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Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping

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@article{1c046ba8435b483b99f38dc0fedaa242,
title = "Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping",
abstract = "BACKGROUND: Direct molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection.RESULTS: In this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5{\%}) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order.CONCLUSIONS: This optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.",
author = "Johnston, {Camille Melissa} and Ulrik Fahn{\o}e and Belsham, {Graham J} and Rasmussen, {Thomas Bruun}",
year = "2018",
month = "8",
day = "1",
doi = "10.1186/s12864-018-4971-8",
language = "English",
volume = "19",
journal = "BMC Genomics",
issn = "1471-2164",
publisher = "BioMed Central Ltd",
number = "600",

}

RIS

TY - JOUR

T1 - Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping

AU - Johnston, Camille Melissa

AU - Fahnøe, Ulrik

AU - Belsham, Graham J

AU - Rasmussen, Thomas Bruun

PY - 2018/8/1

Y1 - 2018/8/1

N2 - BACKGROUND: Direct molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection.RESULTS: In this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5%) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order.CONCLUSIONS: This optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.

AB - BACKGROUND: Direct molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection.RESULTS: In this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5%) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order.CONCLUSIONS: This optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.

U2 - 10.1186/s12864-018-4971-8

DO - 10.1186/s12864-018-4971-8

M3 - Journal article

VL - 19

JO - BMC Genomics

JF - BMC Genomics

SN - 1471-2164

IS - 600

ER -

ID: 54943929