published_or_final_version / Zoology / Master / Master of Philosophy

Background:  The PDZ domain is a peptide sequence of 80-90 amino acids and can be found in e.g. bacteria, animals and plants. These domains are commonly part of the cytoplasmic and membrane adapter proteins and its function are important in protein-protein interactions. The NS1 proteins of influenza A viruses play an important role in inhibiting the IFN-β production in many ways. In the C-terminus of the NS1 protein, a peptide sequence of four amino acids had been demonstrated to bind to the PDZ domain termed as PDZ binding motif (PBM). Objective:  The aim of this study is to determine whether the PBM sequence of the NS1 protein of influenza A virus plays a key roll in the expression of interferon-β. Methods:  The open reading frame of the NS1 protein was amplified and cloned into expressing vector and transfected into A549 cells along with a reporter plasmid containing ISRE promoter, driving expression of firefly luciferase. Dual luciferase reporter assay was performed to measure luciferase activity which represented expression of IFN-β. The assay was performed only once and unfortunately the result can not be trusted since the negative control showed positive value. Therefore, to understand the interaction between the PBM sequence of NS1 proteins and the production of IFN-β, further experiments are needed.

In this study, the expression of HA, a key immunogenic protein of influenza viruses, in insect cells was compared using three baculovirus expression strategies: protein over-expression, surface (GP64) display, and capsid (VP39) display. Further, a recombinant virus expressing NA, another immunogenic influenza virus protein, was generated and fused to an HA epitope-tag. Western immunoblot using various antibodies, including those against HA, demonstrated the expression of HA and NA for all recombinant viruses. HA showed stronger expression when fused to the C-terminus of VP39 than the N-terminus, but unlike other expression methods, there was no observable cleavage of HA in VP39-displayed viruses. Cells infected with only over-expressed and surfaced-displayed HA were biologically active, and capable of hemadsorption and hemagglutination of chicken red blood cells. These results suggest that GP64 display or over-expression are the most efficacious modes of HA-expression for use as antigen to detect anti-HA antibodies in poultry. / NSERC, OGS, OMAFRA, CPRC

Paukščių gripas yra virusinė infekcija, kuri lengvai plintanti pavojinga užkrečiamoji paukščių liga, šia liga serga laukiniai ir naminiai paukščiai. Patys pavojingiausi ir patogeniškiausi yra paukščių gripo H5 ir H7 viruso potipiai. Didžiausią susirūpinimą ir nerimą kelia H5N1 paukščių gripo virusas, kuris priklauso Influenzavirus A viruso atmainai. Svarbiausias antigenas, kuris sukelia apsauginių antikūnų produkciją, o taip pat svarbus viruso prisijungimui prie ląstelių pirmosiose infekcijos stadijose, yra viriono apvalkalo glikoproteinas - hemagliutininas. Pagal hemagliutinino antigenines savybes virusas skirstomas į 16 potipių (H1-H16). Žmonės serga H1, H2 ir H3 potipiais, tačiau visada išlieka galimybė, kad įvykus geno mutacijoms, poslinkiams ar pasikeitus genetinei informacijai pavojingi taps ir likę potipiai. Todėl yra būtina stebėti, kokie A tipo gripo viruso potipiai vyrauja. Darbe yra apžvelgta didžiųjų ančių (Anas platyrhynchos) bendra charakteristika, literatūriniai duomenys apie paukščių gripo virusą, aprašytas polimerazinės grandininės reakcijos metodas, tikro laiko ir atvirkštinės transkripcijos polimerazinės grandininės reakcijos eiga. Skyriuje „Medžiagos ir metodai“ aprašyta, kokia medžiaga buvo naudota tyrimuose, aprašytas RNR išskyrimas ir gryninimas, naudojami rinkiniai. Aprašyta paukščių gripo A viruso H5 ir H7 potipių nustatymas vieno žingsnio realaus laiko atvirkštinės transkripcijos PGR būdu. Iš viso 949 vandens paukščių mėginiai buvo surinkti Lietuvoje... [toliau žr. visą tekstą] / Avian influenza is a viral infection that is easily spread through a dangerous contagious disease of birds that disease sick in wild and domestic birds. The most dangerous and pathogenicity are avian influenza H5 and H7 virus subtypes. The biggest concern and disturbing of the H5N1 avian influenza virus, which belongs to Influenza A Virus species. The aim was to analyze the mallards which was infected by avian influenza virus in Lithuania At work is reviewed mallards (Anas platyrhynchos) general characteristics, the literature results of the avian influenza virus, described polymerase chain reaction method and real time reverse transcriptase polymerase chain reaction process. In chapter „Materials and Methods” describes what material was used in the research described in RNA isolation and purification for use in kits. Described by avian influenza A virus of the H5 and H7 subtypes of setting a one-step real-time reverse transcription PCR. 319 influenza A virus samples from which 22 samples were positive for influenza A virus. Also during the year 2007 to get 630 influenza A virus samples from 43 of the positive samples. After the 2007 and 2011 data analysis reveals that the majority of the positive influenza A samples of migratory birds have been obtained during the cold season. Haemagglutinin subtypes used successfully to identify by the Blast search GenBank database. Avian influenza viruses positive samples were identified exclusively from mallards, hunted in various... [to full text]

