR. Amplicons were purified followed by sequencing in the MiSeq sequencing platform (Illumina). Phylogenetic analyses revealed that the HA and NA genes clustered with influenza viruses of human lineage, whereas the internal genes (PB1, PB2, PA, NP, M and NS) clustered with the A(H1N1)pdm09. The third chapter reports the genetic sequencing of the full genomes of eight swine influenza viruses circulating in the sampled pig population. Two swine human-like subtypes (H1N2 and H3N2) and the A(H1N1)pdm09 virus were identified. The human-like subtypes have the HA and NA genes similar to the human seasonal strains and the internal genes are closely related to the virus A(H1N1)pdm09.

Introduction: Epidemiological studies have indicated that 5-38% of influenza like illnesses (ILI) develop into severe disease due to, among others, factors such as; underlying chronic diseases, age, pregnancy, and viral mutations. There are suggestions that dual or multiple virus infections may affect disease severity. This study investigated the association between co-infection between influenza A viruses and other respiratory viruses and disease severity. Methodology: Datum for samples from North West England tested between January 2007 and June 2012 was analysed for patterns of co-infection between influenza A viruses and ten respiratory viruses. Risk of hospitalization to a general ward ICU or death in single versus mixed infections was assessed using multiple logistic regression models. Results: One or more viruses were identified in 37.8% (11,715/30,975) of samples, of which 10.4% (1,214) were mixed infections and 89.6% (10,501) were single infections. Among patients with influenza A(H1N1)pdm09, co-infections occurred in 4.7% (137⁄2,879) vs. 6.5% (59⁄902) in those with seasonal influenza A virus infection. In general, patients with mixed respiratory virus infections had a higher risk of admission to a general ward (OR: 1.43, 95% CI: 1.2 – 1.7, p = <0.0001) than those with a single infection. Co-infection between seasonal influenza A viruses and influenza B virus was associated with a significant increase in the risk of admission to ICU/ death (OR: 22.0, 95% CI: 2.21 – 219.8 p = 0.008). RSV/seasonal influenza A viruses co-infection also associated with increased risk but this was not statistically significant. For the pandemic influenza A(H1N1)pdm09 virus, RSV and AdV co-infection increased risk of hospitalization to a general ward, whereas Flu B increased risk of admission to ICU/ death, but none of these were statistically significant. Considering only single infections, RSV and hPIV1-3 increased risk of admission to a general ward (OR: 1.49, 95% CI: 1.28 – 1.73, p = <0.0001 and OR: 1.34, 95% CI: 1.003 – 1.8, p = 0.05) and admission to ICU/ death (OR: 1.5, 95% CI: 1.20 – 2.0, p = <0.0001 and OR: 1.60, 95% CI: 1.02 – 2.40, p = 0.04). Conclusion: Co-infection is a significant predictor of disease outcome; there is insufficient public health data on this subject as not all samples sent for investigation of respiratory virus infection are tested for all respiratory viruses. Integration of testing for respiratory viruses’ co-infections into routine clinical practice and R&D on integrated drugs and vaccines for influenza A&B, RSV, and AdV, and development of multi-target diagnostic tests is encouraged.