Le virus influenza de type A est un virus à ARN simple brin de polarité négative qui se réplique dans le noyau des cellules infectées. Son génome se compose de huit segments d'ARN viral (ARNv). Chaque segment est recouvert de multiples copies de nucléoprotéines virales (NP). Chacune des extrémités strictement conservées 3' et 5' des huit segments d'ARNv interagit avec une ARN-polymérase ARN-dépendante. Le complexe entre l'ARNv, NP et l'ARN-polymérase ARN-dépendante forme une particule appelée ribonucléoprotéine (RNP) constituant l'entité fonctionnelle pour la réplication du génome viral et sa transcription en ARN messagers. Dans le contexte de la RNP, ces deux processus sont assurés par l'ARN-polymérase ARN-dépendante formée de trois protéines (PA, PB1 et PB2). L'ARN-polymérase du virus influenza A fait l'objet de nombreuses études. Son étude structurale se heurte à la difficulté d'obtenir ce complexe hétérotrimérique sous forme soluble et en grande quantité, deux conditions qu'impose la biologie structurale. Ainsi, durant les huit dernières années, les données structurales à l'échelle atomique n'ont été obtenues que sur des domaines isolés de l'ARN-polymérase laissant l'essentiel de la structure de PB1 inconnue. Pour contourner ce problème, une nouvelle stratégie s'appuyant sur le système d'expression en cellules d'insecte infectées par baculovirus a été développée. Cette stratégie a permis la production d'une forme tronquée de l'ARN-polymérase du virus influenza de type A qui a été étudiée d'un point de vue structural et fonctionnel. Plusieurs reconstructions en trois dimensions ont été obtenues par microscopie électronique et une structure cristalline à faible résolution (7,7 Å) a pu être résolue. Les études fonctionnelles se sont axées sur les activités portées par l'hétérotrimère tronqué et un accent particulier a été mis sur l'étude des interactions avec l'ARN. Les informations sur les activités catalytiques obtenues in vitro ont mis en évidence un rôle clef de certains ions métalliques. Afin de connaitre l'environnement en ions métalliques et cibler leur rôle à l'échelle cellulaire, leur distribution dans la cellule infectée par le virus influenza A a été étudiée par microscopie rayons-X. Cet aspect de l'infection étant très peu documenté, cette étude s'inscrit dans une démarche originale et a offert l'opportunité d'intégrer les données biochimiques et biophysiques à l'échelle de la cellule entière. / Influenza A virus is a negative single stranded RNA virus that replicates in the nucleus of infected cells. Its genome contains eight single stranded negative-sense RNA segments (vRNA) covered by the viral nucleoprotein (NP). The highly conserved 3' and 5' ends of the vRNA are bound to the RNA-dependent RNA-polymerase (RdRp) which consists of three subunits, PA, PB1 and PB2. The complex between vRNA, NP and the RdRp forms a particle called ribonucleoprotein (RNP). The RNP acts as an independent molecular machine for transcription and replication in the nucleus. Within the context of the RNP, these two processes are mediated by the RdRp. The influenza A RdRp complex has been remarkably intractable to structural analysis and in the last eight years, crystal structures of independent domains covering roughly half of the heterotrimeric RdRp have been determined. In addition, electron microscopy reconstructions have described the RdRp. Nonetheless, a complete model characterizing the RdRp as a whole is still lacking. To overcome this issue, a new strategy has been developed to obtain the RdRp heterotrimeric complex using the baculovirus infected cells expression system. This method has produced a truncated form of the flu A RdRp which has been studied from a structural and functional point of view. Several three-dimensional reconstructions by electron microscopy have been obtained and a crystal structure at low resolution (7,7 Å) has been solved. Functional studies focused on the activities carried by the truncated RdRp and a particular emphasis was placed on the study of the interactions with RNA. In vitro functional data showed highly metal ion-dependent activities. To know more about the subcellular metal context, metallic ions distribution in influenza A infected cells has been studied by X-ray microscopy giving the opportunity to integrate biochemical and biophysical data in the context of the whole cell.

