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Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the family Poxviridae. CPXV is a large complex virus that infects a broad range of animals and can cause zoonotic infections. CPXV has the largest genome and the most complete set of genes among the orthopoxviruses, therefore CPXV has become a popular model for studying poxvirus genetics and pathogenesis. The bacterial artificial chromosome (BAC) clone of CPXV Brighton Red (BR) strain constructed in a previous study has enabled us to efficiently modify the CPXV genome for studying the virus.
In this thesis, a complete knockout library of the BAC clone of CPXV BR was generated and characterized. These mutants allow high-throughput and systematic assessments of the roles of single CPXV genes in virus infection and replication, as well as virus host range determination. Out of 183 mutant BAC clone, 109 mutant viruses were successfully reconstituted, suggesting that the 109 corresponding genes are not necessary for virus replication while the other 74 ORFs are essential.
As all mutant BAC clones contain dual fluorescent markers for early and late viral gene expression virus reconstitution can be monitored easily. Thus it was possible to discriminate between different phenotypes, such as no plaque formation, formation of ‘red fluorescence-only’ plaques, and formation of wild-type-like ’red and green fluorescence’ plaques.
The knockout library was also applied for another systematic screening, the identification of CPXV proteins involved in inducing hemorrhagic lesions (“red pocks”) on chicken chorioallantoic membranes (CAM). Although all orthopoxviruses can infect chicken CAM, only CPXV and rabbit poxvirus induce red pocks. Other orthopoxviruses produce non-hemorrhagic lesions (“white pocks”). Cytokine response modifier A (CrmA) of CPXV proved to be necessary but not sufficient for the induction of red pocks.
Another study showed that kelch-like proteins might also be involved in the hemorrhagic phenotype. By screening all the single knockout mutants and kelch-like deletion mutants, 10 proteins that are required for the formation of hemorrhagic lesions were identified and kelch-like proteins were proved to be not responsible for inducing red pocks.
In conclusion, the first complete targeted BAC knockout library of a large DNA virus was generated in this thesis. Reconstitution of mutant BAC clones provided important information about the importance of single viral genes for viral replication, and led to the identification of new phenotypes of CPXV. By screening CAM infection of the reconstituted mutant viruses, 10 proteins that are involved in inducing hemorrhagic lesions on chicken CAM was identified.
This discovery provided novel insights into the mechanisms of the formation of hemorrhagic lesions. In the future, this knockout library can be used for more high-throughput screens to study various biological characteristics of CPXV.