There are numerous vaccines available for the control of infectious bronchitis virus (IBV) in poultry, however protection is short-lived and poorly cross-protective between strains. The vaccines must currently be grown in embryonated eggs, a cumbersome and expensive process. The ability to grow vaccines on a cell-line such as Vero cells would be highly advantageous. The spike (S) glycoprotein of IBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in determination of cellular tropism and cell-to-cell fusion. The IBV Beaudette strain is able to replicate in both primary chick kidney (CK) cells and Vero cells, whereas the IBV M41 strain replicates in primary cells only. Recombinant IBVs with chimaeric S genes were generated using a reverse genetics system with the genomic background of Beaudette and part of the S gene from M41. Their growth characteristics and cellular tropism were investigated. The S2 subunit of Beaudette was found to be sufficient to confer the ability to grow on Vero cells and swapping just three amino acids with corresponding ones from M41 was sufficient to remove the ability of the Beaudette S glycoprotein for growth on Vero cells. Beaudette was further adapted to syncytia formation on Vero cells by serial passage and isolates were sequenced to identify amino acid changes between parent and Vero-adapted viruses that are potentially involved in cell-to-cell fusion. Understanding the way in which IBV infects host cells is vital in order to rationally design better vaccination and treatment strategies and help to reduce the prevalence of IBV infection in poultry worldwide. Using the IBV reverse genetics system, we now have the potential to grow IBV vaccines on Vero cells.

The aim of this Ph.D. was to develop advanced methods for quantitative proteomics and use these methods to investigate the presence of protein biomarkers of sperm performance, differential expression of sperm membrane proteins and differential expression of E.coli proteins. Quantitative analysis of E.coli generated analytical samples that were analysed with multiple mass spectrometers and with multiple software packages. Through these samples an optimal label free quantitative proteomic workflow was generated and software was thoroughly tested to determine the optimal software to be used for data analysis on varying biological questions. Identification of protein(s) that correlate with increased or decreased fertility would be economically beneficial. Currently semen samples are subject to quality control where general movement and morphological defects are studied, but this does not always correlate with the ejaculate passing a post cryopreservation quality control check or that specific bull generating offspring. Identification of a protein or set of proteins with abundance variation in bulls of known high or low fertility would allow lower fertility bulls to be removed from the breeding programme at an early age, reducing rearing costs, and would allow longitudinal health monitoring of individual bulls. Discovery of differentially expressed proteins in the membrane of sperm with the X or Y chromosome would allow the generation of a method to separate the two sperm populations. This will be beneficial as most livestock farmers would prefer offspring of a specific sex, either to sell or replenish animal stock. Quantitative analysis of proteins present in bovine seminal plasma led to the identification and quantitative comparison of the seminal plasma proteins present in two breeds of bull, Holstein and Belgian Blue and a quantitative comparison of the seminal plasma from two domestic farm animal species, bovine and porcine. Intra species comparisons determined no quantitative variation between the two breeds, while the inter species comparison determined variation between the proteins present in both species seminal plasma and the corresponding amounts of proteins present in both species. A quantitative comparison was performed to determine the expression of proteins from two strains of E.coli, a wild type strain (MG1655) and a genome depleted strain (MDS66), this led to the confirmation of gene deletions in the genome depleted strain due to their lack of protein products in mass spectrometric analysis, and the identification of proteins that were differentially expressed due to pleiotropic effects of these genome deletions. To investigate the proteins expressed in the sperm membrane a mass spectrometer compatible enrichment method was generated and membrane proteins were identified, quantified and compared between sperm expressing X and Y chromosomes. This study did not lead to the determination of any proteins with differential expression in the X or Y bearing sperm.