The highly pathogenic Avian Influenza subtype H5N1 can lead to 100 % mortality in chickens. The main issue in prevention of H5N1 is the development of efficient poultry vaccines. Influenza haemagglutinin (HA) derived recombinant polypeptides would not elicit an immune response against internal viral proteins. Thus HA polypeptide use facilitates differentiation between infected and vaccinated animals (DIVA). Serological tests using recombinant immune-dominant proteins devoid of non-specific moieties present in whole cell preparations might have higher sensitivity and specificity. In the present study, four non-overlapping sequences of different functional domains of influenza A virus subtype H5 virus (A / Thailand / 1 (Kan-1) / 2004) designated P1, P2, P5 and rHA1 were cloned and expressed in Pichia pastoris for vaccination and diagnosis purposes. The four polypeptides were expressed successfully in P. pastoris using peptone methanol (1 % (w/v) yeast extract, 2 % (w/v) peptone, 2 % (v/v) methanol). P1, P2 and rHA1 polypeptides were purified using nickel affinity chromatography, whereas, P5 was purified using lectin affinity chromatography. Correct expression was analysed by SDS-PAGE and western blot, glycosylation analysis and MALDI-TOF.The immune responses of P1, P2 and rHA1 polypeptides were assessed in BALB/C mice. To enhance antibody response, recombinant polypeptides were mixed with the Gerbu adjuvant and injected subcutaneously. Vaccination of mice induced high subtype specific antibody titres in mice as analysed by Elisa (using recombinant antigens or whole H5N1 antigen) and Immunofluorescence assay (IFA) performed on Vero cells infected with H5 (A / Thailand / 1 (Kan-1) / 2004). The immunogenicity of P1, P2, P5 and rHA1 polypeptides was determined in commercial layer chickens. Results showed that P1, P2 and rHA1 polypeptides induced high subtype specific antibody titres in chickens as analysed by Elisa (using recombinant antigens or whole H5N1 antigen), IFA (performed on Vero cells infected with H5N1 A / Thailand / 1 (Kan-1) / 2004) and microneutralization test (µNT). However, P5 polypeptide was not immunogenic in chickens. Neutralizing antibodies could be detected in chicken sera immunized with P1, P2 and rHA1 polypeptides as analyzed with microneutralization test. IgY was analysed in egg yolk of chickens immunized with recombinant polypeptides. The IgY of chicken immunized with P1 and rHA1, transferred to the egg yolk was proportional to maternal serum IgY. However, IgY could not be detected in egg yolk of chickens immunized with P2 and P5 recombinant polypeptides. The more immunogenic polypeptides P1 and rHA1 were used in an recombinant Elisa (rElisa) for detection of influenza A subtype H5 in chickens and duck sera.The optimal antigen for the concentrations of rHA1, P1 was 50 ng / well, 50 ng / well. Analysis of 25 positive sera and 25 negative sera to H5 antibodies revealed that, the sensitivity of Western blot, whole H5N1 Elisa, agar gel immunodiffusion test (AGID), P1-Elisa and rHA1-Elisa was 100 %, 100 %, 52 %, 80 % and 100 %, respectively, while the specificity was 100 %, 100 %, 100 %, 72 %, and 100 %, respectively. Moreover, duck sera, with haemagglutination inhibiting titer ranged from 4 - 8 log2, were tested positive by rHA1 Elisa compared with negative duck sera. Further analysis of 179 serum samples with rHA1-Elisa in comparison with haemagglutination inhibition (HI) and commercial Elisa proved to be highly sensitive and specific. The agreement ratio between rElisa and HI was 84.9 % and between commercial Elisa (Flock check) and HI was 76.5 %. In conclusion, P. pastoris may allow development of an effective recombinant influenza vaccine based on truncated sequences of HA that might provide broader protection against H5 influenza viruses. The possibilities to use rHA1, P1 and P5 recombinant polypeptides as a vaccine against H5 