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Invasion and Replication Strategies of Pathogenes
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SP B8

RNA-Recombination of Pestiviruses


PD Dr. Paul Becher
Institut für Virologie (FB 10), Justus-Liebig-Universität, Gießen


Summary

In addition to point mutations due to the error-prone nature of viral RNA dependent RNA polymerases (RdRp), RNA recombination significantly contributes to genetic changes of RNA viruses which may alter host range and virulence of important human and animal pathogens. With regard to pestiviruses (being closely related to human Hepatitis C virus and classical flaviviruses like Dengue fever virus, West Nile virus, and Tick-borne encephalitis virus), cytopathogenic (cp) viruses emerge from noncytopathogenic (ncp) viruses via nonhomologous RNA recombination. In the case of bovine viral diarrhea virus (BVDV) the cp viruses play a crucial role in the pathogenesis of a lethal disease.
The major goal of the project is to study the molecular basis of RNA recombination using pestiviruses as a model. Analyses of cp pestiviruses led to the identification of sequence characteristics possibly involved in RNA recombination. To study fundamental aspects of RNA recombination we recently established an efficient RNA recombinaton system based on transfection of cells with in vitro transcribed RNAs. This system allows the quantification of the recombination events, the introduction of defined mutations in both recombination partners, as well as the efficient generation of recombinant viruses that are characterized at the molecular level. Using this system we could unequivocally proof that RNA recombination also occurs in the absence of a viral RdRps. It is currently not known whether the observed recombination events are due to replication independent reactions or, alternatively, result from a replicative mechanism mediated by cellular enzymes.

Future studies will concentrate on the following aspects:

(i)    identification and characterization of cellular and/or viral factors involved in RNA recombination

(ii)   identification and analysis of sequence and structural elements influencing the sites of RNA
       recombination (e.g. heteroduplex structures, base pairing)

(iii)  impact of the genetic relatedness between both recombination partners on frequency and
       mode of RNA recombination (homologous versus nonhomologous)

(iv) generation of recombinant pestiviruses for selected applications by the use of the established
      recombination system (e.g. expression of heterologous genes)

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