INTRODUÇÃO: Indivíduos infectados pelo HIV apresentam maior risco de quadros graves de infecção por influenza sazonal e, portanto, devem receber doses anuais da vacina contra gripe. No entanto, a capacidade dos indivíduos responderem às vacinas com títulos apropriados de anticorpos depende de variáveis como tipo de antígeno vacinal, idade e grau de comprometimento imunológico no momento da imunização. OBJETIVOS: 1) Avaliar a imunogenicidade da vacina contra influenza sazonal em 37 pacientes infectados pelo HIV, em comparação com 29 indivíduos não infectados pelo HIV 2) Realizar a vigilância dos episódios de infecções respiratórias durante o período de acompanhamento após a vacinação. MÉTODOS: Ambos os grupos receberam a vacina contra o vírus da influenza sazonal recomendada para o hemisfério sul em 2008. A resposta de anticorpos contra os antígenos H1N1, H3N2 e B foi medida em amostras de sangue extraídas 1-2h antes da vacinação (T0), após 1 mês (T1) e após 6 meses (T6; apenas no Grupo HIV). A vigilância dos sintomas respiratórios foi realizada através de telefonemas semanais, durante 6 meses após a vacinação. Em indivíduos sintomáticos para infecções respiratórios foram coletadas amostras de lavado nasofaríngeo para pesquisa de vírus respiratórios por Imunofluorescência e PCR: influenza A e B, parainfluenza 1, 2 e 3, adenovírus, metapneumovírus, vírus sincicial respiratório, rinovírus e coronavírus. RESULTADOS: A idade mediana da população de estudo foi de 12 (10-18) anos. No momento T1, ambos os grupos mostraram aumento significativo nos TMGs para todos os antígenos. Contudo, o grupo controle apresentou valores mais elevados para os antígenos A/H1N1 e A/H3N2 (p = 0,002 e 0,001, respectivamente). Houve maior aumento na porcentagem de indivíduos não infectados pelo HIV com títulos protetores A/H1N1 (96,6%) em comparação aos infectados pelo HIV (67,6%). No T1 (p=0,004). A porcentagem de indivíduos do grupo controle com aumento de quatro vezes ou mais nos títulos de anticorpos para A/H1N1 e A/H3N2 foram mais elevadas que no grupo HIV (p = 0,03 e 0,01, respectivamente). Agentes virais foram detectados em 39/60 (65%) dos episódios de infecção respiratória no grupo HIV e em 17/32 (53,1%) no grupo controle. Os vírus diagnosticados no grupo HIV e grupo controle foram respectivamente: adenovirus (8,6%), metapneumovirus (1,2%), rinovirus (16,8%), coronavirus (14,0 %) e influenza B (0,1%).CONCLUSÕES: A vacina sazonal contra os vírus da influenza foram imunogenicas em ambos os grupos. Ocorreram diferença nas taxas de soroproteção entre os grupos somente para o antígeno H1, que foi mais elevadas no grupo controle. O grupo controle também mostrou valores mais altos nos TMGs para os antígenos H1 e H3 depois da imunização. Os rinovirus (27,7%) e coronavirus (22,5%) foram os agentes mais prevalentes identificados no grupo infectado pelo HIV. No grupo controle, os vírus mais freqüentes foram os rinovirus (24,2%) e adenovirus (21,2%) / INTRODUCTION: Individuals infected with HIV are at higher risk for severe cases of seasonal influenza infection and therefore should receive annual doses of influenza vaccine. However, the ability to respond to vaccines respond appropriate antibodies titres depends on variables such as vaccine antigen, age and degree of immune impairment at immunization. OBJECTIVES: 1)To evaluate the immunogenicity of a seasonal influenza vaccine in 37 HIV-infected patients (HIV Group), compared to 29 uninfected individuals (Control Group) 2) To carry out a clinical and virological surveillance of influenza in this population during a follow-up period of six months. METHODS: Both groups received the vaccine against seasonal influenza virus recommended for the southern hemisphere in 2008. The antibody response against the antigens H1N1, H3N2 and B were measured in blood samples drawn at vaccination (T0), after 30 days (T1) and after 6 months (T6; only for HIV Group). Antibody titres >1:40 were considered protective against influenza infection A surveillance of respiratory symptoms was performed weekly by telephone calls for a post-vaccination follow-up period of 6 months. Samples were collected (nasal wash) if respiratory symptoms. DFA and real time PCR was used to diagnose influenza A virus (FLU A) and B (FLU B), respiratory syncytial virus (RSV), parainfluenza virus types 1, 2 and 3 ( Paraflu 1, 2 or 3), adenovirus, coronavirus, rhinovirus, metapneumovirus and bocavirus. RESULTS: The median age of the study population was 12 (10-18) years. At T0, there were no significant differences in the antibody geometric mean titres (GMTs) against all vaccine antigens between groups. One month after vaccination (T1), both groups showed significant