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

As doenças respiratórias são muito comuns na produção intensiva de suínos, já em javalis são escassas informações sobre prevalência, etiologia e apresentação clínico-patológica destas enfermidades. No entanto, a presença de patógenos respiratórios comuns entre javalis selvagens e confinados e suínos domésticos já foi relatada. Este trabalho descreve as principais lesões macroscópicas e histológicas de pneumonias de javalis e os agentes comumente envolvidos. Foram examinados pulmões de javalis, ao abate, provenientes de criatórios comerciais e a principal lesão macroscópica foi consolidação crânio-ventral dos lobos craniais e médios e lesões crônicas cursando com hiperplasia linfóide na histologia. O principal agente bacteriano detectado foi o Mycoplasma hyopneumoniae (58,6%). Outros patógenos bacterianos detectados foram Actinobacillus pleuropneumoniae (48,8%), Haemophilus parasuis (49,6%), Mycoplasma hyorhinis (41,3%), Pasteurella multocida (9,1%) e Streptococcus suis (9,1%). Na segunda parte do trabalho, a pesquisa de patógenos virais foi direcionada para o Vírus da influenza suína (VIS) com objetivo de estudar o envolvimento em pneumonias de javalis de criatórios e a relação com agentes bacterianos encontrados. O vírus pandêmico A/H1N1/2009 foi detectado em 18,3% (11/60) e sua identidade foi confirmada por sequenciamento. A carga viral para H1N1 clássico variou de 4,58 a 6275 cópias/μL e para o H1N1 pandêmico, de 4,65 a 3863 cópias/μL. Nenhuma amostra apresentou título viral após a inoculação em ovos embrionados. As lesões histológicas principais foram broncopneumonia crônica difusa e pneumonia intersticial mononuclear leve, além de hiperplasia linfóide. As amostras positivas por RT-PCR para o VIS para o pH1N1 foram testadas por IHQ, sendo todas negativas para influenza A, mas todas eram positivas para M. hyopneumoniae. Quando testadas por bacteriologia, 18,2% das amostras foram positivas para P. multocida. O estudo mostrou que as pneumonias em javalis de criatório apresentaram lesões e patógenos associados similares aos encontrados em suínos domésticos ao abate. Este é o primeiro relato da infecção pelo vírus pH1N1 em javalis no Brasil. / Respiratory diseases are very common in swine intensive production, although in wild-boars the knowledge of the prevalence, etiology and clinic-pathological presentation of these diseases are very limited. However, the presence of common respiratory pathogens among wild-boar, captive wild-boar and domestic pigs has been reported. This paper describes the main macroscopic and histologic pneumonic lesions of captive wild-boars and pathogens commonly involved. Captive wild-boar lungs at slaughter were examined and the main macroscopic lesion observed was cranio-ventral consolidation of cranial and middle lobes and chronic lesions associated with lymphoid hyperplasia by histology. The main bacterial pathogen detected was Mycoplasma hyopneumoniae (58.6%). Other bacterial pathogens detected were Actinobacillus pleuropneumoniae (48.8%), Haemophilus parasuis (49.6%), Mycoplasma hyorhinis (41.3%), Pasteurella multocida (9.1%) and Streptococcus suis (9.1%). In the second part of this work, the survey of viral pathogens was directed to swine influenza virus (SIV) in order to study the involvement in captive wild-boar pneumonias and the relation with bacterial pathogens. The A/H1N1/2009 pandemic virus was detected in 18.3% (11/60) and its identity was confirmed by sequencing. The classical H1N1 viral load ranged from 4.58 to 6275 copies/uL and the pandemic H1N1, from 4.65 to 3863 copies/uL. No samples had viral titers after inoculation in embryonated eggs. The main histological lesions were chronic diffuse bronchopneumonia and interstitial mononuclear pneumonia as well as mild lymphoid hyperplasia. Samples positive to pH1N1 were assayed by IHC for SIV, all with negative results, and to M. hyopneumoniae, all were positive. When assayed by bacteriology, 18.2% of samples were positive to P. multocida. This study showed that pneumonia in captive wild-boar had similar lesions and associated pathogens were similar to those found in domestic pigs at slaughter. This is the first report of pH1N1 virus infection in captive wild-boars in Brazil.

