Transcriptomic Profiling of Equine and Viral Genes in Peripheral Blood Mononuclear Cells in Horses during Equine Herpesvirus 1 Infection

Abstract

Equine herpesvirus 1 (EHV-1) affects horses worldwide and causes respiratory disease, abortions, and equine herpesvirus myeloencephalopathy (EHM). Following infection, a cell-associated viremia is established in the peripheral blood mononuclear cells (PBMCs). This viremia is essential for transport of EHV-1 to secondary infection sites where subsequent immunopathology results in diseases such as abortion or EHM. Because of the central role of PBMCs in EHV-1 pathogenesis, our goal was to establish a gene expression analysis of host and equine herpesvirus genes during EHV-1 viremia using RNA sequencing. When comparing transcriptomes of PBMCs during peak viremia to those prior to EHV-1 infection, we found 51 differentially expressed equine genes (48 upregulated and 3 downregulated). After gene ontology analysis, processes such as the interferon defense response, response to chemokines, the complement protein activation cascade, cell adhesion, and coagulation were overrepresented during viremia. Additionally, transcripts for EHV-1, EHV-2, and EHV-5 were identified in pre- and post-EHV-1-infection samples. Looking at micro RNAs (miRNAs), 278 known equine miRNAs and 855 potentially novel equine miRNAs were identified in addition to 57 and 41 potentially novel miRNAs that mapped to the EHV-2 and EHV-5 genomes, respectively. Of those, 1 EHV-5 and 4 equine miRNAs were differentially expressed in PBMCs during viremia. In conclusion, this work expands our current knowledge about the role of PBMCs during EHV-1 viremia and will inform the focus on future experiments to identify host and viral factors that contribute to clinical EHM.

Identification of Host Factors Associated with the Development of Equine Herpesvirus Myeloencephalopathy by Transcriptomic Analysis of Peripheral Blood Mononuclear Cells from Horses

“…We observed an upregulation of CXCL10 in EHM horses, but not in non-EHM horses. This is in contrast to our previous study where we found a statistically significant in-crease in CXCL10 expression in PBMCs following EHV-1 CH [91]. Additionally, infection of PBMCs in vitro has been shown to increase CXCL10 expression [121].…”

“…We found very little EHV-1 transcription in PBMCs prior to EHV-1 CH, and only two pre-CH samples (0/6 non-EHM horses and 2/8 EHM horses) were positive for EHV-1 transcripts. This is in contrast to our previous study, where we found low-level transcription in five out of seven horses prior to EHV-1 CH, which likely indicated latent infection [91]. It is possible that the previously described horses were latently infected with EHV-1, while the majority of the horses in the present study were not.…”

“…These genes tend typically to be related to the innate immune response to viruses. Many of these ISGs were upregulated in both EHM and non-EHM horses, which is in agreement with our previous observations [91]. However, EHM horses appeared to have more significantly upregulated genes related to the antiviral defense and type 1 interferon response than the non-EHM group, such as the pattern recognition receptor, TLR3, the interferon regulatory factors IRF7 and IRF9, and interferon stimulated genes, such as RSAD2.…”

“…It is possible different factors between the herds contributed to modulation of EHV-1 depth of latency in the PBMCs. We additionally identified transcripts from the gamma herpesviruses EHV-2 and EHV-5 in several samples both pre and post EHV-1 CH with no apparent effect of EHV-1 CH, which is what we observed previously [91]. We did observe that EHV-2 transcripts were more prevalent in the non-EHM group-which may indicate that younger horses are more likely to have active transcription of EHV-2.…”

“…In this study, we found two genes related to fibrin clot formation (THBS1 and FN1) as well as MMP9, which is associated with increasing permeability of blood vessels, to be downregulated in PBMCs of EHM horses compared to non-EHM horses. Previously, we also found upregulation of genes encoding proteins involved in coagulation during EHV-1 viremia in PBMCs [91]. In humans, decreases of THBS1 expression by endothelial cells infected with hantavirus is linked to hemorrhagic disease, and elevated serum levels have been seen in stroke patients [92,93].…”