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

Fahnøe U, Pedersen AG, Dräger C, Orton RJ, Blome S, Höper D, Beer M, Rasmussen TB - PLoS ONE (2015)

Bottom Line: To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones.Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo.Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

View Article: PubMed Central - PubMed

Affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark; Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Phenotypic and mutational distribution of the cDNA clones.A) Fraction of cDNA clones found to be infectious, replication competent, or non-functional. B) Number of mutations of different types (silent, missense, insertion/deletion and untranslated region), in the three functional cDNA classes (infectious, replication competent, and non-functional). The average number per cDNA clone is shown; error bars depict standard deviations. Non-functional cDNAs were found to have significantly more missense mutations than infectious cDNAs (p = 3.3E-5, Student's t-test). They also displayed significantly more mutations in total (p = 0.0002).
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pone.0140912.g001: Phenotypic and mutational distribution of the cDNA clones.A) Fraction of cDNA clones found to be infectious, replication competent, or non-functional. B) Number of mutations of different types (silent, missense, insertion/deletion and untranslated region), in the three functional cDNA classes (infectious, replication competent, and non-functional). The average number per cDNA clone is shown; error bars depict standard deviations. Non-functional cDNAs were found to have significantly more missense mutations than infectious cDNAs (p = 3.3E-5, Student's t-test). They also displayed significantly more mutations in total (p = 0.0002).

Mentions: Eighty-four unique complete cDNAs directly cloned into bacterial artificial chromosome (BAC) vectors were generated from full-length RT-PCR products obtained using RNA extracted from a fifth passage of the CSFV “Roesrath” isolate (termed CSFV_Roesrath_P5). RNA transcripts were produced from individual cloned cDNAs and were tested for replication competence in PK-15 cells. Transcripts from 15 of the cDNA clones (18%) were scored as functional and replicated in PK-15 cells whereas 69 cDNAs (82%) were non-functional without any indication of RNA replication (Fig 1A). For the 15 replicating cDNA clones differences in replication efficiency were observed with varying phenotypes ranging from all cells producing viral proteins to only a few small foci of infected cells. In order to address these differences, harvests from cells displaying CSFV protein production following introduction of the viral RNA transcripts were passaged once on PK-15 cells to establish whether infectious virus had been produced. Ten cDNA clones (12%) were identified as producing virus progeny after this additional passage and were classified as “infectious” whereas the cDNA clones yielding non-infectious RNA transcripts, although producing detectable viral protein, were termed “replication competent” (Fig 1A).


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

Fahnøe U, Pedersen AG, Dräger C, Orton RJ, Blome S, Höper D, Beer M, Rasmussen TB - PLoS ONE (2015)

Phenotypic and mutational distribution of the cDNA clones.A) Fraction of cDNA clones found to be infectious, replication competent, or non-functional. B) Number of mutations of different types (silent, missense, insertion/deletion and untranslated region), in the three functional cDNA classes (infectious, replication competent, and non-functional). The average number per cDNA clone is shown; error bars depict standard deviations. Non-functional cDNAs were found to have significantly more missense mutations than infectious cDNAs (p = 3.3E-5, Student's t-test). They also displayed significantly more mutations in total (p = 0.0002).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4613144&req=5

pone.0140912.g001: Phenotypic and mutational distribution of the cDNA clones.A) Fraction of cDNA clones found to be infectious, replication competent, or non-functional. B) Number of mutations of different types (silent, missense, insertion/deletion and untranslated region), in the three functional cDNA classes (infectious, replication competent, and non-functional). The average number per cDNA clone is shown; error bars depict standard deviations. Non-functional cDNAs were found to have significantly more missense mutations than infectious cDNAs (p = 3.3E-5, Student's t-test). They also displayed significantly more mutations in total (p = 0.0002).
Mentions: Eighty-four unique complete cDNAs directly cloned into bacterial artificial chromosome (BAC) vectors were generated from full-length RT-PCR products obtained using RNA extracted from a fifth passage of the CSFV “Roesrath” isolate (termed CSFV_Roesrath_P5). RNA transcripts were produced from individual cloned cDNAs and were tested for replication competence in PK-15 cells. Transcripts from 15 of the cDNA clones (18%) were scored as functional and replicated in PK-15 cells whereas 69 cDNAs (82%) were non-functional without any indication of RNA replication (Fig 1A). For the 15 replicating cDNA clones differences in replication efficiency were observed with varying phenotypes ranging from all cells producing viral proteins to only a few small foci of infected cells. In order to address these differences, harvests from cells displaying CSFV protein production following introduction of the viral RNA transcripts were passaged once on PK-15 cells to establish whether infectious virus had been produced. Ten cDNA clones (12%) were identified as producing virus progeny after this additional passage and were classified as “infectious” whereas the cDNA clones yielding non-infectious RNA transcripts, although producing detectable viral protein, were termed “replication competent” (Fig 1A).

Bottom Line: To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones.Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo.Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

View Article: PubMed Central - PubMed

Affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark; Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus