A) Predicted location of Hin dIII restriction sites and respective expected restriction fragment sizes (not shown to scale). Ethidium bromide stained agarose gel electrophoresis of specific amplification of BoHV-4 DNA fragment from LVR, DN599 reference strains and the BoHV-4-U isolate. PCR amplified 2,538-bp fragment (Uncut) and digested with HindIII restriction enzyme (Cut). Lanes (-) correspond to negative controls and La to 1 kb ladder molecular size marker. B) Diagram showing the genomic region containing the BoHV-4 IE2 gene (not to scale), where the exon II contains most of the IE2 ORF, except the translation initiation codon contained into the exon I. During splicing, an intron is removed and the two exons (I and II) are joined together to generate the IE2 ORF. The position of the primers used to amplify the region containing IE2 are shown by arrows. The selected primers generate an amplicon of 1400 bp from the viral IE2 genomic region, while the amplicon is only 386 bp in length from the spliced product of IE2 transcript, thus allowing distinction between viral genomic DNA and the reverse transcribed IE2 RNA. Ethidium bromide stained agarose gel electrophoresis of RT-PCR or PCR of RNA and DNA from LVR, DN599 reference strains and the new isolate BoHV-4-U infected cells.