Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. Includes bibliographical references.

Orientador: Hélio José Montassier / Banca: Antonio Carlos Paulillo / Banca: Ricardo Luiz Morode Sousa / Resumo: Existem diversos trabalhos publicados sobre métodos de diagnóstico da Doença de Newcastle (DN) em aves comerciais da espécie Gallus gallus. Entretanto, é escassa a literatura sobre o desenvolvimento de técnicas para o diagnóstico laboratorial da infecção pelo vírus da Doença de Newcastle (VDN) em espécies de aves não-galiformes. O presente trabalho foi delineado com o objetivo de desenvolver e aplicar a técnica de bloqueio de fase líquida de ELISA com a concanavalina A (BFL-Con A-ELISA) para a detecção e quantificação de anticorpos contra o VDN em soros de pombos de vida livre e da técnica de Semi-nested-RTPCR para detecção do vírus da DN (VDN) em suabes cloacais das mesmas aves. Na comparação entre o BFL-Con A-ELISA e o HI para a detecção de anticorpos em 107 amostras de soros de pombos, foram obtidos uma correlação significativa com o teste de HI (r = 0,875), bem como valores elevados de sensibilidade (100%), especificidade (95,8%), acurácia (96,3%) e concordância (κ = 0,83). Os resultados obtidos na técnica de semi-nested-RT-PCR mostraram que nove das 101 amostras analisadas foram positivas para o VDN. Na análise comparativa dos resultados da técnica do semi-nested-RT-PCR com os obtidos nas técnicas sorológicas, foram observados índices de especificidade relativa de 93,9% e 96,8% em relação aos métodos de HI e BFL-Con A-ELISA, respectivamente; além de uma máxima sensibilidade relativa (100%), comparativamente a esses testes. Esses dados demonstram que tanto o BFL-Con A-ELISA como o semi-nested-RT-PCR desenvolvidos no presente estudo foram eficientes no diagnóstico da DN, ou detectando anticorpos anti-VDN, ou esse agente viral, respectivamente, e ambas as técnicas apresentaram um grande potencial para serem usadas com vantagens no diagnóstico da infecção pelo VDN em pombos e em outras espécies de aves não-galiformes. / Abstract: A number of studies were done on diagnostic methods of Newcastle disease in commercial poultry (Gallus gallus, species). However the literature is scarce on the development of techniques for the diagnosis of Newcastle disease (ND) infection in non-galiforme and free-living birds. The present study aims to develop and apply a liquid-phase blocking ELISA with Concanavalin A (BFL-Con AELISA) for the detection and quantification of antibodies against ND virus (NDV) in sera of free-living pigeons and a semi-nested-RT-PCR technique for detection of NDV in cloacal swabs collected from these birds. The results of BFL-Con A-ELISA and HI were compared for the detection of antibodies in serum samples from 107 pigeons, and a significant correlation with the HI test was obtained (r = 0.875) as well as high values of relative sensitivity (100 %), specificity (95.8%), accuracy (96.3%) and agreement (k = 0.83). The results of semi-nested-RT-PCR showed that nine of the 101 samples were positive for the presence of NDV. By comparing the seminested- RT-PCR with serological tests, high relative specificity values of 93.9% and 96.8% were obtained with regard to HI and BLF-Con A-ELISA tests, respectively, as well as a maximum relative sensitivity (100%) was observed regarding these serological tests. These data demonstrate that the BFL-Con A-ELISA and seminested- RT-PCR developed in this study were effective in the diagnosis of NDV infection, detecting either specific antibodies, or this virus, respectively, and have a great potential to be used advantageously in the diagnostic of NDV infection in pigeons and other species of non-galliforme birds. / Mestre

Please read the abstract in the secton 00front of this document. / Thesis (PhD (Zoology))--University of Pretoria, 2007. / Zoology and Entomology / unrestricted