influenza should be further studied. Also our study demonstrates the potential utility of recombinant Elisa as a tool for improvement of serological diagnosis of influenza A subtype H5 in chickens and ducks. / Die hochpathogene aviäre Influenza des Subtyps H5N1 erreicht beim Ausbruch von Infektionen in Nutzgeflügelbeständen Mortalitätsraten von bis zu 100 %. Effektive und kostengünstige Impfstoffe werden benötigt, die möglichst auch eine Differenzierung zwischen geimpften Tieren und mit Wild-Virus infizierten Tieren zulassen. In diesem Zusammenhang könnten Peptid-Vakzine eine mögliche Alternative zu den herkömmlichen Impfstoffen darstellen, bei denen unter Verwendung des Vollvirus Antikörper gegen mehrere Virusproteine induziert werden. Außerdem, könnten rekombinante Antigene in serologischen Tests zur Diagnose von H5 Virus in Nutzgeflügel eingesetzt werden. Von dem Einsatz spezifischer rekombinanter Antigene ist eine Verbesserung der Serodiagnostik zu erwarten. In dieser Arbeit, wurden vier verkürzte Sequenzen des Hämagglutinins (P1, P2, P5 und rHA1) von Subtyp H5 (A / Thailand / 1 (Kan-1) / 2004) rekombinant in Pichia Pastoris exprimiert. Dazu erfolgten zunächst eine Klonierung in der Expressionsvektor pAOX und die Transformation von Pichia Pastoris. Die Expression wurde durch Methanol induziert. Der Nachweis der rekombinanten Fusionspeptiden mit C-terminalen Histidin-Tag erfolgte durch SDS-PAGE, Western Blot, Glycolysierungsanalyse, und MALDI-TOF. Der Histidin-Tag ermöglichte die Reinigung von P1, P2 und rHA1 mit Metall-Affinitätschromatographie. Polypeptid P5 hingegen wurde mittels Lectin-Affinitäts- chromatographie gereinigt. Balb/c Mäuse wurden mit Polypeptid P1, P2 bzw. rHA1, versetzt mit Gerbu Adjuvans, immunisiert. Zur Untersuchung der Immunantwort wurden die murinen Seren mittels Elisa (unter Verwendung rekombinanter Antigene oder Voll-H5N1 Antigen) sowie IFA (durchgeführt in Vero- Zellen infiziert mit A / Thailand / 1 (Kan-1) / 2004) analysiert. Dabei wurde die präferentielle Induktion von H5-spezifischen Antikörpern detektiert. Die Immunogenität der P1, P2, P5 und rHA1-Polypeptide wurde in kommerziellen Legehennen bestimmt. Seren wurden mit ELISA, IFA, und Mikroneutralizationstest (μNT) analysiert. Die ELISA-Ergebnisse zeigten, dass die Polypeptide P1, P2 und rHA1 hohe Subtyp-spezifische Antikörpertiter in Hühnern induzierten. Im µNT konnte nur ein niedriger neutralisierender Antikörpertiter nachgewiesen werden. Das P5- Polypeptid ist bei Hühnern nicht immunogen. Im Eigelb von Hühnern, die mit den rekombinanten Polypeptiden P1 und rHA1 immunisiert wurden, konnten H5-spezifische IgY Antikörper detektiert werden. Hühner, die mit P2 und P5 immunisiert wurden, zeigten keine IgY im Eigelb. Die rekombinanten Antigene P1 und rHA1 wurden im ELISA auf ihre potenzielle Eignung für die Serodiagnostik untersucht. Die optimale Antigenkonzentration war 50 ng / well. Die serologische Analyse von 25 positiven und 25 negativen Seren auf Antikörper gegen H5 zeigte, dass Sensitivität und Spezifität von Western Blot, Voll-H5N1 ELISA und rHA1-ELISA bei jeweils 100 % lagen. Bei Agargel- Immunodiffusiontest (AGID) lagen Sensitivität und Spezifität bei 52 % und 100 %, während im P1-Elisa lediglich eine Sensitivität von 80 % und eine Spezifität von 72 % erreicht wurden. Somit eignet sich rHA1 für die Anwendung in der Serodiagnostik. Bei der serologischen Untersuchung von 175 Hühnerseren wurde eine Überbestimmung zwischen rHA1-ELISA und Hämagglutinationshemmungstest (HAI) 84.9 % festgestellt, während diese zwischen dem kommerziellen ELISA (Flock Check) und HAI 76.5 % betrug. Die Ergebnisse zeigten, dass das Expressionssystem P. pastoris als Produktionssystem rekombinanter Antigene für die Serodiagnostik von H5 Influenza geeignet ist. Challenge-Versuche sind nötig, um die Eignung von rekombinanten Antigenen als möglichen Impfstoff gegen H5 Influenza zu untersuchen.

