Avian paramyxoviruses (APMVs) consist of twelve distinct serotypes (APMV-1 to -12) isolated from a wide variety of domestic and wild birds. APMV-1/Newcastle disease virus (NDV) is the most characterized and globally important avian pathogen, because of the huge economic loss associated with the disease. However, very little information is known about the pathogenicity of APMV 2-12. APMV expresses six structural and two accessory proteins. The functions of APMV accessory proteins (V and W) are not fully established. Only the function of V protein in NDV is studied so far. V protein was found to be an IFN antgonist and a major virulent determinant of NDV. In this study, we tested for the potential functions of W protein in NDV and fuctions of V protein in other APMV serotypes. Vaccination failure is a major cause for NDV outbreak in developing and tropical countries, because of thermolabile nature of vaccine strains. Thermostable and thermolabile NDV strains exhibit difference in W protein length. In the first part of our study, we mutated the genome of a thermolabile NDV strain to express W protein of different lengths, rescued recombinant viruses by reverse genetics system and tested for thermostability. Our results showed that W protein does not confer thermostability to NDV. In the second part of study, we constructed plasmids expressing APMV -2, -3 and -6V proteins and tested for IFN antagonism by a dual luciferase reporter assay. Our results showed that APMV-3V acts as IFN antagonist by blocking IFN induction and thereby may play an important role in the evasion of innate immunity. / Master of Science

The aim of this study is to improve our understanding of the ecology, spatiotemporal patterns, and risk of infectious pathogens of migratory waterfowl, using the blue-winged teal (Anas discors, BWTE), as a model. From 2007-2010, 1,869 BWTE were sampled in the prairie provinces (Alberta, Saskatchewan and Manitoba, Canada) to examine infection status and/or evidence of previous exposure to avian influenza virus (AIV), West Nile virus (WNV), and avian paramyxovirus-1 (APMV-1), in relation to host demographic variables (age, sex, body condition, exposure to other pathogens), other ecological variables such as local waterfowl breeding population density and local pond density, and year. The probability of AIV infection depended on an interaction between age and AIV antibody status. Hatch year birds with antibodies to AIV were more likely to be infected, suggesting an antibody response to an active infection. After hatch year birds with antibodies to AIV were less likely to be infected, suggesting immunity resulting from previous exposure. AIV infection was positively associated with local BWTE density, supporting the hypothesis of density dependent transmission. Exposure to WNV and APMV-1 were also associated with age and year. Furthermore, the probability of WNV exposure was positively associated with local pond density rather than host population density, likely because ponds provide suitable breeding habitat for mosquitoes, the primary vectors for transmission. We also investigated large-scale spatiotemporal trends in apparent prevalence of AIV across Canada and the United States throughout the year, using data from national avian influenza surveillance programs in Canada and the US in 2007-2010. Our analyses revealed that age, sex, year of sampling, flyway, latitude, and season (categorized by stages of the BWTE annual life cycle) were all important variables in predicting probability of AIV infection. There was an interaction between age and season. During late summer staging (August) and fall migration (September-October), hatch year birds were more likely to be infected than after hatch year birds, however there was no difference between age categories for the remainder of the year (winter, spring migration, and breeding season). Probability of infection increased non-linearly with latitude, and was highest in summer, corresponding to the beginning of fall migration when densities of birds and the proportion of susceptible hatch year birds in the population are highest. Birds in the Pacific, Central and Mississippi flyways were significantly more likely to be infected compared to those in the Atlantic flyway. Observed trends in seasonal, annual, and geographic patterns of AIV infection in BWTE across Canada and the US were primarily driven by the dynamics of AIV infection in hatch year birds. Our results demonstrate demographic as well as seasonal, latitudinal and flyway trends across Canada and the US. This research provided further evidence for the role of wild dabbling ducks, particularly BWTE, in the maintenance and ecology of AIV. This improved understanding of the role of BWTE as natural hosts, and the geographic, demographic and temporal variables that affect infection and transmission parameters, moves us closer to deciphering the overall ecology of the virus and its transmission and transportation pathways at the individual, population and continental levels. This knowledge, in turn, will permit development of better tools to predict and perhaps to prevent possible outbreaks in domestic animals as well as in humans.

