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

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

Y1 - 2018

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.

KW - Bacterial artificial chromosome

KW - Genome

KW - Haplotyping

KW - Pestivirus

KW - RNA

KW - RNA virus

U2 - 10.1186/s12864-018-4971-8

DO - 10.1186/s12864-018-4971-8

M3 - Journal article

C2 - 30092775

AN - SCOPUS:85054930890

SN - 1471-2164

VL - 19

JO - BMC Genomics

JF - BMC Genomics

M1 - 600

ER -