Orientador: Hélio José Montassier / Banca: Liana Brentano / Banca: Manoel Victor Franco Lemos / Resumo: A técnica de imunocaptura associada com a reação de transcrição reversa e reação em cadeia da polimerase (IC-RT-PCR) executadas tanto pelos procedimentos convencional como em tempo real foram testadas para a detecção rápida do gene da glicoproteína de Matriz (M) do vírus de influenza aviária (AIV) em amostras de líquido cório-alantóide (LCA) e em suabes traqueais e cloacais. O presente trabalho teve como objetivo desenvolver e otimizar a técnica de IC-RT-PCR para o diagnóstico do vírus da Influenza aviária. Os resultados obtidos foram comparados com um sistema empregando "beads" magnéticas em microplacas (AMBION), que é o método padrão de extração de RNA usado no laboratório de referência para diagnóstico de influenza aviária, o National Veterinary Services Laboratory - Ames, EUA (USDA), acrescido ainda de outros métodos de extração tradicionalmente usados nos laboratórios de referência para AIV, como os procedimentos com o uso do solvente orgânico Trizol® (Invitrogen) e com um sistema robotizado e que utiliza "beads" magnéticas (MagNA Pure - ROCHE). A técnica de IC-RT-PCR em tempo real neste estudo detectou a estirpe H2N2 do AIV, sem que nenhum outro dos RNA-vírus heterólogos testados fossem detectados (vírus das doença de Gumboro, de Newcastle e da bronquite infecciosa aviária). Os limites de detecção do IC-RTPCR foram iguais aos obtidos na técnica de extração com o kit da AMBION e menores do que aqueles que foram observados para os métodos de extração com Trizol® (Invitrogen) e com o MagNA Pure. O IC-RT-PCR demonstrou ser um sistema de diagnóstico capaz de conciliar simplicidade operacional e um menor custo com sensibilidade e especificidade analíticas iguais às do procedimento padrão atualmente adotado, podendo ser inclusive por laboratórios dotados de uma infra-estrutura mais simples / Abstract: The polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), including real-time RT-PCR have been used for the rapid detection of Matrix glycoprotein gene (M gene) Avian influenza virus (AIV). Despite the availability of various RNA extraction methods for using in RT-PCR, isolation and detection of viral RNA are still difficult due to the unstable nature of viral RNA molecules and the presence of PCR inhibitory substances. In this study, a simple method using immune-capture (IC) to recover viral RNA from H2 AIV samples was developed and compared to one standard and two others reference methods used for viral RNA extraction, such as Ambion MagMAXTM kit and Trizol® (Invitrogen) and Magnapure kit (Roche), respectively, with subsequent analysis by real-time RT-PCR. The real-time IC-RT-PCR developed in was able to detect specifically H2N2 AIV strain, without detecting non-related avian RNA-virus pathogens, such as Newcastle disease virus, avian infectious bronchitis virus and Gumboro disease virus. Comparable detection limits were found for IC and the standard RNA extraction method using Ambion MagMAXTM kit, either for the detection of AIV in allantoic fluid suspension or in seeded tracheal and cloacal swab samples by conventional or real time RT-PCR techniques. These methods were less sensitive than Trizol® (Invitrogen) and Magnapure kit (Roche) procedures. Thus, IC was rapid and as sensitive and specific as current standard AIV RNA extraction method for real time or conventional RT-PCR, besides it conciliated simplicity and lower cost and can be applied simultaneously for direct detection of AIV in a large number of samples, including less-equipped laboratories / Mestre