Thesis (D.P.H.)--University of Michigan.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 78-89) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 78-89) Also available online.

Dissertation (D.P.H.)--University of Michigan.

abstract: Avian influenzas are zoonoses, or pathogens borne by wildlife and livestock that can also infect people. In recent decades, and especially since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in 1996, these diseases have become a significant threat to animal and public health across the world. HPAI H5N1 has caused severe damage to poultry populations, killing, or prompting the culling of, millions of birds in Asia, Africa, and Europe. It has also infected hundreds of people, with a mortality rate of approximately 50%. This dissertation focuses on the ecological and socioeconomic drivers of avian influenza risk, particularly in China, the most populous country to be infected. Among the most significant ecological risk factors are landscapes that serve as “mixing zones” for wild waterfowl and poultry, such as rice paddy, and nearby lakes and wetlands that are important breeding and wintering habitats for wild birds. Poultry outbreaks often involve cross infections between wild and domesticated birds. At the international level, trade in live poultry can spread the disease, especially if the imports are from countries not party to trade agreements with well-developed biosecurity standards. However, these risks can be mitigated in a number of ways. Protected habitats, such as Ramsar wetlands, can segregate wild bird and poultry populations, thereby lowering the chance of interspecies transmission. The industrialization of poultry production, while not without ethical and public health problems, can also be risk-reducing by causing wild-domestic segregation and allowing for the more efficient application of surveillance, vaccination, and other biosecurity measures. Disease surveillance is effective at preventing the spread of avian influenza, including across international borders. Economic modernization in general, as reflected in rising per-capita GDP, appears to mitigate avian influenza risks at both the national and sub-national levels. Poultry vaccination has been effective in many cases, but is an incomplete solution because of the practical difficulties of sustained and widespread implementation. The other popular approach to avian influenza control is culling, which can be highly expensive and raise ethical concerns about large-scale animal slaughter. Therefore, it is more economically efficient, and may even be more ethical, to target the socio-ecological drivers of avian influenza risks, including by implementing the policies discussed here. / Dissertation/Thesis / Doctoral Dissertation Biology 2018

