The Transcriptional Landscape of Marek’s Disease Virus in Primary Chicken B Cells Reveals Novel Splice Variants and Genes

Abstract

Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and poses a serious threat to poultry health. In infected animals, MDV efficiently replicates in B cells in various lymphoid organs. Despite many years of research, the viral transcriptome in primary target cells of MDV remained unknown. In this study, we uncovered the transcriptional landscape of the very virulent RB1B strain and the attenuated CVI988/Rispens vaccine strain in primary chicken B cells using high-throughput RNA-sequencing. Our data confirmed the expression of known genes, but also identified a novel spliced MDV gene in the unique short region of the genome. Furthermore, de novo transcriptome assembly revealed extensive splicing of viral genes resulting in coding and non-coding RNA transcripts. A novel splicing isoform of MDV UL15 could also be confirmed by mass spectrometry and RT-PCR. In addition, we could demonstrate that the associated transcriptional motifs are highly conserved and closely resembled those of the host transcriptional machinery. Taken together, our data allow a comprehensive re-annotation of the MDV genome with novel genes and splice variants that could be targeted in further research on MDV replication and tumorigenesis.

Pervasive differential splicing in Marek’s Disease Virus can discriminate CVI-988 vaccine strain from RB-1B virulent strain in chicken embryonic fibroblasts

“…Whether this is due to limited sensitivity or to the fact that such isoforms are specific to other experimental conditions – for instance, (23) used MSB1 cells rather than CEFs – the conclusion can only be that the potential size of the MDV transcriptome is even larger than what emerges from our study. That is confirmed by the novel transcripts identified in (15). Again, this is entirely similar to what can be seen for other herpesviruses – in previously studied CMV infected cells, the authors were unable to identify some of the previously annotated genes (13).…”

“…The assay was proven to be reproducible. As far as we know such a striking result had not been previously reported in the literature, and it is not reported in (15) either. Not surprisingly, our preliminary analysis of the host transcriptome (data not shown) indicates that the splicing of host transcripts also depends on the virulence of the infected strains.…”

“…In the past, some MDV transcripts had been determined through northern blot analysis and the nucleotide sequencing of a limited number of cDNA products, most notably the spliced variants of MDV’s oncogene Meq (16), vIL8 (17) and glycoprotein C (19). Very recently, a study of MDV infection in B cells has identified novel transcripts and genes (15). To expand on this, we report on the viral transcriptome of MDV using high-throughput short-read RNA sequencing on RNA isolated from CEF cells infected with MDV virulent strain – RB-1B (30) – and MDV attenuated vaccine strain – CVI-988 (31).…”

“…Detailed knowledge of the viral transcriptomes can also potentially shed a much better light on the different mechanisms taking place during virus-host interaction. In fact while our manuscript was under review, a study conducted by Betzbach and colleagues appeared in press reporting novel genes and a comprehensive annotation of MDV in primary B cells inoculated with BAC-derived MDV strains RB-1B or CVI-988 (15). Their study confirms that the community is increasingly interested in better understanding the complexity of viral transcriptomes that arise as a response to infection by closely related strains.…”

Marek’s Disease Virus Infection of Natural Killer Cells

“…Briefly, spleens were homogenized using a 70 µM cell strainer (Beckton Dickinson (BD); Franklin Lakes, NJ, USA) to obtain a single cell suspension. Viable cells were then purified by Ficoll-Paque density gradient centrifugation [25], resuspended in NK medium and used directly or stored in liquid nitrogen until further use.…”