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

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

African swine fever virus (ASFV) is the causative agent of African swine fever, a lethal haemorrhagic \ndisease of domestic swine and wild boar to which there is no vaccine available. ASFV is the only \nmember of the Asfarviridae family though additionally belongs to the nucleocytoplasmic large DNA \nvirus (NCLDV) superfamily, which also includes the Poxviridae. ASFV has a large, double-stranded \nDNA genome that encodes over 150 proteins and has a complex replication cycle that occurs mainly \nin cells of the monocyte/macrophage lineage. ASFV can survive and replicate in these cells by \nmodulation of several host-cell pathways. This thesis investigates the interplay between ASFV and \nthe small non-coding RNA system to determine if manipulation of sncRNAs could be used to optimise \nASFV live-attenuated vaccines. \n \nSmall non-coding RNAs (sncRNAs) are involved in a wide range of cellular processes. Consequently, \nmany viruses have evolved to manipulate them. Vaccinia virus (VACV), the prototypic poxvirus and \nNCLDV member induces widespread polyadenylation and decay of host microRNAs (miRNAs), a type \nof sncRNA. Using Northern blotting, no evidence was found that ASFV shares this ability with VACV, \nsuggesting it is not a consistent feature of the NCLDV superfamily. \n \nIn order to investigate the interplay between ASFV and sncRNAs in-depth, sequencing of small RNAs \nextracted from ASFV-infected primary porcine macrophages was undertaken. The data revealed that \nASFV infection does not lead to widespread miRNA disruption, inducing the differential expression of \nonly 6 out of 200 detected miRNAs. Closer analysis of the sequencing data identified a novel small \nRNA encoded by ASFV, which when overexpressed in cells infected with ASFV led to a small but \nsignificant reduction in ASFV growth. This work revealed that ASFV utilises a virally-encoded small \nRNA to regulate its own replication. \n \nThe results indicated that the miRNA system remains intact during ASFV infection in macrophages; \ntherefore, the potential to attenuate the virus using host miRNAs was investigated. Examination of \nthe small RNA sequencing identified the miRNA, miR-142-3p to be heavily expressed in porcine \nmacrophages. Addition of miR-142-3p target sites in the 3’ UTR of the essential ASFV gene E183L \n(P54) led to a reduced expression of P54 in a plasmid system. \n \nA recombinant ASFV with 2x miR-142- \n3p target sites on the 3’ UTR of P54 was then generated. \nASFV replicates only in primary macrophages in vitro, hindering the development of live attenuated \nvaccines. The ability of ASFV to replicate in stem-cell-derived porcine macrophages and Red River \nHog (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 \nreplication was detected in the stem-cell-derived RRH macrophages, mimicking the in vivo infection \nkinetics. \n \nOverall, this study has revealed, in detail, the interaction between ASFV and the sncRNA system and \nthe potential of this system to be utilised in the development of novel live attenuated ASFV \nvaccines.