Cross-species analysis of transcriptomic response to alphaherpesvirus infection in human, bovine and equine cells

Comparative transcriptomics offers a powerful approach to elucidate host–virus interactions across related pathogens, yet systematic evaluations across spe-cies-matched cellular systems remain limited. Here, we performed a cross-species RNA-sequencing analysis of respective species' cells infected with three alphaherpesviruses - herpes simplex virus 1 (HSV-1), bovine al-phaherpesvirus 1 (BHV-1), and equid alphaherpesvirus 1 (EHV-1) - to dissect conserved and virus-specific transcriptional responses. We show that certain orthologous genes and orthologous pathways are differentially regulated upon infection among the three species like pathways related to translation rRNA processing and TNF-alpha signaling. We find that the earliest sampled timepoint of infection, 2 hours post infection (hpi), shows the most commonly enriched pathways among the three species compared to later timepoints. At 6h and 9h post infection, BHV-1- and EHV-1-infections have more in common with each other in terms of enriched pathways than with HSV-1 infections. Moreover, we provide a comprehensive analysis of temporal viral gene expression for all three herpesviruses. Together, these findings provide a comparative framework for understanding alphaherpesvirus–host interactions and reveal both conserved core responses and species-specific transcriptional signatures. This work estab-lishes a foundation for identifying broadly acting antiviral targets as well as vi-rus-specific vulnerabilities that may inform host-directed therapies and cross-species disease management. Overall design: RNA-sequencing was performed for MDBK cells infected with BHV-1, EDerm cells infected with EHV-1 and HFF cells infected with HSV-1 in the following conditions: Mock, 2hpi, 6hpi and 9hpi. The HFF cells have the following additional conditions: 6hpi + PAA treatment, 9hpi + PAA treatment and 16hpi.