increases in the antibody GMTs for all antigens. However, healthy controls showed higher values for antigens A/H1N1 and A/H3N2 (p = 0.002 and 0.001, respectively). There was a higher increase in the percentage of HIVuninfected subjects with protective A/H1N1 antibodies (96.6%) comparing to HIVinfected vaccinees (67.6%) at T1 (p = 0.004). The percentage in subjects control group with a fourfold or greater increase of A/H1N1 and A/H3N2 antibody titres was higher than that found in HIV group (p = 0.03 and p = 0.01, respectively. Viral agents were identified in 39/60 (65%) episodes of respiratory infections in HIV-infected group and in 17/32 episodes (53.1%) from the control group (P=0.273). The virus diagnosed in HIV group and control group were, respectively: Adenovirus (8;6), Metapneumovirus(1;2) Rinovirus(16;8), Coronavirus(14 ;0); Influenza B(0;1). CONCLUSIONS: The seasonal influenza vaccine was immunogenic in both groups. There were differences in seroprotection rates between groups only for AgH1, which was higher in the control group. The control group also showed a greater increase in GMTs for H1 and H3 antigens after immunization. Viral agents were identified in respiratory symptoms during the follow-up: Rhinoviruses (27.7%) and coronavirus (22.5%) were the most prevalent agents identified in HIV-infected individuals. In the control group, the viruses most frequently found were rhinoviruses (24.2%) and adenovirus (21.2%)

Influenza aviaire est une maladie infectieuse contagieuse, due à des virus de la famille des Orthomyxoviridae, les virus Influenza A, touchant les oiseaux et susceptible d'entraîner une mortalité extrêmement élevée, notamment chez les volailles. Les souches hautement pathogènes des virus influenza aviaire provoquent une maladie sévère et mortelle chez les volailles et constituent un risque pour la santé publique. En février 2006, la France a été touchée par une épizootie d'influenza aviaire H5N1 hautement pathogène, qui s'est répandu dans l'avifaune sauvage de la zone humide la Dombes. La Dombes est une zone humide, mosaïque de plus de mille étangs, et une zone d'importance ornithologique internationale, abritant des milliers d'oiseaux d'eau. C'est une zone à risque pour la propagation de l'influenza aviaire de part à la fois la présence des populations d'oiseaux sauvages sur les étangs et de la proximité de ces populations aux élevages de volailles qui parsèment la région. L'objectif principal de ce travail est la modélisation du risque influenza aviaire dans la région de la Dombes. Ce travail est axé sur l'avifaune sauvage et sur le risque d'infection et de transmission de l'infection par les oiseaux sauvages. Nous avons, dans un premier temps, identifié les caractéristiques particulières à la Dombes, pouvant influer sur l'épidémiologie de l'influenza aviaire dans la région. Les trois acteurs principaux de l'écosystème de la Dombes sont les étangs, les populations d'oiseaux sauvages et les élevages de volailles. Ensuite, le risque influenza aviaire dans la Dombes a été décliné à deux niveaux : 1) dans l'écosystème d'un seul étang ; et 2) au sein d'un réseau d'étangs comme caractérisés en 1), où la propagation de l'infection peut se faire d'étang à étang. Au niveau étang, le risque influenza aviaire est modélisé, d'une part, par le temps de persistance des virus influenza aviaire dans l'eau de l'étang, et d'autre part, par la dynamique de l'infection au sein des populations d'oiseaux sauvages par transmission directe entre oiseaux et indirecte via l'eau de l'étang. Au niveau réseau d'étangs, l'analyse du risque permet d'identifier des groupements spatiaux d'étangs à risque élevé d'infection aux virus influenza aviaire. Quant au risque d'exposition des élevages aux virus influenza aviaire d'origine de l'avifaune sauvage, il est calculé en tenant compte à la fois de la proximité des élevages aux étangs susceptibles d'être infectés et de la probabilité de ces étangs à être infectés en cas d'épizootie. / Avian influenza is a contagious infectious disease caused by viruses of the family Orthomyxoviridae, influenza A viruses, affecting birds and known to cause extremely high mortality, especially in poultry. Highly pathogenic strains of avian influenza viruses cause severe and fatal disease in poultry and pose a risk to public health. In February 2006, France was hit by a highly pathogenic H5N1 avian influenza outbreak, affecting aquatic wildfowl in Dombes wetland. The Dombes is a wetland mosaic of more than a thousand ponds and an area of international ornithological importance, home to thousands of waterfowl. This is an area at risk for the spread of avian influenza viruses due to both the presence of wild