Objective:
The purpose of this project is to assess the role of the Classical Swine Fever Virus 3'UTR RNA conserved secondary structures in virus infectivity. CSV is a highly infectious disease of swine. Pigs vaccinated with live attenuated vaccines are indistinguishable from those infected with CSF, thus posing regulatory problems. These vaccines may not be fully attenuated and safe for use in immunosuppressed pigs. Rational engineering of new marker live-attenuated CSF vaccines require knowledge of the genetic basis of viral virulence and host range, and identification of nonessential regions of the viral genome suitable for introducing modifications. Additionally, we will assess the role of glycolsylation of Classical Swine Fever Virus envelope proteins E0, E1 and E2.

Approach:
To identify these biological significant genetic determinants, an infectious clone of a pathogenic CSF strain and an infectious clone of a non-pathogenic CSF vaccine strain have been developed at PIADC. Chimeric full-length constructs between these two clones have been successfully developed, and important determinants of virulence have been mapped utilizing this methodology. An alternative approach to assess the role of each viral protein and un-translated regions (UTRs) in CSFV virulence is to develop deletion mutants. Partial or complete deletions of those areas in a virulent CSFV will determine the role of viral proteins and UTRs on infectivity and growth kinetics. A striking difference between the LAV and virulent strains used in these studies is the insertion of 12 nucleotides in the 3'UTR of the attenuated virus. A computer alignment of these CSFV sequences, albeit slightly different, predicts the presence of three conserved stem-loops in the 3'untranslated region in both 3'UTRs. Investigating how the differences in CSFV 3'UTR between virulent and avirulent viruses affect virus infectivity and growth kinetics will be helpful to understand the evolution from virulent to avirulent viruses. Furthermore, since it has been shown that Pestivirus 3'UTRs exhibit a rather extensive RNA folding it is important to analyze the role of those structure in the cycle of CSFV. To address the role of the conserved secondary structures of the 3'UTR in CSFV cycle, we have begun a structural mapping of the 3'UTR. The entire 3'UTR was deleted from a clone encompassing the last 4000 nt of the CSFV genome. This clone will be used to rebuild the 3'UTR by adding oligonucleotides encompassing each of the conserved secondary structures predicted by computer modeling. The obtained clones will be then submitted to PIADC for assembly of full-length clones. Viruses obtained from those clones will be evaluated for genetic stability, ability to replicate in swine macrophages, growth kinetics, plaque morphology, and infectivity in swine. Glycosylation mutants of the three CSFV envelope proteins will be obtained by site directed mutagenesis of DNA plasmids containing partial sequence of the infectious DNA of virulent CSFV strain Brescia. PIADC will then assemble full-length infectous clones. Modified DNA encoding for these proteins will be cloned into the backbone of the full-lenght DNA clone pBIC. Viral RNA obtained in vitro will be used to transfect swine kidney cells, and rescued virus will be sequenced for assessing the presence of the introduced changes.