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

• Published in: PloS one Vol. 10; no. 10; p. e0140912
• Main Authors: Fahnøe, Ulrik, Pedersen, Anders Gorm, Dräger, Carolin, Orton, Richard J, Blome, Sandra, Höper, Dirk, Beer, Martin, Rasmussen, Thomas Bruun
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20.10.2015; Public Library of Science (PLoS)
• Subjects: Analysis; Animal welfare; Artificial chromosomes; Bioinformatics; Cell culture; Classical Swine Fever Virus - genetics; Cloning; Cloning, Molecular; Computer applications; DNA, Complementary - genetics; DNA, Viral - genetics; Evolution; Evolution, Molecular; Fever; Fitness; Gene sequencing; Genetic aspects; Genome, Viral; Genomes; Genomics; Haplotypes; Hog cholera; Hogs; In vivo methods and tests; Infections; Life sciences; Livestock; Mutation; Mutation rates; Phylogenetics; Phylogeny; Population; Reconstruction; Reproductive fitness; Ribonucleic acid; RNA; RNA viruses; RNA, Viral - genetics; Socio-economic aspects; Software; Sus scrofa; Swine; Swine production; Virology; Virulence; Virulence (Microbiology); Viruses; West Nile virus; United States; United Kingdom > UK; Denmark; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0140912

RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion 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 a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic 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 ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.