Diseña y analiza la generación de VLPs con membrana lipídica para un Adenovirus, que no posee membrana. Aprovecha la capacidad que tienen las principales proteínas estructurales del virus de la enfermedad de Newcastle (NDV), de tomar parte de la membrana del hospedero y autoensamblarse en viriones, para generar VLPs con envoltura conteniendo a la proteína Fibra-2 de FAdV-4. El primer paso es estandarizar la técnica de transfección con polietilenimina de 25 kDa (PEI25) en células DF-1. La eficiencia media máxima de transfección, medida en porcentaje de células que expresan EGFP, fue de 61.07%, la cual se obtiene utilizando 0.53 μg DNA más 1.59 μg de PEI25 por cm2 de células sembradas en monocapa. Luego, se expresa simultáneamente las proteínas Matriz (M) y Nucleoproteína (N) de NDV, con la proteína quimérica hnFib2. Esta última compuesta de la proteína Fibra-2 de FAdV-4 fusionada en su extremo N-terminal a los dominios citoplasmático y transmembrana de la proteína Hemaglutinina-Neuraminidasa (HN) de NDV, permitiendo la interacción de la Fibra-2 con la proteína M lo que facilita el ensamblaje de los viriones. Los VLPs purificados son evaluados por Western blot, obteniéndose bandas de ~40 kDa y ~55kDa positivas a suero anti-NDV correspondientes a las proteínas M y N, respectivamente; y a suero anti epítope CDSATMGNRPGDLNS de Fibra-2 obteniendo una banda de ~63 kDa. Esta investigación es el primer trabajo de la obtención de VLP para FAdV, dando un paso importante en el desarrollo de la siguiente generación de vacunas contra este patógeno. Estos VLP necesitan ser probados a nivel inmunológico para determinar su eficiencia como vacuna candidata contra FAdV-4. / Tesis

Newcastle disease (ND), caused by Newcastle disease virus, is an acute, contagious and pathogenic infection of pet, free living and domestic birds. ND is an epidemic disease and it is responsible for high economic losses due to up to 100 % mortality. The control of ND in the intensive commercial poultry farms is largely dependent on prophylactic immunisation using conventional vaccines. The ND V4 vaccine and its derivative ND V4-HR vaccine were selected originally for use in village chickens, due to their immunogenicity, thermostability, transmissibility and ease of administration. The efficacy of V4 and V4HR vaccines have been established in many Asian and African countries in their ability to challenge a wide range of recognised and local velogenic NDV. Therefore, ND V4 was tested for efficacy against B1172 challenge NDV isolated in south Africa in 1993. Twenty-eight one day-old replacement pullets were vaccinated by eye-drop route at 21 and 49 days old. Chickens vaccinated by eye-drop route were left to mingle with the unvaccinated in-contact chickens. At 63 days all chickens including the unvaccinated control group were individually challenged with B1172 NDV. Serological monitoring of NDV antibody response was done using HI and ELISA tests. The ND V4 vaccine induced full protection against B1172 NDV in chickens vaccinated by eye-drop vaccination and in 55 % of chickens vaccinated by the in-contact method. No association was seen between NDV antibody titer at pre-challenge and the ability to withstand B1172 challenge NDV. A fair to good agreement was seen between the HI and ELISA test in monitoring NDV antibody response during the experiment. Although, the ELISA showed a higher sensitivity and specificity than the HI test, further studies are required using this method of comparison. / Dissertation (MMed Vet (Poultry diseases))--University of Pretoria, 2002. / Production Animal Studies / unrestricted

Since 2002, the South African poultry industry has experienced outbreaks of Newcastle disease (ND) caused by a newly introduced virus (NDV) strain belonging to lineage 5d/VIId (“goose paramyxovirus” - GPMV). Control of the disease has proved difficult with commercially available vaccines appearing ineffective. In the first of two studies, broilers chicks were vaccinated with VG-GA vaccine (lineage II), then challenged with both GPMV and a “classic” challenge virus (RCV) of lineage 3d/VIII to compare the efficacy of the vaccine against both strains. In the second study, commercial and SPF hens in lay were vaccinated with La Sota vaccine and challenged with GPMV isolate, and immunohistochemistry staining used to determine the distribution pattern of viral antigen in the oviduct of the hens. The second study also compared the efficacy of cloacal and ocular routes of vaccination. The first study did not detect any statistically significant difference in protection offered by the vaccine against the GPMV strain in comparison to the RCV strain. The protection offered by the vaccine against challenge with both viruses was found to be dosedependant with 106.0 EID50 producing a 100% protection and 94.44% and 13.89% for 104.5 EID50 and 103.0 EID50 vaccination doses respectively. Protected birds did not manifest clinical signs, but still had macropathological lesions in some organs at necropsy. The computed protective doses (PD50 and PD90) for the VG-GA vaccine were 103.51 and 104.38 for GPMV and 103.79 and 104.43 for RCV. Results from the second study showed no clear difference in the protection of the oviduct from challenge with GPMV by either the cloacal and ocular routes of vaccination. Vaccinated birds were fully protected (100%) against challenge by La Sota vaccine, but not against infection and replication of the virus, as birds showed varying degrees of macropathology with numerous stained viral antigens in the oviducts demonstrated by immunohistochemistry. The susceptibility and colonisation of the oviduct of laying hens by both the lentogenic La Sota and the virulent NDV isolates was confirmed, with the uterus being more susceptible than magnum and isthmus. Necrosis and apoptosis of cells of the oviduct were not detected but cellular infiltration, gland dilatation and interstitial oedema were observed. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2009. / Production Animal Studies / unrestricted