birds on the ponds and the proximity of these populations to poultry farms that are present in the area. The main objective of this work is the modeling of the avian influenza risk in the Dombes region. This work is focused on the wildfowl and the risk of infection and transmission of infection by wild birds. As a first step, we have identified the specific characteristics of the Dombes area, which may affect the epidemiology of avian influenza in the region. The three main players in the Dombes ecosystem are the ponds, the wildfowl populations and the poultry farms. Secondly, the avian influenza risk in the Dombes was declined at two levels: 1) in the ecosystem of a single pond; and 2) within a network of ponds as characterized by 1), where the infection is spread from pond to pond. At the pond level, the avian influenza risk is modeled, on the one hand, by the persistence time of avian influenza viruses in the water of the pond, and, on the other hand, by the infection dynamics within wild bird populations by direct transmission between birds and indirectly through the water of the pond. At the ponds network, the risk analysis identifies spatial clusters of ponds at high risk of infection with avian influenza viruses. Concerning the risk of exposure of poultry farms to avian influenza viruses of wildfowl origin, it is calculated taking into account both the proximity of the farms to ponds likely to be infected and the probability of these ponds to become infected during an outbreak.

Los virus de influenza aviar de alta patogenicidad (IAAP) causan una enfermedad muy severa en pollos, los cuales frecuentemente inducen lesiones en el sistema nervioso central (SNC). Esta tesis recoge los resultados de tres estudios que se llevaron a cabo para determinar el mecanismo de patogénesis y neurotropismo, así como establecer la ruta de entrada al SNC para un virus H7N1 IAAP. En el primer estudio se estableció un modelo animal de infección en pollos libres de patógenos específicos, que consistía en la inoculación intranasal con el virus H7N1 IAAP. Para establecer este modelo, se utilizaron tres diferentes dosis del virus, obteniendo que las dosis más altas producen una enfermedad similar a la reportada para otros virus de IAAP. Además, se observó que las dosis más bajas causan infección demostrada porque con las dosis más bajas, el virus es hallado en muestras de tejido, muestras de heces y secreciones respiratorias. Adicionalmente, se pudo comprobar el alto neurotropismo del virus, ya que aún en pollos inoculados con bajas dosis el RNA viral es hallado en el CNS. La viremia fue detectada a un día post infección (dpi), sugiriendo que está podría ser la vía de diseminación al SNC. En el segundo estudio, se determinó la distribución topográfica del antígeno viral en el SNC durante las primeras horas post infección, lo cual permitió determinar que el virus se disemina de forma simétrica y bilateral en núcleos neurales del diencéfalo, mesencéfalo y rombencéfalo. La distribución del antígeno viral indica que el bulbo olfatorio y los nervios periféricos están involucrados en el proceso de invasión del SNC. El hallazgo de receptores aviares y humanos en las células endoteliales explica porque estas células son tan sensibles a la infección. El RNA viral fue hallado en el líquido cerebro espinal el primer dpi, lo que indica que el virus atraviesa la barrera hemato-encefálica (BHE). En el tercer estudio, la alteración de la BHE inducido por el virus H7N1 IAAP fue demostrado usando tres diferentes métodos que incluye la perfusión intracardial de Azul de Evans, la detección de la extravasación de la proteína del suero IgY, y evaluación del patrón de tinción con el marcador de las uniones fuertes de la BHE, ZO-1 y claudin-1. El antígeno viral fue observado a las 24 hpi en las células endoteliales, mientras que el daño de la BHE fue observado a las 36 hpi y 48 hpi. En resumen, se puede afirmar que el virus H7N1 IAAP se disemina por la vía hematógena durante las primeras horas pi, posiblemente favorecido por la presencia de receptores en las células endoteliales del sistema nervioso central, y poco después daña la BHE durante las primeras horas de infección como se demuestra por la presencia de extravasación del azul de Evans and IgY del suero. / Highly pathogenic avian influenza viruses (HPAIV) cause a very severe systemic disease in chickens, in which is also frequent to find central nervous system (CNS) lesions. In this thesis, three studies were undertaken in order to determine the mechanism of pathogenesis, the neurotropism and establish the route of entry into the CNS use for a H7N1 HPAI virus. In the first study, an animal model was set up that