RNAs expression analyses in commercially used Marek's disease vaccine viruses to identify their Latency associated transcripts splicing patterns
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 investigate their role in viral infections, specifically Mardivirus Gallidalpha 2 (GaHV-2, MDV) infection. In a previous study focused on the virulent GaHV-2, RB-1B strain, we extensively cataloged circRNAs produced from virulence genes, notably from the MEQ-vIL-8 locus and the Latency Associated Transcripts (LATs) gene. Building upon this groundwork, our current investigation uncovers novel loci expressing viral circRNAs in distinct stages of GaHV-2 infection. Furthermore, we extend our focus to viral circRNA signatures in three commonly used MDV vaccines, the avirulent GaHV-2 (CVI988- Rispens strain), non-oncogenic Mardivirus Gallidalpha 3 (GaHV-3), and non-oncogenic Mardivirus Meleagridalpha 1 (MeHV-1) commercially called Herpesvirus of Turkey (HVT). In these vaccine viruses, we identified viral circRNA expression from a locus antisense to the immediate early ICP4 gene a conserved feature across the three species. This region has been characterized herein for the first time in term of candidate LATs exons and introns for GaHV-3 and MeHV-1. Potential LATs circRNAs were then deeply analyzed and we observed both similarities and distinctions when compared to 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 expression were found from the meq locus between virulent and avirulent GaHV-2 strains. Our findings highlight a conserved pattern of viral derived circRNA expression in these related avian alphaherpesviruses. This conservation underscores the potential significance of these transcripts in completing the viral cycle and facilitating viral spread. Overall design: To identify Latency associated transcripts splicing pattern from three viruses used in Marek's Disease vaccination program, we used in vitro  lytic infection of ESCDL-1 cells with GaHV-2 CVI-988 (Acc. n° DQ530348), GaHV-3 SB-1(Acc. n° NC_002577), and MeHV-1 FC-126 (Acc. n° NC_002641) viral strains . We extracted RNAs from the infected cell lines and send them for paired-end RNA sequencing at Novogene. We processed the data through vCircTrappist to identify spliced RNAs (sense and antisense).