Abstract
BackgroundPseudorabies virus is a widely-studied model organism of the Herpesviridae family, with a compact genome arrangement of 72 known coding sequences. In order to obtain an up-to-date genetic map of the virus, a combination of RNA-sequencing approaches were applied, as recent advancements in high-throughput sequencing methods have provided a wealth of information on novel RNA species and transcript isoforms, revealing additional layers of transcriptome complexity in several viral species.ResultsThe total RNA content and polyadenylation landscape of pseudorabies virus were characterized for the first time at high coverage by Illumina high-throughput sequencing of cDNA samples collected during the lytic infectious cycle. As anticipated, nearly all of the viral genome was transcribed, with the exception of loci in the large internal and terminal repeats, and several small intergenic repetitive sequences. Our findings included a small novel polyadenylated non-coding RNA near an origin of replication, and the single-base resolution mapping of 3′ UTRs across the viral genome. Alternative polyadenylation sites were found in a number of genes and a novel alternative splice site was characterized in the ep0 gene, while previously known splicing events were confirmed, yielding no alternative splice isoforms. Additionally, we detected the active polyadenylation of transcripts earlier believed to be transcribed as part of polycistronic RNAs.ConclusionTo the best of our knowledge, the present work has furnished the highest-resolution transcriptome map of an alphaherpesvirus to date, and reveals further complexities of viral gene expression, with the identification of novel transcript boundaries, alternative splicing of the key transactivator EP0, and a highly abundant, novel non-coding RNA near the lytic replication origin. These advances provide a detailed genetic map of PRV for future research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0470-0) contains supplementary material, which is available to authorized users.
Highlights
Pseudorabies virus is a widely-studied model organism of the Herpesviridae family, with a compact genome arrangement of 72 known coding sequences
Assessment of the Pseudorabies virus (PRV) transcriptome by total RNA sequencing and polyadenylation sequencing (PA-Seq) For the investigation of the lytic PRV transcriptome, porcine kidney (PK-15) epithelial cells were infected with a high dose (10 pfu) of PRV strain Ka
In our modified polyadenylation sequencing (PA-Seq) protocol [14], total RNA was reverse-transcribed by using custom designed oligo(T10-VN) anchored primers containing standard Illumina strand-specific adaptor sequences
Summary
Pseudorabies virus is a widely-studied model organism of the Herpesviridae family, with a compact genome arrangement of 72 known coding sequences. PCR is inconvenient for the detection of novel transcripts Coding sequences and their related transcripts have been widely studied in PRV [5, 7], together with the microRNA expression in both the lytic and latent phases of the viral life cycle [8, 9], whereas other sources of non-coding transcription, alternative transcript termination and alternative splicing have not yet been analyzed at a genome-wide level. Transcriptome-wide profiling has led to the discovery of novel regulatory RNAs and an accurate assessment of their expression in several members of the Herpesviridae (human cytomegalovirus: [10], anguillid herpesvirus 1: [11]) These studies have discovered highly abundant long non-coding RNAs (lncRNAs), while in addition, the characterization of the MAT ncRNA in murine cytomegalovirus has shown its role as a lncRNA, and coding for an ORF with potential regulatory functions [12]. The PA sites have been categorized in terms of relative expression levels by determining the overall frequency of proximal and distal PA-site usage per gene
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