consisted of SPF chickens inoculated intranasally with the H7N1 HPAI virus. To do that, three different doses were used, obtaining that the highest dose induced a disease similar to the produce by other HPAI viruses, moreover, it was also observed that very low doses also cause infection demonstrated because viral RNA was found in tissues samples, faeces and respiratory secretions. Besides, the high neurotropism of this virus was demonstrated because still in chickens inoculated with low doses, viral RNA is found in the brain. Viremia was detected at one dpi, which indicated that the bloodstream is the pathway of viral spreading to the brain. In the second study, the topographical distribution study of the viral antigen during the first dpi was determined, which allow to determine that the virus disseminates showing a symmetrical and bilateral pattern in the diencephalon, mesencephalon and rhombencephalon, whereas in the telencephalon and cerebellum it was multifocal and random. Viral antigen distribution indicates that the olfactory bulb (OB) and peripheral nerves are not involved in the process of virus invasion into the brain. Avian and human influenza receptors were found in endothelial cells which explain why these cells are so sensitive to the infection. Viral RNA was found in cerebrospinal fluid (CSF) at one dpi, indicating that the virus was able to cross blood brain barrier (BBB). In the third study, the disruption of the BBB induce by the H7N1 HPAI was demonstrated using three different methods that include the intracardial perfusion of the tracer Evans blue (EB), detection of the extravasation serum IgY, and evaluation of the pattern of staining of the tight junction proteins ZO-1 and claudin-1. Viral antigen can be observed as early as 24 hpi in the endothelial cells, whereas disruption was detected at 36 and 48 hpi. In summary, it can be asserted that this H7N1 HPAIV disseminates via the haematogenous route early during the infection, favored by the presence of abundant receptors on the CNS endothelial cells, and soon after it disrupts the BBB during the first hours of infection as demonstrated by the presence of EB and serum IgY extravasation.

Abstract AI (Avian Influenza) it is originally a kind of epidemic disease infecting among the poultry, once the poultry are infected with the Avian Influenza, will cause high death rate and even the poultry do not die and recover may cause productivity drop : laying hens decrease egg production, breeders reduce hatchery, broilers have poor weight gain, therefore increase the farmers producing cost. And in order to prevent Avian Influenza farmers have to avoid infecting and use the right vaccines. But the Taiwan environment is open disinfectant can do not much . The directly effective method is to use the vaccines.But Taiwan no one produces the vaccines at present, the government has not allowed vaccines to import yet either. Even government allow to import but there are too many serum types of the Avian Influenza, and the types do not have cross protect , let farmers have no effective method to prevent the Avian Influenza. once the avian got infection the farmer losses much. The Avian Influenza happens for many years in the poultry, with the time and mutation of the virus, the Avian Influenza become to high pathogenic Avian Influenza (High pathogen Avian Influenza, is abbreviated as HPAI) and infect to the human being . The poultry industry is very important in the animal husbandry of Taiwan, occupy the important position in agricultural production. NowTaiwan farmers not only have to face the internal Avian Influenza infection problem but also to face the step that opens with the internationalization of accession to the WTO. The animal husbandry of Taiwan has produced and faced the serious impact. Though high pathogenic Avian Influenza did not happen in Taiwan yet, but some parts of the world release the case of infecting successively, some cases even cause death. World Health Organization (WHO, World Health Origanization) monitor and control tightly, and prophesy Avian Influenza will lead to the fact that prevails greatly. This research studies the past events in the animal livestock and finds the effect and change of the market. Also use SWOT to know present animal livestock situation.And then to know the changes of high pathogenic Avian Influenza happened in Taiwan (HPAI, High Pathogen Avian Influenza), Finally gives some recommend and strategy to G company. Key words: AI (Avian Influenza), HPAI, High Pathogen Avian Influenza, WTO, Animal production, Strategy

The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4 % in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.

