Memoria para optar al Título Profesional de Médico Veterinario / El virus influenza A (IAV) de origen porcino posee gran importancia en salud pública y salud animal. IAV posee gran variabilidad genética y antigénica, lo cual es muy importante dentro de su patogenia. Este estudio consistió en determinar la variabilidad genética del gen de la neuraminidasa (NA) en virus aislados de planteles porcinos de producción intensiva entre los años 2013 y 2014. Para esto, se analizaron 1167 muestras desde 25 granjas mediante aislamiento viral, RT-PCR en tiempo real y secuenciación por Next Generation Sequencing, obteniéndose 41 secuencias genómicas de NA pertenecientes al 50% de las empresas muestreadas. Se determinó la presencia de los subtipos H1N1, H1N2 y H3N2 circulando en los planteles chilenos, específicamente en la Región de Valparaíso, Región Metropolitana, Región del Libertador General Bernardo O’Higgins, Región del Maule y Región del Biobío, confirmando la gran diseminación de IAV en porcinos de Chile. Del total de secuencias de NA, 17 correspondieron a N1, las que fueron agrupadas dentro del clúster N1 pandémico, evidenciando múltiples introducciones del virus a la especie porcina. Por otro lado, 24 secuencias correspondieron a N2. La mayoría de éstas se agruparon en un clúster monofilético, que fue designado como “N2 chileno”, el cual resultó ser distinto a aquellos encontrados en otros países y evidenciaban una introducción única a la población de cerdos chilenos. Se observó también la presencia de distintas variantes virales dentro de una misma empresa y la presencia de una misma variante en empresas distintas. Estos resultados permitieron conocer la realidad de IAV y específicamente la diversidad de NA. / Swine origin influenza A virus (IAV) is an important concern in both public and animal health. IAVs have shown high genetic and antigenic diversity, which is very important for the viral pathogenesis. The aim of this study was to determine the neuraminidase gene (NA) genetic variability in IAVs obtained from intensive swine farms, during 2013 to 2014. One thousand one hundred sixty-seven samples from 25 swine farms were tested by viral isolation, real time RT-PCR and sequenced by Next Generation Sequencing. A total of 41 NA sequences were obtained, representing 50% of the swine farms in study. H1N1, H1N2 and H3N2 subtypes were detected in Valparaíso, Metropolitan, Libertador General Bernardo O´Higgins, Maule and Biobío Regions, confirming the wide spreading of IAV in intensive swine farms in Chile. Seventeen viral sequences belonged to N1 subtype. All of them were grouped into the pandemic N1 cluster, and the tree topology suggests multiple introductions of the virus into swine population. The rest of the sequences belonged to N2 subtype. Most of them were grouped into a monophyletic cluster, named “Chilean N2”. This cluster was not close related with viruses previously described, suggesting a unique introduction into the swine population. At farm level, was observed the presence of more than one IAV variant and the presence of a determined variant circulating in several farms. These results could help to understand the influenza A virus dynamic in swine in Chile. / Financiamiento: Proyecto Fondef ID14I10201.

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Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...

Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...

Influenza is an infectious disease caused by the influenza virus. This virus causes a severe viral infection that spreads easily from person to person in yearly pandemics. Vaccination is the most effective way to prevent the infection, however, due to the high rate in mutations of the virus, the vaccine needs to be often reformulated. Another option to combat influenza is based on administration of antiviral drugs. Clinical studies of isolated influenza strains ("avian flu" H5N1, 2004; "swine flu" H1N1, 2009) revealed resistance towards known influenza neuraminidase inhibitors (zanamivir, oseltamivir). The resistance is caused by structural changes close to the enzymatic site. This calls for the development of new neuraminidase inhibitors as well for development of inhibitors targeting different influenza enzymes. This Thesis is focused on design and synthesis of new inhibitors of influenza neuraminidase and RNA-dependent RNA polymerase, namely PA subunit and the assembly of PA-PB1 heterodimer enzymes (Scheme 1). Influenza neuraminidase inhibitors were prepared by C-5 derivatization of oseltamivir followed by subsequent extension of its structure with binders of 150-cavity. Binding potencies of new oseltamivir derivatives against two influenza strains were determined. The next part contributed to...