Columbiformes sinantrópicos podem ter um importante papel na epidemiologia de patógenos com potencial zoonótico ou de impacto econômico para a indústria avícola. Dentre eles destacam-se: Mycoplasma spp., Salmonella spp., paramixovírus aviário tipo 1 (APMV-1), inseridos no Programa Nacional de Sanidade Avícola (PNSA) e a Chlamydophila psittaci, agente de uma das principais zoonoses relacionada com aves silvestres. Dentro desse contexto, este trabalho objetivou pesquisar, sazonalmente, a ocorrência destes patógenos em pombos-domésticos (Columba livia) em dois entrepostos no Estado de São Paulo. Ao longo de um ano, mensalmente 10 pombos foram capturados em cada entreposto para a colheita de amostras de suabe cloacal e sangue. A técnica de soroaglutinação rápida em placa (SAR) foi utilizada para a detecção de anticorpos anti-M. synoviae, anti-M. gallisepticum e anti-S.Pullorum/Gallinarum; para a confirmação dos sororeagentes foram utilizadas a prova de inibição da hemaglutinação e soroaglutinação lenta, respectivamente. Para a detecção do DNA de C. psittaci e RNA de AMPV-1 foram utilizados métodos moleculares, PCR e RT-PCR. Para investigação de anticorpos anti-APMV-1 foi empregada a técnica de HI. Na SAR, 3,3% dos soros foram reagentes para M. synoviae; 2,5% para M. gallisepticum e 0,4% para S. Pullorum/Gallinarum. No entanto, essas amostras foram negativas nas técnicas confirmatórias. A ocorrência do APMV-1 não foi detectada. O DNA de C. psittaci foi detectado em 13,3% das amostras sendo 10,8% provenientes de aves capturadas na estação seca e 15,8% na estação chuvosa. Tais resultados são relevantes, pois demonstram que a C. psittaci ocorre em pombos presentes em áreas públicas frequentadas por um grande número de pessoas. Frente à escassez de pesquisas realizadas em Columbiformes no país, novos estudos são necessários para a determinação do real risco que pombos-domésticos podem representar quanto à transmissão de patógenos para aves comerciais e a influência da sazonalidade na disseminação desses microrganismos. / Columbiformes may play an important role in the epidemiology of pathogens with zoonotic potential or economic impact in the poultry industry. Among these pathogens there are Mycoplasma spp., Salmonella spp., Avian Paramyxovirus type 1 (APMV-1), included in the National Poultry Health Program (PNSA) and Chlamydophila psittaci, etiologic agent of an important zoonosis associated with wild birds. Thus, the aim of this study was to investigate, seasonally, the occurrence of the pathogens listed above in feral pigeons (Columba livia) in two warehouses in São Paulo State. During one year, 10 birds were captured monthly in each locality and cloacal swabs and blood samples were collected from each pigeon. The rapid seroagglutination test was performed for the detection of antibodies against M. synoviae, M. gallisepticum and Salmonella Pullorum/Gallinarum. Positive results were submitted to the hemagglutination inhibition and slow seroagglutination test, respectively. For the C. psittacis DNA and APMV-1s RNA diagnosis, molecular techniques PCR and RT-PCR were performed. Hemagglutination inhibition test was also performed in order to detect antibodies against APMV-1. From the serum samples analyzed by rapid seroagglutination test, 3.3% were positive for M. synoviae, 2.5% for M. gallisepticum and 0.4% for S. Pullorum/Gallinarum. However, none of these samples was positive on the confirmatory tests. APMV-1 was not detected in any of the laboratory tests used. C. psittacis DNA was detected in 13.3% of the samples being, 10.8% from pigeons captured during the dry season and 15.8% in the rainy season. These results are relevant since they indicate that C. psittaci occurs in birds living in public areas frequented by a large number of people. The occurrence of the other pathogens was not detected. Nevertheless, due to lack of information about the pigeons sanitary status in the country, additional researches are necessary to determine the risk that feral pigeons can pose in the transmission of pathogens for poultry and the influence of each season in the spread of these microorganisms.

