2026-09-24T23:22:54.931637702Zhttps://repository.nwu.ac.za/server/oai/request

oai:repository.nwu.ac.za:10394/380082021-11-26T09:27:06Zcom_10394_26463col_10394_26478

Bezuidenhout, C.C. Jarvis, M.A. Lebea, P.J. Maremagae, Ramaesela Sharon 12540110 - Bezuidenhout, Cornelius Carlos (Supervisor) 2021-11-26T08:27:37Z 2021-11-26T08:27:37Z 2021 https://orcid.org/0000-0002-7632-3602 http://hdl.handle.net/10394/38008 PhD (Microbiology), North-West University, Potchefstroom Campus Avian influenza (AI) is an infectious viral disease in birds caused by a type A influenza virus of the family Orthomyxoviridae. These viral outbreaks usually cause significant economic losses to the poultry industry. Conventional strategies for AI detection and control using vaccination both start by growing the viruses and isolating them in embryonated chicken eggs. This entire process takes 6 to 8 months from viral detection to the release of the vaccine for that particular strain. A rapid and cost effective diagnostic method for the early detection of AI viruses, as well as a more effective vaccine that can be produced quickly, are urgently needed as tools for AI outbreak management. Therefore, the aim of this study was to develop a strategy for the point-of-care (POC) detection of AI and to develop a vaccine that is not only effective in the treatment of such outbreaks, but can also be produced quickly enough to manage AI viruses before they spread and cause an outbreak. Loop-mediated isothermal amplification (LAMP), which is a rapid nucleic acid amplification assay that takes 30 to 60 minutes, was used for POC diagnosis of the AI viruses. Three LAMP assays were designed − one for the detection of general AI strains and the other two for the specific detection of the H5 and H7 AI subtypes. The device used to make the detection of these viruses at the POC possible was the Axxin T16 isothermal instrument. This device is ideal for POC assays as it is portable, the results can be detected in real time, and it operates on rechargeable batteries. Using the designed LAMP assays, the identification of the AI samples that were tested was successfully achieved within 30 minutes at the POC without having to transport the samples to a laboratory. The Adenovirus vectors are known as suitable recombinant vaccine vectors because they are capable of infecting a wide variety of cell types, can grow to high titres in vitro, and cannot integrate in the host genome. Due to these features, the Adenovirus was used in this study to design a safe and effective vaccine that can be produced quickly. However, policies of the Department of Agriculture, Land Reform and Rural Development (DALRRD) formerly known as the Department of Agriculture, Forestry and Fisheries (DAFF) prohibit vaccination against AI as conventional vaccines previously interfered with surveillance programmes that did not allow any differentiation between infected and non-infected poultry that had been vaccinated using diffierentiation of infected and vaccinated animal (DIVA) strategies. To prove that the strategy proposed by this study will address the challenges that conventional AI vaccines pose, the Newcastle disease virus (NCDV) was used as a target virus in the design of the Adenovirus vector-based vaccine. The vaccine was designed by inserting the Matrix gene of the NCDV into the Adenovirus vector. The functionality and propagation of the recombinant Adenovirus were performed in 293T human embryonic kidney (HEK) cells. This was followed by testing the recombinant Adenovirus in vitro in chicken embryo fibroblasts (CEFs) that were prepared inhouse. The results demonstrated the successful expression of the recombinant Adenovirus in the CEFs, which suggests that the vaccine design strategy that was developed in this study may be successful for future application as the vaccine showed the desired expression in vitro. These results also suggest that the same strategy may be used for the design of Adenovirus-based vaccines against AI and any other viral diseases in chickens. Even more noteworthy is that the current strategy takes only 5 weeks from viral detection to vaccine production, while the conventional strategy takes up to 6 or 8 months for the entire process to be completed. However, despite these exciting results, clinical trials using live chickens will have to be conducted in future studies to check how safe and effective these vaccines are. This strategy may even be efficacious in solving the COVID-19 crisis that the world is currently facing. Doctoral en North-West University (South Africa) Avian influenza Adenovirus Chicken embryo fibroblasts Loop-mediated isothermal amplification Point-of-care Polymerase chain reaction Vaccine Strategy for speedy field diagnosis and control of avian influenza viruses (AIV) outbreak at the point of contact/care (POC) Thesis open.access