Turkey adenovirus 3 (TAdV-3), Siadenovirus gallopavotertii, historically referred to as turkey hemorrhagic enteritis virus (THEV), causes immunosuppression and hemorrhagic enteritis in turkey poults. Although an avirulent TAdV-3 strain is used as a live vaccine, its immunosuppressive properties remain poorly understood at the molecular level. Here, we employed Rapid Amplification of cDNA Ends (RACE) and Sanger sequencing to generate a transcriptomic map of TAdV-3 infected MDCTRP-19 cells at 24 hours post infection.

Using RACE and virus-specific primers, we obtained full-length annotation of 14 out of 23 predicted viral transcripts. Analysis of late structural genes revealed a conserved tripartite leader (TPL) sequence shared across all late transcripts, providing new insights into their transcription start sites, splicing events, and promoter architecture.

Additionally, we identified alternative leader exons—for instance, DBP mRNA contains a bipartite leader, whereas Hyd mRNA utilizes the TPL—and observed coordinated 3′ end processing via shared polyadenylation signals between certain transcripts (e.g., ORF1 and Hyd). This precise annotation of TAdV-3 mRNAs lays the groundwork for linking transcript structure to protein function, enhancing our understanding of TAdV-3’s immunosuppressive strategies and informing future development of TAdV-3 based gene delivery platforms.