Functional analysis of replication determinantsin classical swine fever virus

Johanne Hadsbjerg

    Research output: Book/ReportPh.D. thesis

    307 Downloads (Pure)

    Abstract

    The ability to efficiently produce new viral progeny is an essential process in the viral life cycle.Virus replication is a complex process involving viral proteins, interactions within the viral genome and assistance from host proteins. Identification of replication determinants of important human and animal pathogens should facilitate finding new approaches for efficient disease control. The principal aim of this thesis is to characterise determinants involved in the replication of classical swine fever virus (CSFV). Classical swine fever is a highly contagious virus disease of domestic pigs and wild boar with great socio-economic importance for countries, like Denmark, that have a significant trade in agricultural production. CSFV has, like other RNA viruses, a high mutation rate allowing the virus to adapt to changes in selection pressure. Hence obtaining a more thorough knowledge of sequence variation during virus replication is crucial for the design of effective vaccines and for diagnostic purposes.This thesis is comprised of four parts: An overview consisting of a general introduction (part 1) and a discussion (part 4) which provides a review of CSFV biology with an emphasis on the viral life cycle and structural RNA elements important for virus replication. This establishes at heoretical background and provides perspectives for the work performed during this Ph.D.,which is outlined in part 2 that summarises the project aims and findings. These findings are described in-depth in part 3 comprising the three manuscripts included in the thesis. Manuscript 1 has focused on adaptations that occurred in the CSFV genome following introduction of mutations into the internal ribosome entry site (IRES) and after serial passaging in cell culture and when tested in vivo. Sequence adaptations occurred in the coding regions for NS2 and NS5B that restored the coding sequence to that of the parental field strain. When rescued viruses, containing mutant IRES elements, were introduced into pigs no induction of clinical disease could be observed and only limited levels of viral RNA were detected. In contrast, inoculation with the parental virus caused similar clinical symptoms as that observed with the parental field strain. Analysing the adaptation of the viruses throughout the entire viralgenome during virus replication allowed a more comprehensive understanding of the virusproperties beyond what can be explained by knowing just the consensus sequence. Manuscript 2 examines replication determinants by analysing a sequence motif in the codingregion of NS5B which has the possibility to form long-range RNA-RNA interactions. When synonymous mutations were introduced into this motif in a CSFV replicon (a self-replicatingbut non-infectious genome) reduced RNA replication was observed compared to a wild type replicon. However, this effect was not observed in the context of the infectious virus. Interestingly, an adaptive silent mutation in a conserved part of the structural CSFV protein E1 occurred upon virus passaging in cell culture. Knowledge of these sequence variations and putative long-range interactions will provide valuable insights into mechanisms underlying virustranslation and replication. In manuscript 3, a selection marker has been inserted into a CSFV-based replicon making it suitable for screening of antiviral drugs that block virus translation or RNA replication. The neomycin phosphotransferase coding sequence was chosen as a selection marker which confers resistance to neomycin. The neomycin-selectable replicon was demonstrated to be functional and designed with the aim of facilitating the maintenance of replicons by selection within mammalian cells. This might prove to be useful for the maintenance and expression of specific viral antigens in an efficient manner in order to produce more effective and safer prototype vaccine candidates.
    Original languageEnglish
    PublisherTechnical University of Denmark
    Number of pages98
    Publication statusPublished - 2016

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