Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Genes involved in hemorrhagic lesions on infected chicken chorioallantoic membranes (CAMs)

This thesis consists two parts. In the first part, a knockout library of the genome of CPXV strain Brighton Red was constructed, based on a previously described full-length BAC clone termed pBRF (92). Mediated by Red recombination in E. coli single single open reading frames (ORF) of CPXV BR were deleted by the insertion of a sequence containing stop codons and a kanamycin resistance gene. Upon reconstitution of each of the mutant virus genomes, different viral phenotypes were characterized and genes essential or non-essential for CPXV replication were identified. In the second part, single deletion viruses reconstituted in the first part and kelch-like deletion mutants were used to screen for factors involved in the red pock phenotype on CAM. Most of the mutant viruses produce hemorrhagic red pocks on infected CAM, while 10 single deletion mutants induced non-hemorrhagic white pocks.