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-13Bitstream added on 2014-06-13T20:16:39Z : No. of bitstreams: 1 dipillo_f_me_jabo.pdf: 266438 bytes, checksum: 248a318a5e5422d95006058e8dd1370a (MD5) / A técnica de imunocaptura associada com a reação de transcrição reversa e reação em cadeia da polimerase (IC-RT-PCR) executadas tanto pelos procedimentos convencional como em tempo real foram testadas para a detecção rápida do gene da glicoproteína de Matriz (M) do vírus de influenza aviária (AIV) em amostras de líquido cório-alantóide (LCA) e em suabes traqueais e cloacais. O presente trabalho teve como objetivo desenvolver e otimizar a técnica de IC-RT-PCR para o diagnóstico do vírus da Influenza aviária. Os resultados obtidos foram comparados com um sistema empregando “beads” magnéticas em microplacas (AMBION), que é o método padrão de extração de RNA usado no laboratório de referência para diagnóstico de influenza aviária, o National Veterinary Services Laboratory – Ames, EUA (USDA), acrescido ainda de outros métodos de extração tradicionalmente usados nos laboratórios de referência para AIV, como os procedimentos com o uso do solvente orgânico Trizol® (Invitrogen) e com um sistema robotizado e que utiliza “beads” magnéticas (MagNA Pure - ROCHE). A técnica de IC-RT-PCR em tempo real neste estudo detectou a estirpe H2N2 do AIV, sem que nenhum outro dos RNA-vírus heterólogos testados fossem detectados (vírus das doença de Gumboro, de Newcastle e da bronquite infecciosa aviária). Os limites de detecção do IC-RTPCR foram iguais aos obtidos na técnica de extração com o kit da AMBION e menores do que aqueles que foram observados para os métodos de extração com Trizol® (Invitrogen) e com o MagNA Pure. O IC-RT-PCR demonstrou ser um sistema de diagnóstico capaz de conciliar simplicidade operacional e um menor custo com sensibilidade e especificidade analíticas iguais às do procedimento padrão atualmente adotado, podendo ser inclusive por laboratórios dotados de uma infra-estrutura mais simples / The polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), including real-time RT-PCR have been used for the rapid detection of Matrix glycoprotein gene (M gene) Avian influenza virus (AIV). Despite the availability of various RNA extraction methods for using in RT-PCR, isolation and detection of viral RNA are still difficult due to the unstable nature of viral RNA molecules and the presence of PCR inhibitory substances. In this study, a simple method using immune-capture (IC) to recover viral RNA from H2 AIV samples was developed and compared to one standard and two others reference methods used for viral RNA extraction, such as Ambion MagMAXTM kit and Trizol® (Invitrogen) and Magnapure kit (Roche), respectively, with subsequent analysis by real-time RT-PCR. The real-time IC-RT-PCR developed in was able to detect specifically H2N2 AIV strain, without detecting non-related avian RNA-virus pathogens, such as Newcastle disease virus, avian infectious bronchitis virus and Gumboro disease virus. Comparable detection limits were found for IC and the standard RNA extraction method using Ambion MagMAXTM kit, either for the detection of AIV in allantoic fluid suspension or in seeded tracheal and cloacal swab samples by conventional or real time RT-PCR techniques. These methods were less sensitive than Trizol® (Invitrogen) and Magnapure kit (Roche) procedures. Thus, IC was rapid and as sensitive and specific as current standard AIV RNA extraction method for real time or conventional RT-PCR, besides it conciliated simplicity and lower cost and can be applied simultaneously for direct detection of AIV in a large number of samples, including less-equipped laboratories

A influenza aviária é uma enfermidade viral causada por vírus influenza A que acomete várias espécies de aves. No Brasil, a influenza aviária é considerada uma doença exótica, uma vez que os subtipos H5 e H7 nunca foram detectados. O Ministério da Agricultura, Pecuária e Abastecimento realiza vigilância permanente para esta enfermidade nos plantéis comerciais de produção avícola e também em aves de subsistência criadas no entorno de sítios de aves migratórias com o intuito de detectar precocemente a introdução dos subtipos H5 e H7 no país. No entanto, se desconhece a situação sanitária das aves de subsistência no que se refere à infecção por outros subtipos do vírus da