Conserved and distinct expression of circular RNAs in commercially used Marek’s disease vaccine viruses

Circular RNAs (circRNAs) are covalently closed RNA molecules, supporting a wide diversity of functions. While aberrant circRNA expression stands as a recognized hallmark of cancer development, our attention has turned to investigating their role in viral infections, specifically <i>Mardivirus Gallidalpha 2</i> (GaHV-2, Marek's disease virus) infection. In a previous study focused on the virulent GaHV-2 strain, RB-1B, we extensively catalogued circRNAs produced from virulence genes, notably from the MEQ-vIL-8 <i>locus</i> and the latency-associated transcripts (LATs) gene. Building upon this groundwork, our current investigation uncovers novel <i>loci</i> expressing viral circRNAs in distinct stages of GaHV-2 infection. Furthermore, we extend our focus to viral circRNA signatures in three commonly used Marek's disease vaccines, the avirulent GaHV-2 (CVI988/Rispens strain), non-oncogenic <i>Mardivirus Gallidalpha 3</i> (GaHV-3) and non-oncogenic <i>Mardivirus Meleagridalpha 1</i> (MeHV-1) commercially called herpesvirus of turkey. In these vaccine viruses, we identified viral circRNA expression from a <i>locus</i> antisense to the ICP4 immediate early gene, a conserved feature across the three species. This region has been characterized herein for the first time in terms of candidate LATs' exons and introns for GaHV-3 and MeHV-1. LATs' circRNAs were then deeply analysed, and we observed both similarities and distinctions when compared with those of the virulent GaHV-2. Another conserved gene, encoding the DNA packaging protein, was identified as a source of circRNAs in all three species. Eventually, different levels of circRNAs were found to be expressed from the <i>meq locus</i> between virulent and avirulent GaHV-2 strains. Our findings highlight a conserved pattern of virus-derived circRNAs in these related avian alphaherpesviruses. This conservation underscores the potential significance of these transcripts in completing the viral cycle and facilitating viral spread.