Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay

Dóra Tombácz, J. Tóth, Pál Petrovszki, Z. Boldogkői

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Background: Pseudorabies virus (PRV), a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection. Results: In this study, we have developed and applied a quantitative reverse transcriptase-based real-time PCR technique in order to profile transcription from the whole genome of PRV after lytic infection in porcine kidney cells. We calculated the relative expression ratios in a novel way, which allowed us to compare different PRV genes with respect to their expression dynamics, and to divide the PRV genes into distinct kinetic classes. This is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qRT2-PCR. We additionally established the kinetic properties of uncharacterized PRV genes and revised or confirmed data on PRV genes earlier examined by traditional methods such as Northern blot analysis. Our investigations revealed that genes with the same expression properties form clusters on the PRV genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. Further, we detected inverse relationships as concerns the expressions of EP0 and IE180 mRNAs and their antisense partners. Conclusion: Most (if not all) PRV genes begin to be expressed from the onset of viral expression. No sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral DNA synthesis. The expressions of the PRV genes were analyzed, categorized and compared by qRT2-PCR assay, with the average of the minimum cycle threshold used as a control for the calculation of a particular R value. In principle, this new calculation technique is applicable for the analysis of gene expression in all temporally changing genetic systems.

Original languageEnglish
Article number1471
Pages (from-to)491
Number of pages1
JournalBMC Genomics
Volume10
DOIs
Publication statusPublished - Oct 23 2009

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Suid Herpesvirus 1
Real-Time Polymerase Chain Reaction
Genome
Gene Expression
Herpesviridae
Genes
Nested Genes
Swine
Overlapping Genes
Polymerase Chain Reaction
RNA-Directed DNA Polymerase
Viral DNA
Infection
Life Cycle Stages
Northern Blotting
Publications
Cultured Cells
RNA
Kidney
Messenger RNA

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay. / Tombácz, Dóra; Tóth, J.; Petrovszki, Pál; Boldogkői, Z.

In: BMC Genomics, Vol. 10, 1471, 23.10.2009, p. 491.

Research output: Contribution to journalArticle

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