Sequence adaptations during growth of rescued classical swine fever viruses in cell culture and within infected pigs.

Classical swine fever virus (CSFV) causes an economically important disease of swine. Four different viruses were rescued from full-length cloned cDNAs derived from the Paderborn strain of CSFV. Three of these viruses had been modified by mutagenesis (with 7 or 8 nt changes) within stem 2 of the subdomain IIIf of the internal ribosome entry site (IRES) that directs the initiation of protein synthesis. Rescued viruses were inoculated into pigs. The rescued vPader10 virus, without modifications in the IRES, induced clinical disease in pigs that was very similar to that observed previously with the parental field strain and transmission to in-contact pigs occurred. Two sequence reversions, in the NS2 and NS5B coding regions, became dominant within the virus populations in these infected pigs. Rescued viruses, with mutant IRES elements, did not induce disease and only very limited circulation of viral RNA could be detected. However, the animals inoculated with these mutant viruses seroconverted against CSFV. Thus, these mutant viruses were highly attenuated in vivo. All 4 rescued viruses were also passaged up to 20 times in cell culture. Using full genome sequencing, the same two adaptations within each of four independent virus populations were observed that restored the coding sequence to that of the parental field strain. These adaptations occurred with different kinetics. The combination of reverse genetics and in depth, full genome sequencing provides a powerful approach to analyse virus adaptation and to identify key determinants of viral replication efficiency in cells and within host animals.