RESEARCH ARTICLE Creation of Functional Viruses from Non- Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

RNA viruses have the highest known mutation rates. Consequently it is likely that a high pro-portion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from amoderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeco-nomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12 % produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ances