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 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, functiona...
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Public Library of Science (PLoS)
2015-01-01
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| Online Access: | https://doi.org/10.1371/journal.pone.0140912 |
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| author | Ulrik Fahnøe Anders Gorm Pedersen Carolin Dräger Richard J Orton Sandra Blome Dirk Höper Martin Beer Thomas Bruun Rasmussen |
| author_facet | Ulrik Fahnøe Anders Gorm Pedersen Carolin Dräger Richard J Orton Sandra Blome Dirk Höper Martin Beer Thomas Bruun Rasmussen |
| author_sort | Ulrik Fahnøe |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-fb1d9387425d44f9bc9e8c48871a29fc |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-fb1d9387425d44f9bc9e8c48871a29fc2025-08-20T02:22:37ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-011010e014091210.1371/journal.pone.0140912Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction.Ulrik FahnøeAnders Gorm PedersenCarolin DrägerRichard J OrtonSandra BlomeDirk HöperMartin BeerThomas Bruun RasmussenRNA 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.https://doi.org/10.1371/journal.pone.0140912 |
| spellingShingle | Ulrik Fahnøe Anders Gorm Pedersen Carolin Dräger Richard J Orton Sandra Blome Dirk Höper Martin Beer Thomas Bruun Rasmussen Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction. PLoS ONE |
| title | Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction. |
| title_full | Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction. |
| title_fullStr | Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction. |
| title_full_unstemmed | Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction. |
| title_short | Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction. |
| title_sort | creation of functional viruses from non functional cdna clones obtained from an rna virus population by the use of ancestral reconstruction |
| url | https://doi.org/10.1371/journal.pone.0140912 |
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