A recombinant classical swine fever virus with a marker insertion in the internal ribosome entry site

Based on an infectious cDNA clone of classical swine fever virus (CSFV) strain Alfort/187 (Ruggli et al., J Virol 70, 3478-3487, 1996) a full-length cDNA was constructed harbouring a nonviral 44 base insertion in the internal ribosome entry site (IRES) within the 5' nontranslated region (5'NTR) of the genome. Genome size RNA transcribed in vitro served as a positive control in routine RT-PCR used to detect CSFV RNA in diagnostic material. Unexpectedly this RNA proved to be infectious upon transfection into susceptible cells. The replication kinetics of the resulting virus vA187-Ins44 were characterized and found to be indistinguishable from its parent virus. However, a deletion mutant with 29 of the 44 inserted bases missing was detected after multiple cell culture passages. RNA secondary structure analysis of the 5'NTR showed that the 44 base insertion destroyed a stem-loop structure and a pseudoknot previously described to be essential for virus replication, demonstrating that insertions within this functionally essential IRES element are tolerated by CSFV.