influenza aviária. Considerando a importância deste tipo de criação como fonte de alimentos e de rendimentos em comunidades de baixa renda e o risco que provavelmente representa para a introdução da influenza aviária no Brasil, o objetivo deste trabalho foi monitorar as criações de aves de subsistência criadas no entorno de sítios de aves migratórias no período de 2011 a 2015, a fim de verificar a circulação de todos os subtipos do vírus da influenza aviária (AIV). Foram colhidas amostras de soro e suabes de traqueia e cloaca de 2816 aves criadas em onze diferentes sítios de aves migratórias localizados em sete estados brasileiros, totalizando 391 explorações de fundo de quintal amostradas. As amostras de soro (n = 2716) foram submetidas a triagem para pesquisa de anticorpos para a proteína NP do influenza A utilizando-se um kit comercial de ELISA competitivo, com posterior subtipificação das amostras positivas pela técnica de inibição da hemaglutinação para os dezesseis subtipos do vírus influenza A. Os suabes de cloaca e traqueia foram submetidos à prova de PCR em tempo real para detecção do RNA do vírus influenza A. Não foram detectados anticorpos para os subtipos H5 e H7 do AIV, mas anticorpos para os subtipos H1, H3, H4, H6, H8, H9, H10, H12, H13 e H16 foram identificados em aves oriundas de nove dos onze sítios. O RNA do AIV foi detectado em apenas três amostras pertencentes a uma mesma propriedade e nenhum vírus hemaglutinante foi isolado a partir deste material. Os resultados obtidos permitem concluir que os vírus de influenza aviária de baixa patogenicidade circulam em aves de subsistência criadas no entorno de sítios de aves migratórias no Brasil e alertam para a importância da ampliação da vigilância ativa nesta população / Avian influenza is a viral disease caused by an influenza A virus and affects various avian species. Avian influenza is considered an exotic disease in Brazil, since H5 and H7 notifiable subtypes have never been detected. The Ministry of Agriculture, Livestock and Food Supply carries out permanent surveillance for avian influenza in commercial poultry production flocks and also in backyard poultry raised in the vicinity of concentration areas of migratory birds with the purpose of detecting the early introduction of H5 and H7 subtypes in the country. However, the health status of backyard poultry in relation to infection by other avian influenza subtypes is unknown. Considering the importance of this kind of family production system as a source of food and revenue for low-income communities and the risk it probably represents for the introduction of avian influenza in Brazil, the aim of this work was monitoring backyard poultry raised in the vicinity of concentration areas of migratory birds from 2011 to 2015 to verify the circulation of all avian influenza virus (AIV) subtypes. Serum samples and cloacal and tracheal swabs were sampled from 2816 birds raised in eleven migratory birds concentration areas located in seven Brazilian states, totaling 391 backyard poultry flocks harvested. Serum samples (n = 2716) were screened using a commercial competitive ELISA kit to detect specific antibodies for the NP protein of the influenza A virus and afterwards the positive samples were subtyped through inhibition hemagglutination assay for the sixteen subtypes of influenza A viruses. Cloacal and tracheal swabs were tested by real-time PCR for detection of the influenza A virus RNA. No antibodies for H5 and H7 subtypes were detected, but antibodies for subtypes H1, H3, H4, H6, H8, H9, H10, H12, H13 e H16 have been identified in birds from nine out of eleven areas. The influenza A virus RNA was detected only in three samples from one flock and no hemagglutinating viruses were isolated from these specimens. Results obtained in this work suggest that low pathogenic avian influenza viruses are circulating in backyard poultry flocks raised in concentration areas of migratory birds in Brazil and alert to the importance of the expansion of active surveillance in this population