As doenças respiratórias são muito comuns na produção intensiva de suínos, já em javalis são escassas informações sobre prevalência, etiologia e apresentação clínico-patológica destas enfermidades. No entanto, a presença de patógenos respiratórios comuns entre javalis selvagens e confinados e suínos domésticos já foi relatada. Este trabalho descreve as principais lesões macroscópicas e histológicas de pneumonias de javalis e os agentes comumente envolvidos. Foram examinados pulmões de javalis, ao abate, provenientes de criatórios comerciais e a principal lesão macroscópica foi consolidação crânio-ventral dos lobos craniais e médios e lesões crônicas cursando com hiperplasia linfóide na histologia. O principal agente bacteriano detectado foi o Mycoplasma hyopneumoniae (58,6%). Outros patógenos bacterianos detectados foram Actinobacillus pleuropneumoniae (48,8%), Haemophilus parasuis (49,6%), Mycoplasma hyorhinis (41,3%), Pasteurella multocida (9,1%) e Streptococcus suis (9,1%). Na segunda parte do trabalho, a pesquisa de patógenos virais foi direcionada para o Vírus da influenza suína (VIS) com objetivo de estudar o envolvimento em pneumonias de javalis de criatórios e a relação com agentes bacterianos encontrados. O vírus pandêmico A/H1N1/2009 foi detectado em 18,3% (11/60) e sua identidade foi confirmada por sequenciamento. A carga viral para H1N1 clássico variou de 4,58 a 6275 cópias/μL e para o H1N1 pandêmico, de 4,65 a 3863 cópias/μL. Nenhuma amostra apresentou título viral após a inoculação em ovos embrionados. As lesões histológicas principais foram broncopneumonia crônica difusa e pneumonia intersticial mononuclear leve, além de hiperplasia linfóide. As amostras positivas por RT-PCR para o VIS para o pH1N1 foram testadas por IHQ, sendo todas negativas para influenza A, mas todas eram positivas para M. hyopneumoniae. Quando testadas por bacteriologia, 18,2% das amostras foram positivas para P. multocida. O estudo mostrou que as pneumonias em javalis de criatório apresentaram lesões e patógenos associados similares aos encontrados em suínos domésticos ao abate. Este é o primeiro relato da infecção pelo vírus pH1N1 em javalis no Brasil. / Respiratory diseases are very common in swine intensive production, although in wild-boars the knowledge of the prevalence, etiology and clinic-pathological presentation of these diseases are very limited. However, the presence of common respiratory pathogens among wild-boar, captive wild-boar and domestic pigs has been reported. This paper describes the main macroscopic and histologic pneumonic lesions of captive wild-boars and pathogens commonly involved. Captive wild-boar lungs at slaughter were examined and the main macroscopic lesion observed was cranio-ventral consolidation of cranial and middle lobes and chronic lesions associated with lymphoid hyperplasia by histology. The main bacterial pathogen detected was Mycoplasma hyopneumoniae (58.6%). Other bacterial pathogens detected were Actinobacillus pleuropneumoniae (48.8%), Haemophilus parasuis (49.6%), Mycoplasma hyorhinis (41.3%), Pasteurella multocida (9.1%) and Streptococcus suis (9.1%). In the second part of this work, the survey of viral pathogens was directed to swine influenza virus (SIV) in order to study the involvement in captive wild-boar pneumonias and the relation with bacterial pathogens. The A/H1N1/2009 pandemic virus was detected in 18.3% (11/60) and its identity was confirmed by sequencing. The classical H1N1 viral load ranged from 4.58 to 6275 copies/uL and the pandemic H1N1, from 4.65 to 3863 copies/uL. No samples had viral titers after inoculation in embryonated eggs. The main histological lesions were chronic diffuse bronchopneumonia and interstitial mononuclear pneumonia as well as mild lymphoid hyperplasia. Samples positive to pH1N1 were assayed by IHC for SIV, all with negative results, and to M. hyopneumoniae, all were positive. When assayed by bacteriology, 18.2% of samples were positive to P. multocida. This study showed that pneumonia in captive wild-boar had similar lesions and associated pathogens were similar to those found in domestic pigs at slaughter. This is the first report of pH1N1 virus infection in captive wild-boars in Brazil.

Le porc est un modèle d’étude présentant à la fois une grande importance agronomique et un fort potentiel comme modèle biomédical pour différentes pathologies respiratoires. Pourtant, son système immunitaire pulmonaire est peu connu, limitant l’approfondissement de ces études. Notamment, les cellules dendritiques (DCs) et macrophages (Mθs) tissulaires, qui sont à l’interface entre immunités innée et acquise, n’étaient pas caractérisés. L’objectif de cette thèse était de décrire ces cellules dans le poumon porcin à l’état basal et durant des infections par différentes souches de virus Influenza A (VIA), un pathogène capable d’infecter le porc et l’Homme. Nous avons caractérisé pour la première fois les DCs conventionnelles (cDCs) et dérivées de monocytes (moDCs), ainsi que des macrophages dérivés de monocytes (moMθs) et les macrophages alvéolaires (AMs) porcins. Les cDC1 et cDC2 se sont révélées proches de leurs équivalentes murines et humaines de par leur phénotype et leurs capacités de migration et de présentation de l’antigène in vitro. Les moDCs sont, comme chez la souris, pro-inflammatoires et recrutées lors d’une infection par le VIA in vivo. Nous avons également étudié des cellules tissulaires fortement représentées dans le poumon porcin