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

Abstract

BackgroundDirect 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.ResultsIn 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.ConclusionsThis optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4971-8) contains supplementary material, which is available to authorized users.

Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants

“…Both phylogenetic reconstructions displayed low degrees of order, most likely due to the use of clonederived virus inocula for the infection experiments. This result is in agreement with those of our earlier studies, in which a low degree of order was seen in the phylogenetic reconstruction of cDNA clones from vKos_SL-infected serum (45), whereas, in contrast, a high degree of order was seen with cDNA clones obtained from a field isolate of the moderately virulent CSFV strain Roesrath (46).…”