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

Camille Melissa Johnston, Ulrik Fahnøe, Graham J. Belsham, Thomas Bruun Rasmussen*

*Corresponding author for this work

    Research output: Contribution to journalJournal articleResearchpeer-review

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    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.
    Original languageEnglish
    Article number600
    JournalB M C Genomics
    Number of pages9
    Publication statusPublished - 2018

    Bibliographical note

    s This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.


    • RNA
    • Genome
    • Bacterial artificial chromosome
    • RNA virus
    • Haplotyping
    • Pestivirus

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