et dont le phénotype est très similaire à celui des AMs : les cellules AM-like. Ces dernières sont pro-inflammatoires lors d’infections in vitro par un virus H3N2 porcin ou une souche Lena du PRRSV. Nous avons enfin étudié ces différents types cellulaires dans le cadre d’infections par deux souches porcines du VIA, l’une du sous-type H3N2 et l’autre du sous-type H1N2, la première étant plus pathologique en élevage et induisant plus d’inflammation. Il se pourrait que les cellules AM-like aient un rôle important dans cette différence de pathogénicité. En conclusion, nous avons mis en place des moyens pour étudier plus précisément le rôle des DCs et Mθs dans le poumon porcin, et ainsi, de mieux comprendre l’inflammation et la réponse immunitaire pulmonaire. Ce travail ouvre des voies, en santés vétérinaire et biomédicale, qui étaient jusqu’à présent réservées au modèle murin. / Pig is a research model with a major agronomic importance and a great potential as a biomedical model for different respiratory pathologies. Yet, its pulmonary immune system is little known, limiting the deepening of these studies. In particular, tissular Dendritic Cells (DCs) and Macrophages (Mθs), which are at the interface between innate and adaptive immune systems, were not characterized. The aim of this thesis was to describe DCs and Mθs in the porcine lung at steady-state and during infections with different strains of influenza virus A (IAV), a pathogen capable of infecting pigs and humans. We characterized for the first time conventional DCs (cDCs) and monocyte derived (moDCs), as well as monocyte-derived macrophages (moMθs) and alveolar macrophages (AMs) in pig. The cDC1 and cDC2 were phenotypically close to their murine and human equivalents. They also had the same in vitro capabilities of migration and antigen presentation. The moDCs are, like murine ones, pro-inflammatory and are recruited during an IAV infection in vivo. We also found a highly represented interstitial population whose phenotype is very similar to that of AMs: AM-like cells. They are pro-inflammatory upon in vitro infection by a swine H3N2 strain or a Lena strain of PRRSV. Finally, we studied these different cell types through infection by two strains of porcine IAV, an H3N2 and an H1N2 strains, the first one being more pathological and inducing more inflammation. The AM-like cells might play an important role in this pathogenicity difference. In conclusion, we found a strategy to study more precisely the roles of DCs and Mθs in the porcine lung, and thus to better understand lung inflammation and immune response. This work opens pathways, in veterinary and biomedical health, which were previously reserved for mouse model.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology. / Influenza-A virus is a single stranded negative sense RNA virus that is a member of a Orthomyxoviridae group. The virus is diverse and consists of 16 haemagglutinin (H) and 9 neuraminidase (N) glycoproteins subtypes that form a serotype. Avian influenza virus (AIV) has been detected in more than 100 bird species from 26 different families, although Anseriformes and Charadriiformes are considered the natural hosts of the virus. A 12-month study was conducted at the African Pride Irene Country Club lodge in Pretoria where the prevalence of AIV was monitored in a community of wild birds. The African Pride Irene Country Club lodge houses a population of wild bird species such as Egyptian geese (Alopochen aegytptiaca), Yellow-billed duck (Anas undulata), Red knobbed coot (Fulica cristata), African sacred ibis (Threskiornis aethiopicus) and Hadeda ibis (Bostrycha hagedash). A total of 3674 faecal samples were collected over a period of 12 months and screened for AIV group using the Matrix gene (M-gene) real time reverse-transcriptase PCR (rRT-PCR). Positive samples were submitted for virus isolation in embryonated chicken eggs. In addition, the RNAs were screened using H5 and H7 subtype specific rRT-PCR and a conventional universal RCR assay that targets the HA gene was also used. Polymerase Chain Reaction (PCR) products were requenced using Sanger sequencing and the viral isolates were subjected to Next Generation sequencing (NGS). Twenty percent of the samples tested positive for the AIV group and four virus subtypes were identified. One virus isolate was identified through NGS as H3N6; two through conventional PCR and Sanger sequencing as H9Nx and H6Nx. Of the twenty percent samples that tested positive for AIV 98% were identified as H7Nx by subtype specific through rRT-PCR. The highest frequency of AIV positive samples was detected between the months of January and February 2017 (20%), with smaller peaks detected in february and March 2016 (0.3%). Lower peaks were also detected between the months July and November 2016 (0.1%), respectively. A high prevalence of AIV was detected in the late summer months with a frequency of 65% positive, although a low prevalence was also detected in the autumn (0.6%) winter (0.6%) and spring 0.08%). Thus, the study provides a valuable insight into the seasonal prevalence of AIV in a heterogeneous wild duck population in Gauteng Province.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19048号 / 農博第2126号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4930(農学部図書室) / 31999 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 植田 和光, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

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.