The specific objectives of this study were to determine the Newcastle disease virus (NDV) antibody titres from the chicken sera collected from various districts and provinces of Zambia and to determine the seroprevalence of ND in Zambian backyard chickens. Results showed that 73.9 % of the birds sampled tested positive for Newcastle disease (ND) antibodies. The seroprevalence of Newcastle disease virus (NDV) in Zambian backyard chicken flocks varied among the five provinces sampled, ranging from 82.6 % in Eastern Province to 48.3 % in Luapula Province. The seroprevalence of the virus also varied among the 11 districts sampled, ranging from 91.3 % in Monze District of Southern Province to 22.8 % in Mufulira District of the Copperbelt Province. The results indicated that the seroprevalence of ND in Zambia has increased since the last survey conducted in 1994. The data generated is expected to contribute towards a more clear understanding of the epidemiology of NDV that would ultimately contribute towards an improved ND control programme to benefit all stakeholders in Zambia. An improved ND control programme is expected to enhance flock numbers and ultimately improve the dietary requirements and income needs of many poor households in the country. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Although Newcastle disease is reported to be endemic in Nigeria, little information exists on the molecular epidemiology and the lineage distribution of the Newcastle disease viruses (NDVs) in the country, especially in the live bird markets. Recent studies reported the identification of three distinct sub-lineages namely; 5f, 5g and 5h in West Africa, particularly sub-lineages 5f and 5g were identified in Nigeria. In this study a total of 41 NDV isolates were analysed. Thirty six NDVs were recovered from a variety of poultry species from live bird markets in the six geo-political zones of Nigeria during active surveillance from 2007 to 2008. Five NDVs recovered from outbreaks in backyard and commercial poultry farms within the same period were also genetically characterized. A commonly used region of the virus genome that spans nucleotide 61 to nucleotide 374 of the Fusion protein, including the cleavage site was targeted. Based on sequence analysis, 39 of the isolates were classified as virulent. Of these, 20 were classified as sub-lineage 5g and 17 were classified as sub-lineage 5f. One isolate differ markedly from all other strains included in the phylogeny. Interestingly, 13 strains from the 5f group formed a distinct cluster that was not identified by other groups in similar studies. Phylogenetic analysis, amino acid sequence determination of the F0 cleavage site sequence analysis, pair wise distance analysis of the partial fusion protein gene sequences and Geographic Information System (GIS) mapping was done. Results showed close genetic similarities and provided evidence for the first time of the epidemiological link between the viruses circulating in the LBMs and those identified in outbreaks in backyard and commercial farms in Nigeria between 2007 and 2008. The emergence and identification of new sub-lineages gives an insight in to the high rate of genetic drift occurring in NDV strains in Nigeria, and raises concerns about the efficacy of current NDV control measures in the country. Thus there is need for continuous surveillance and characterization of NDV from Nigeria to monitor the emergence of new lineages and sub-lineages in the Nigerian poultry industry. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Production Animal Studies / unrestricted

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

Many oncolytic virotherapies have shown great advantages for rapid, rational design through recombinant DNA technology to facilitate the targeting of a broad spectrum of malignancies. Newcastle disease virus (NDV), an avian paramyxovirus, is naturally tumor-selective and inherently oncolytic. Our approach is to develop NDV-based nanoparticles (VBNP) for oncolytic virotherapy. VBNPs are non-infectious and non-replicating and are relatively safe. We obtained VBNPs by co-expressing matrix (M), hemagglutinin (HN), and fusion (F) proteins of NDV in avian/ mammalian cells. The budding characteristics, size and morphology of VBNPs were similar to authentic virions. As a proof of concept, we engineered the apoptin (VP3) gene of chicken anemia virus in VBNPs and specifically targeted them to folate-receptor bearing tumor cells by surface conjugation to folate. The VBNPs killed tumor cells by apoptosis and induced proinflammatory and chemotactic cytokines. The VBNPs, although not curative, were able to limit the progression of xenotransplanted fibrosarcoma and malignant glioma tumors and provided a survival advantage in nude mice. We also engineered NDV M based particles with nipah virus surface glycorporteins to target ephrin B receptors. NDV based nipah Virus BNPs (NiV-ndBNP) were morphologically similar to authentic NiV virions. NiV glycoproteins were incorporated into the NDV M based particles, despite poor sequence homology in the transmembrane domain and cytoplasmic tails of glycoproteins. Our results suggest that VBNPs could be used to deliver small molecules, tumor antigens, anti-tumor/ reporter genes and also aid in generating tumor specific immunity by rational design. / Master of Science