Columbiformes sinantrópicos podem ter um importante papel na epidemiologia de patógenos com potencial zoonótico ou de impacto econômico para a indústria avícola. Dentre eles destacam-se: Mycoplasma spp., Salmonella spp., paramixovírus aviário tipo 1 (APMV-1), inseridos no Programa Nacional de Sanidade Avícola (PNSA) e a Chlamydophila psittaci, agente de uma das principais zoonoses relacionada com aves silvestres. Dentro desse contexto, este trabalho objetivou pesquisar, sazonalmente, a ocorrência destes patógenos em pombos-domésticos (Columba livia) em dois entrepostos no Estado de São Paulo. Ao longo de um ano, mensalmente 10 pombos foram capturados em cada entreposto para a colheita de amostras de suabe cloacal e sangue. A técnica de soroaglutinação rápida em placa (SAR) foi utilizada para a detecção de anticorpos anti-M. synoviae, anti-M. gallisepticum e anti-S.Pullorum/Gallinarum; para a confirmação dos sororeagentes foram utilizadas a prova de inibição da hemaglutinação e soroaglutinação lenta, respectivamente. Para a detecção do DNA de C. psittaci e RNA de AMPV-1 foram utilizados métodos moleculares, PCR e RT-PCR. Para investigação de anticorpos anti-APMV-1 foi empregada a técnica de HI. Na SAR, 3,3% dos soros foram reagentes para M. synoviae; 2,5% para M. gallisepticum e 0,4% para S. Pullorum/Gallinarum. No entanto, essas amostras foram negativas nas técnicas confirmatórias. A ocorrência do APMV-1 não foi detectada. O DNA de C. psittaci foi detectado em 13,3% das amostras sendo 10,8% provenientes de aves capturadas na estação seca e 15,8% na estação chuvosa. Tais resultados são relevantes, pois demonstram que a C. psittaci ocorre em pombos presentes em áreas públicas frequentadas por um grande número de pessoas. Frente à escassez de pesquisas realizadas em Columbiformes no país, novos estudos são necessários para a determinação do real risco que pombos-domésticos podem representar quanto à transmissão de patógenos para aves comerciais e a influência da sazonalidade na disseminação desses microrganismos. / Columbiformes may play an important role in the epidemiology of pathogens with zoonotic potential or economic impact in the poultry industry. Among these pathogens there are Mycoplasma spp., Salmonella spp., Avian Paramyxovirus type 1 (APMV-1), included in the National Poultry Health Program (PNSA) and Chlamydophila psittaci, etiologic agent of an important zoonosis associated with wild birds. Thus, the aim of this study was to investigate, seasonally, the occurrence of the pathogens listed above in feral pigeons (Columba livia) in two warehouses in São Paulo State. During one year, 10 birds were captured monthly in each locality and cloacal swabs and blood samples were collected from each pigeon. The rapid seroagglutination test was performed for the detection of antibodies against M. synoviae, M. gallisepticum and Salmonella Pullorum/Gallinarum. Positive results were submitted to the hemagglutination inhibition and slow seroagglutination test, respectively. For the C. psittacis DNA and APMV-1s RNA diagnosis, molecular techniques PCR and RT-PCR were performed. Hemagglutination inhibition test was also performed in order to detect antibodies against APMV-1. From the serum samples analyzed by rapid seroagglutination test, 3.3% were positive for M. synoviae, 2.5% for M. gallisepticum and 0.4% for S. Pullorum/Gallinarum. However, none of these samples was positive on the confirmatory tests. APMV-1 was not detected in any of the laboratory tests used. C. psittacis DNA was detected in 13.3% of the samples being, 10.8% from pigeons captured during the dry season and 15.8% in the rainy season. These results are relevant since they indicate that C. psittaci occurs in birds living in public areas frequented by a large number of people. The occurrence of the other pathogens was not detected. Nevertheless, due to lack of information about the pigeons sanitary status in the country, additional researches are necessary to determine the risk that feral pigeons can pose in the transmission of pathogens for poultry and the influence of each season in the spread of these microorganisms.