La maladie de Newcastle (MN) et l'influenza aviaire (IA) sont des maladies virales d'importances majeures. L'aviculture malgache est dominée par de petits élevages familiaux. Plusieurs facteurs de risque d'IA et de MN décrits dans la littérature sont présents à Madagascar. Les objectifs de cette thèse sont de déterminer les facteurs de risque associés à la transmission des virus de la MN et de l'IA ; d'évaluer leur niveau de circulation et leur incidence clinique ; d'étudier le rôle des circuits de commercialisation des volailles dans leur diffusion et de proposer des mesures de surveillance adaptées.Les sites d'étude étaient Antananarivo et son pourtour, et le Lac Alaotra. Une étude exploratoire des facteurs de risque a été effectuée, suivie d'une.surveillance épidémiologique événementielle et d'une épidémiologie participative pour estimer l'incidence clinique des maladies. Le rôle des circuits commerciaux a été évalué par des méthodes d'analyse de réseaux sociaux.La circulation des 2 virus était plus importante au Lac Alaotra, caractérisé par les rizières, les eaux de surface et les palmipèdes. Les élevages de basse-cour et les élevages de poulets à haut niveau relatif de protection sanitaire étaient les plus à risque à la MN Antananarivo. Leurs profils de facteurs de risque étaient l'absence de mesure de biosécurité et un contact avec plusieurs marchés pour le premier ; et une densité élevée, les visites des auxiliaires d'élevage et des collecteurs d'œufs et un contact avec d'autres acteurs pour le second. Il n'y a pas de circulation d'IAH, la MN est la maladie dominante avec un taux de mortalité de 44%. Le rôle du circuit de commercialisation a été confirmé. Les villages ayant les plus fortes connexions commerciales étaient les plus exposés à la MN mais constituaient aussi des super-diffuseurs de virus. L'association de plusieurs méthodes de surveillance a été discutée pour en améliorer la sensibilité, la faisabilité pratique et l'efficacité. / Newcastle disease (ND) and avian influenza (AI) are major poultry viral diseases. Poultry production in Madagascar is dominated by smallholder family system. Several risk factors for both diseases described in the literature are present in Madagascar: The objectives of this thesis were to determine the risk factors associated with transmission of AI and ND viruses; evaluate the intensity of their transmission and their clinical incidence; study the role of poultry trade network in their spread and suggest surveillance adapted to local conditions.Study sites were: Antananarivo and its surrounding and the Lake Alaotra. An exploratory study of risk factors was carried out. Passive surveillance and participatory surveys were combined to assess the incidence and clinical impact of AI and ND. The role of poultry-trade network was assessed using social network analysis methods. Higher circulating level for both viruses was observed in Lake Alaotra which is characterized by large paddy fields and surface water, and a high density of domestic waterfowl. At the farm level, backyard chicken and chicken farms with some biosecurity awareness were associated with higher risk of ND in Antananarivo. Risk factors associated with them were lack of biosecurity measures and contact with many markets for the former, and a high density of poultry, high exposition to the visits of animal health workers, egg collectors and multiple interactions with other poultry stakeholders for the second. There was no evidence of HPAI circulation The major infection was ND with an overall mortality rate of 44%, within observed outbreaks. The role of poultry trade in the spread of these viruses was confirmed. Villages with the highest commercial connections were the most exposed to ND. They were also super-spreaders of these viruses. The relevance of the specific combination of several methods of surveillance was discussed to improve their sensitivity, practical implementation, and effectiveness.