Influenza B virus causes a respiratory disease in people with a compromised immune system. The NS1B protein of influenza B virus is essential for virus growth and plays a crucial role in inhibiting the anti-viral responses mounted by the infected host cell. The N terminal 104 amino acids of NS1B bind a cellular protein called ISG15. ISG15 is an interferon induced 'ubiquitin-like' protein, and upon interferon induction, is conjugated to hundreds of targets. It has been found that both ISG15 and its conjugation inhibit many viruses. The focus of the current study was to characterize the interaction between NS1B and ISG15. Study of a recombinant influenza B virus which encoded a mutant NS1B protein that is unable to bind ISG15 revealed that ISG15 is mis-localized in cells infected with wild type but not the mutant influenza B virus. Further, such a mutant virus is attenuated in growth as compared to wild type virus in human cell lines but is not attenuated in canine cell lines. This result led to the discovery of the species specific nature of the interaction between NS1B and ISG15. Specifically, NS1B was found to bind ISG15 homologs from human and old world monkeys like Rhesus macaques and African green monkeys but not those from mouse or canines. These findings were extended by identifying the hinge between the N and C terminal domains of ISG15 as one of the major determinants of species specificity. These results highlight the importance of using human or primate cell culture models to study the effect of ISG15 on influenza B virus, and raises new possibilities on differences in the function of the ISG15 system in different species. / text

published_or_final_version / Public Health / Master / Master of Public Health

published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

Highly pathogenic avian influenza H5N1, which is panzootic in poultry, continues to spread and becomes endemic in Asia, Africa, and Europe. It causes human disease with high fatality (about 60%) and continues to pose a pandemic threat. The pathological lesions associated with human H5N1 disease is Acute Respiratory Distress Syndrome (ARDS). The biological basis underlying the development of ARDS in human H5N1 disease remains controversial. Clinical, animal and in vitro studies suggested that the differences between H5N1 influenza viruss and low pathogenic influenza viruses in regard to viral replication, tissue tropism and cytokine dysregulation may contribute to disease pathogenesis. We previously found delayed onset of apoptosis in influenza H5N1 virus infected human peripheral blood monocyte-derived macrophages. This may allow a longer survival time for the virus in target cells for prolonged viral replication, which may contribute to the pathogenesis of H5N1 disease. As human bronchial and alveolar epithelial cells are target cells of influenza virus, I explored if influenza H5N1 and H1N1 virus infected human respiratory epithelial cells displayed differential apoptotic response and dissected the apoptotic pathways triggered by influenza virus infection. In this study, the apoptotic response in highly pathogenic influenza H5N1 viruses, A/HK/483/97 and A/Vietnam/1203/04, their precursor avian influenza H9N2 virus, A/Quail/HK/G1/97, and seasonal H1N1 virus, A/HK/54/98 infected primary human alveolar and bronchial epithelial cells was compared by TUNEL. A delayed onset of apoptosis in influenza H5N1 viruses and avian H9N2 virus infected alveolar epithelial cells was observed; the pattern was similar in bronchial epithelial cells. Concomitantly, by Western blotting, a delay in caspase 3 activation in H5N1 virus (A/HK/483/97) infected alveolar epithelial cells compared to H1N1 virus (A/HK/54/98) infected cells was shown. Also, influenza H5N1 and H1N1 virus induced apoptosis through both intrinsic and extrinsic pathways in human alveolar epithelial cells. Chemokine IP-10 was differentially up-regulated in influenza H5N1 virus infected alveolar epithelial cells, but its relationship to the delayed onset of apoptosis requires further studies. TRAIL, an upstream signaling molecule of extrinsic apoptotic pathway, mRNA was up-regulated in influenza H5N1 infected alveolar epithelial cells but not in influenza H1N1 infected cells. Using recombinant viruses, I showed that the 627 amino acid residue on PB2 of H5N1 virus and mutation of amino acids on 253 and 591 residues on PB2 of H9N2 virus contribute to the TRAIL upregulation. Immunohistochemical staining of physiologically relevant ex vivo model of human bronchus showed that influenza H5N1 (A/Vietnam/3046/04) and H9N2 (A/Quail/HK/G1/97) virus did not infect human bronchi as well as human H1N1 (A/HK/54/98) virus. Profiling of apoptosis related genes showed that TRAIL tends to be up-regulated in H5N1 virus infected bronchi ex vivo. This study demonstrated the delayed onset of apoptosis by H5N1 virus infected respiratory epithelial cells may be a mean for influenza virus to have prolonged replication within the human respiratory tract and contribute to disease severity. The results generated provide a robust research agenda, yielding critical information that elucidate molecular mechanisms, such as TRAIL up-regulation, that may contribute to the virulence and pathogenesis in human H5N1 disease. / HKU 3 Minute Thesis Award, 2rd Runner-up (2011) / published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy