Characterizing the Transcriptome of Turkey Hemorrhagic Enteritis Virus
Characterizing the splice map of turkey hemorrhagic enteritis virus (THEV) is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used an RNA-sequencing experiment to characterize the transcriptome of THEV for the first time, providing key insight into the THEV gene expression and mRNA structures. Researchers previously annotated the genome of THEV as encoding 23 open reading frames (ORFs). In this work we identified 29 spliced transcripts all of which consisted of novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. We performed PCR amplification of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions. During validation we identified five additional unique transcripts, a subset of which were further validated by 3' rapid amplification of cDNA ends (3' RACE) experiments. Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs (ORF1, E3, 33K, ORF8, IVa2, and protease) to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated. Similar to other adenoviruses (AdVs), THEV also produces multiple distinctly spliced transcripts that code for the same proteins across its genome. Our data show that all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters like other AdVs. Overall design: To understand the genetic basis by which THEV causes immunosuppression (IS) in infected turkeys, we performed an RNA-seq experiment to characterize the transcriptome (structure and splicing patterns) of its genes to set the stage for further experimentation with specific viral genes that may mediate IS. A THEV vaccine was used to infect a turkey cell line (MDTC-RP19) and harvested at 4-, 12-, 24-, and 72-hpi in triplicates. RNA-seq was done for polyA-tailed mRNA Reads mapping to THEV's genome were filtered out as new BAM files. The mapped reads were assembled into transcripts using StringTie. StringTie set to expression estimation mode was used to calculate FPKM scores for all transcripts after which Ballgown in R was used to perform the statistical analysis on the transcript expression levels