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The key for the survival of a virus is to copy its own genome into progeny genomes that allows continued reproduction. The mechanism behind this "copy function" or "replication" is a wellorganized process that involves the formation of a replication complex in the cell and interactions between the viral proteins. The replication process in single-stranded RNA viruses of positive polarity requires a particular enzyme, an RNA dependent RNA polymerase, that has no direct counterpart elsewhere in nature. The variable nature of rapidly evolving viral genomes, pose a constant challenge to the host, and in depth knowledge of the traits that determine the fitness of the virus in this regard are highly valuable. Recent advances in the field of molecular virology with methods to manipulate viral genomes have significantly helped to uncover these core mechanisms responsible for exploitation of the host. This includes aspects of the infection, evasion from host antiviral defense, genome replication and viral assembly. With special reference to a particular RNA virus, Classical swine fever virus (CSFV), this thesis deals with the elucidation of traits involved in replication of the viral genome. This is accomplished via the application of precisely bioengineered viral constructs and through the use of state-of-the-art virological methods. The presence of full-length cDNA sequences of RNA viruses within stable vectors has been the “holy grail” for the reverse genetics approaches, and for the rescue of bioengineered mutants. The availability, in our lab, of bacterial artificial chromosomes (BACs) containing full-length cDNA sequences which can be used to rescue three different CSFV strains with a spectrum of virulence, have been a central resource for this work. The thesis is composed of four parts: Part 1, gives a general introduction to RNA viruses, with the focus on viruses classified within the Flaviviridae. Next, pestiviruses are described with special attention to classical swine fever virus and the disease it is responsible for. A brief history of types of viral vaccines is provided, finishing with a description of the molecular methods used for viral cDNA manipulation, bio-engineering approaches, description of viral reporters and so forth. Part 2, "Pestiviruses: Infection and requirements for viral RNA replication " is meant as a walk through the literature describing Pestivirus/Classical swine fever virus replication determinants, including a thorough presentation of the viral proteins, and the involvement of these in the infection progress. Part 3, "The manuscripts", includes the papers published and submitted on this work. These describe the outcome of experiments performed during the three years. Manuscript I is a coauthored paper that describes a summary of the work I have been doing in my thesis dealing with the application of the Red/ET mediated homologous recombination method to modify viral cDNA. For proof of this method, CSFV/BDV chimeric clones were produced and characterized (Submitted paper, BMC genomics). Manuscript II describes the generation of replicons that express two different types of luciferases (Rluc and Gluc), and their application as a tool for easy monitoring of replication competence (published paper, Journal of General Virology (94), 1739-1748). Manuscript III describes the properties of chimeric replicons and infectious clones that include a RNA dependent RNA polymerase (NS5B) from one of three different CSFV strains with distinct virulence properties. The entire NS5B proved to influence replication competence and key residues for replication competence was identified as judged by reporter protein expression kinetics and from using infectious clones. Furthermore, evidence is provided that these specific single amino acid substitutions in the NS5B fingertip region, can influence the rate of viral RNA replication and virus spread. Part 4, is a summary and discussion of the general and overall conclusions and a walk through the milestones that have been achieved. Future perspectives and work that should be carried out are addressed as well.
|Place of Publication||Frederiksberg C|
|Publisher||National Veterinary Institute|
|Number of pages||135|
|Publication status||Published - 2013|
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- 1 Finished
Risager, P. C., Larsen, L. E., Becher, P., Lindberg, M., Belsham, G. & Rasmussen, T. B.
01/01/2010 → 18/12/2013