L’Influenza Aviaire Hautement Pathogène H5N1 (IAHP à H5N1) a été déclaré en Thaïlande lors de plusieurs vagues épidémiques puis lors de cas sporadiques entre 2004 et 2008, et le risque persiste encore en Thaïlande. La plupart des foyers confirmés d’IAHP à H5N1 en Thaïlande ont eu lieu dans les populations de volailles de basse-cour. Les poulets de basse-cour sont élevés pour de nombreuses raisons, y compris pour un revenu supplémentaire. Ce commerce est géré informellement par les collecteurs de volailles qui peuvent être classés en collecteur-abatteur (TS), sous-collecteur (HT) et collecteur de collecteurs (TT). Ces collecteurs se déplacent entre villages avec les mêmes véhicules et matériel non nettoyés. Nous avons mesuré leurs activités commerciales dans l'espace et le temps. Nous avons développé un modèle spatial dynamique compartimental et stochastique de la filière de poulet de basse-cour dans une province. Nos résultats indiquent que ces échanges commerciaux peuvent contribuer à la propagation d’IAHP à H5N1 grâce au chevauchement des zones de collecte et aux distances parcourues. Des variations temporelles ont été observées lors de certaines grandes fêtes rituelles tel le Nouvel An chinois. Nous avons développé un modèle SIR couplé au modèle de la filière de poulets. Nous avons supposé que la maladie peut se propager par deux moyens : localement entre villages voisins et par les échanges commerciaux. Nous avons utilisé ce modèle infectieux de base pour tester plusieurs mesures de contrôle connexes. Nos résultats suggèrent que lors d’un foyer une interdiction totale et rapide de tous les échanges de volailles devrait être mise en oeuvre ainsi qu’une campagne de désinfection. / Highly pathogenic avian influenza H5N1 (HPAI H5N1) was recognized in Thailand by multiple epidemic waves and some sporadic cases between 2004 and 2008 but the risk of disease remerging in Thailand still remains up to present. Most of HPAI H5N1 confirmed outbreaks in Thailand occurred in backyard chicken populations. Backyard chickens are reared for many purposes including for additional cash income. Backyard chicken trade is informally managed by poultry traders which can be categorized into trader–slaughterhouse (TS), household trader (HT) and trader of trader (TT). These traders roam around different villages with the same unclean vehicle and facilities. Thus, their trade patterns in space and time are necessary to be elaborately studied. In our study, we developed a spatial compartmental stochastic dynamic model of backyard chicken trade network in a province of Thailand. Our model results indicated that the structure of poultry networks may contribute to HPAI H5N1 spread through overlapped catchment areas and long distance trades. Also, temporal variations of live poultry movements were observed during some major ritual festivals especially Chinese New Year. Subsequently, we developed an SIR model upon the dynamic model of backyard chicken trade network. It was assumed in this study that the disease can spread by two means: local spread to neighboring uninfected villages and spread by poultry traders. Then, we used this baseline infectious model to test multiple related control measures. Our ultimate results suggested that a complete ban of all poultry traders should be promptly implemented with poultry area disinfection campaign once the outbreak occurs.