O vírus de influenza aviária H5N1 tem se disseminado rapidamente por diversos países e continentes nos últimos anos, gerando grandes perdas econômicas e de vidas humanas. Existe a possibilidade de a doença chegar ao Brasil, o que provocaria elevado impacto sobre a economia, especialmente sobre o setor avícola. A ausência e a necessidade de estimativas de impacto econômico no país, derivadas de surtos de gripe aviária em território brasileiro, motivaram a presente pesquisa. Visando gerar tais estimativas e analisá-las, foram simulados três cenários utilizando-se de um modelo aplicado de equilíbrio geral inter-regional, denominado TERM-BR. O primeiro cenário (Cenário I) contempla um foco da doença no Rio Grande do Norte, região Nordeste do país. O segundo (Cenário II) simula a presença de diversos focos da doença no estado de São Paulo. O terceiro (Cenário III) considera o surgimento de diversos focos presentes nos estados Rio Grande do Norte, Rondônia, São Paulo e Rio Grande do Sul. Os resultados sinalizam impactos de maior dimensão na medida em que os focos da doença surgem próximos aos mercados produtores, exportadores e consumidores, como é o caso da região Sul e Sudeste. Na região Sul, em função da dimensão da avicultura na economia local, a crise do setor avícola acaba refletindo negativamente e acentuadamente sobre o conjunto da economia local. Nas regiões Norte e Nordeste, as restrições sobre a aquisição de aves vivas pelas famílias têm grande peso na retração da atividade econômica do setor avícola, já que a atividade de abate é pouco representativa e parcela importante das aves é adquirida diretamente pelas famílias, o que não acontece na mesma dimensão nas demais regiões do país. Observa-se também que o aumento do consumo de carne bovina e suína contribui para reduzir a crise na economia estadual nos estados produtores, e é também a fonte de crescimento para os estados em que a bovinocultura se destaca. O choque de demanda doméstica de produtos avícolas, comparado com os demais choques, revela-se como o principal responsável pelo comportamento na produção em quase todos os estados. Já a redução das exportações tem grande peso sobre o comportamento da produção avícola quando o foco da doença é na região exportadora ou próxima a ela. No estado de Santa Catarina, o choque de exportações prepondera sobre a queda do consumo doméstico quando do fechamento quase total dos mercados externos para carne de aves. Por último, o choque de oferta, via mortalidade das aves e destruição de ovos, pouco influencia a magnitude da queda na produção dos produtos da avicultura. / In the past few years, the bird flue virus H5N1 spread rapidly through various countries and continents, causing great economic and human losses. There is also the possibility of the disease arriving in Brazil, which would have a substantial impact on the country\'s economy, particularly on its poultry sector. The present study addresses the lack of estimates of the potential economic consequences of a bird flu outbreak on commercial poultry production in Brazil. The analysis consists of three simulations using a interregional general equilibrium model called TERM-BR. The first scenario focuses on an outbreak in the state of Rio Grande do Norte in the Northeastern part of the country. The second scenario simulates an outbreak at various places in the State of Sao Paulo, and the third scenario assesses the consequences of a bird flue outbreak in various states simultaneously, Rio Grande do Norte, Rondônia, São Paulo and Rio Grande do Sul. The results indicate greater economic impact when the outbreak occurs close to points of production and consumption, which is the case in the Southern and Southeastern regions of Brazil. In the South, where the poultry sector constitutes a larger share of the local economy, a potential avian flu outbreak will also have a greater negative economic impact. In the Northern and Northeastern region, live birds are generally purchased and slaughtered directly by individual families living in suburban and small town settings rather then by large scale processors and packinghouses. This characteristic explains why potential restrictions on these small purchases of live birds will have a very large negative economic impact on the poultry sector in the affected states. A possible reduction in poultry supply could however be offset by an increase in beef and pork consumption, thereby softening the economic affect of a bird flue outbreak by promoting growth of the beef and pork industry. In most states, this fall of domestic poultry demand is the primary cause for a shift in production. In contrast, the fall in export demand only weighs heavily on the local economy when the outbreak occurs close to exporting regions. In the state of Santa Catarina, for example, the effect of a fall in export demand dominates the effect of a fall in domestic demand, as export markets are almost completely shutdown. Finally, the reduction in poultry supply through death of infected birds and destruction of eggs, has little affect on the decrease of poultry production.