Role of small non-coding RNA in African swine fever virus infection

African swine fever virus (ASFV) is the causative agent of African swine fever, a lethal haemorrhagic disease of domestic swine and wild boar to which there is no vaccine available. ASFV is the only member of the Asfarviridae family though additionally belongs to the nucleocytoplasmic large DNA virus (NCLDV) superfamily, which also includes the Poxviridae. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins and has a complex replication cycle that occurs mainly in cells of the monocyte/macrophage lineage. ASFV can survive and replicate in these cells by modulation of several host-cell pathways. This thesis investigates the interplay between ASFV and the small non-coding RNA system to determine if manipulation of sncRNAs could be used to optimise ASFV live-attenuated vaccines. Small non-coding RNAs (sncRNAs) are involved in a wide range of cellular processes. Consequently, many viruses have evolved to manipulate them. Vaccinia virus (VACV), the prototypic poxvirus and NCLDV member induces widespread polyadenylation and decay of host microRNAs (miRNAs), a type of sncRNA. Using Northern blotting, no evidence was found that ASFV shares this ability with VACV, suggesting it is not a consistent feature of the NCLDV superfamily. In order to investigate the interplay between ASFV and sncRNAs in-depth, sequencing of small RNAs extracted from ASFV-infected primary porcine macrophages was undertaken. The data revealed that ASFV infection does not lead to widespread miRNA disruption, inducing the differential expression of only 6 out of 200 detected miRNAs. Closer analysis of the sequencing data identified a novel small RNA encoded by ASFV, which when overexpressed in cells infected with ASFV led to a small but significant reduction in ASFV growth. This work revealed that ASFV utilises a virally-encoded small RNA to regulate its own replication. The results indicated that the miRNA system remains intact during ASFV infection in macrophages; therefore, the potential to attenuate the virus using host miRNAs was investigated. Examination of the small RNA sequencing identified the miRNA, miR-142-3p to be heavily expressed in porcine macrophages. Addition of miR-142-3p target sites in the 3’ UTR of the essential ASFV gene E183L (P54) led to a reduced expression of P54 in a plasmid system. A recombinant ASFV with 2x miR-142- 3p target sites on the 3’ UTR of P54 was then generated. ASFV replicates only in primary macrophages in vitro, hindering the development of live attenuated vaccines. The ability of ASFV to replicate in stem-cell-derived porcine macrophages and Red River Hog (RRH) macrophages was therefore compared. ASFV replicated to similar levels in the stem-cellderived porcine macrophages as in the primary macrophage controls. In contrast, only low levels of replication was detected in the stem-cell-derived RRH macrophages, mimicking the in vivo infection kinetics. Overall, this study has revealed, in detail, the interaction between ASFV and the sncRNA system and the potential of this system to be utilised in the development of novel live attenuated ASFV vaccines.