The H9N2 avian influenza virus (AIV) has emerged, spread and established itself in poultry globally, in just under 30 years. During this time, multiple reassortants of H9N2 with increased zoonotic potential have been isolated in poultry and humans, causing a major threat to the economy and global health. Curiously, H9N2 appears to be compatible with multiple Hemagglutinin (HA) and Neuraminidase subtypes, in nature. Here, the aim was to investigate the HA reassortment dynamics of the poultry adapted H9N2 AIV, in a laboratory setting. Firstly, HA subtypes from wild bird isolates were cloned, before being co-transfected with the backbone of a chicken H9N2 AIV. The rescued H9N2 reassortants were titred on cells before the replication kinetics of a subset of the HA reassortants was assessed. The cDNA sequence of seven HA subtypes induced extensive recombination in E. coli, but ultimately ten out of eleven available HA subtypes were successfully cloned. Further, the chicken H9N2 AIV was compatible with all ten HA subtypes, producing infectious viral particles after co-transfection. However, all HA reassortants displayed decreased replicative fitness in MDCK-2 cells, compared to the wild-type virus. Interestingly, HA subtypes with similar genotypes cluster into distinct HA clades and groups, but these HA clades did not correlate with the replicative fitness of the reassortants. This study suggests that poultry adapted H9N2 AIV is compatible with many HA subtypes, highlighting the importance of reducing its spread in poultry, to reduce reassortment opportunities.

No description available.

Les travaux de recherche effectués dans le cadre de cette thèse ont été guidés par le manque d’information et une compréhension limitée des mécanismes épidémiologiques à l’origine de l’émergence et de la diffusion de la grippe aviaire hautement pathogène, souche H5N1 en Chine du Sud, aussi reconnue comme l’épicentre potentiel de l’émergence des virus influenza aviaires à caractères pandémiques. <p>Dans ce cadre, des données spatio-temporelles relatives aux foyers de la maladie ainsi que des données de surveillance virologiques (isolement du virus effectué dans le cadre du système de surveillance nationale) ont été collectées sur une période de quatre ans et analysées afin d’éxplorer les facteurs de risque relatifs à l’émergence et persistence de la maladie dans certaine zones de production du sud de la Chine. Les analyses ainsi effectuées ont permis d’identifier, à travers l’utilisation de méthodes statistiques robustes ayant fait leur preuve dans le domaine de la santé ou de l’écologie (la régression logistique classique et les arbres de regression logistique), des facteurs de risque liés à certains types de production de volailles (canards élevés en plein air, zones riches en eau et par extension associées à la riziculture) ou des facteurs associés à l’activité humaine. A travers une représentation cartographique des facteurs ainsi identifiés, des cartes de risque ont été produites permettant ainsi de visualiser d’une part les zones à haut risque de persistence de l’infection virale et d’autre part les zones vulnérables à l’apparition de foyers de la maladie, donnant aux autorités nationales la possibilité de mieux cibler leurs politiques de surveillance et de contrôle. <p>Dans un second temps, notre étude s’est portée sur les marchés à volailles traditionnels du sud de la Chine qui représentent un risque permanent de persistence, d’évolution et de diffusion des virus influenza aviaires, ainsi qu’un risque important en matière de santé publique. La dynamique de ces marchés et les liens qui les unissent ont été étudiés à travers des outils d’analyse empruntés à la sociologie tels que l’Analyse des Réseaux Sociaux (Social Network Analysis). Grace à cette approche, l’importance de l’hygiène de ces marchés et notamment du nettoyage et de la désinfection des cages dans la persistence du virus a été mise en évidence. Enfin, des enquêtes effectuées auprès des vendeurs de volailles ont permis d’identifier l’origine et la destination des animaux vendus et de reconstruire des réseaux plus ou moins intriqués de liens commerciaux qui unissent ces marchés entre eux dans trois provinces du sud de la Chine. L’analyse de ces réseaux et de leurs configurations ont permis d’identifier des marchés à plus haut risque de persistence de l’infection du fait de leur position centrale au sein de ces réseaux. De même qu’il est indispensable de cibler la surveillance et le contrôle de la maladie dans des zones écologiquement favorables à la persistence des virus influenza aviaires, cette étude révèle l’importance de certaines pratiques hygiéniques et commerciales dans la persistence de la maladie et la nécessité de cibler la surveillance et le contrôle au niveau de certains de ces marchés situés au centre d’un réseau dense et connecté, pour pouvoir in fine mieux contrôler la maladie au niveau national.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

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