Detection of Viral RNA Splicing in Diagnostic Virology

This chapter is the first one to introduce the detection of viral RNA splicing as a new tool for clinical diagnosis of virus infections. These include various infections caused by influenza viruses, human immunodeficiency viruses (HIV), human T-cell leukemia viruses (HTLV), Torque teno viruses (TTV), parvoviruses, adenoviruses, hepatitis B virus, polyomaviruses, herpesviruses, and papillomaviruses. Detection of viral RNA splicing for active viral gene expression in a clinical sample is a nucleic acid-based detection. The interpretation of the detected viral RNA splicing results is straightforward without concern for carry-over DNA contamination, because the spliced RNA is smaller than its corresponding DNA template. Although many methods can be used, a simple method to detect viral RNA splicing is reverse transcription-polymerase chain reaction (RT-PCR). In principle, the detection of spliced RNA transcripts by RT-PCR depends on amplicon selection and primer design. The most common approach is the amplification over the intron regions by a set of primers in flanking exons. A larger product than the predicted size of smaller, spliced RNA is in general an unspliced RNA or contaminating viral genomic DNA. A spliced mRNA always gives a smaller RT-PCR product than its unspliced RNA due to removal of intron sequences by RNA splicing. The contaminating viral DNA can be determined by a minus RT amplification (PCR). Alternatively, specific amplification of a spliced RNA can be obtained by using an exon-exon junction primer because the sequence at exon-exon junction is not present in the unspliced RNA nor in viral genomic DNA.