La influenza es una enfermedad altamente infectocontagiosa de la cual se tienen registros históricos descriptivos desde la época griega y de certeza etiológica tan solo hace casi ocho décadas atras. Su agente causal es el virus del influenza de los que se conoce exiten tres grandes tipos: A B y C. El tipo A tiene la propiedad de circular por diversos reservorios biológicos, tales como el hombre, los cerdos y las aves, entre otros. Representa además una elevada variabilidad genética lo que le permite continuos cambios o derivas antigénicas menores responsables de la influenza humanas epidémicas y a veces reordenamientos amplios con cambios antigénicos mayores los que originan la temida influenza pandémica. En un mundo cada vez mas globalizado, con una población superior a los seis mil millones de personas, marcado por grandes inequidades sociales y con cambios climáticos evidentes, los virus de la influenza serán un riesgo permanente para la seguridad de la humanidad. La clínica de los diversos subtipos virales pueden balancearce desde las formas inaparentes hasta las formas graves de gripe o influenza, dependiendo de la virulencia del subtipo viral infectante y del huesped. Aunque nuestra tecnología diagnóstica y de inmunización ha avanzado sorprendentemente, la preparación y disposición de los nuevos kits diagnósticos suelen tardar al principio y en el caso de las vacunas estas no están disponibles para el un nuevo subtipo viral pandémico en el momento que se las necesita. El desarrollo de antivirales contra la gripe no es notorio, contándose aprobados para uso humano los amadantanes como la amantadina y la rimantadina y los inhibidores de la neuraminidasa como oseltamivir y sanamivir principalmente. Claramente los más vulnerables en este contexto son los países en vías de desarrollo y en especial aquellos mas pobres, hecho que nos debería llamar a una profunda reflexión. / Influenza is a highly contagious disease. There are some historical descriptions of this condition by ancient Greek physicians, and the etiological agents have been known only for the last eight decades. The causative agent is the influenza virus, which has three main types: A, B, and C. Type A is capable of circulating within many different biological reservoirs, including humans, swine, and birds. It also has high genetic variability, which allows it to have minor antigenic drifts or mutations which are responsible of epidemics in humans. Sometimes changes are quite marked, leading to pandemics. In a globalized world, with more than 6 billion inhabitants, with many social inequities and evident climate changes, influenza viruses are a permanent risk for mankind. Clinical features for the different viral subtypes may vary from subtle infections to full blown and severe, life-threatening forms. Event with the great advances in diagnostics and immunization, the manufacture and distribution of new diagnostic kits may take some time, and new vaccines are not always readily available. Specific therapies against influenza are not well developed. There are two groups of drugs, the so called adamantane derivatives, such as amantadine and rimantadine, and the neuraminidase inhibitors, such as oseltamivir and zanamivir. Most vulnerable areas for the new flu pandemics include developing countries, particularly the poorest ones, so that the greatest effort must be made for helping these areas.

Cystic fibrosis (CF) is one of the relatively common inherited diseases caused by a mutation in the gene encoding for CFTR protein, which forms a chloride channel that significantly affects ion homeostasis and the associated fluid management of the cell. This disease mainly affects the respiratory and digestive systems, being the most life-threatening in the respiratory tract. Patients with CF suffer from frequent and recurrent respiratory infections that lead to the development of chronic inflammation and gradual destruction of lung tissue. These lung infections, which are caused mostly by the opportunistic pathogen Pseudomonas aeruginosa, are the most common cause of morbidity and mortality in these patients. At present, antibiotics are used in the treatment of Pseudomonas aeruginosa infections, but new methods of antibacterial therapy need to be found to overcome the development of resistance. In addition to active immunization of CF patients against Pseudomonas aeruginosa, their passive immunization with specific chicken antibodies directed against this pathogen offers promising possibilities. This dissertation thesis is aimed to verify the prophylactic potential of hen IgY antibodies against the virulence factor Pseudomonas aeruginosa - lectin PAIIL, and to further develop an experimental...