Equine herpesvirus myeloencephalopathy-associated mutations in equine herpesvirus 1 DNA polymerase confer the ability to replicate at elevated temperatures

Equine herpesvirus myeloencephalopathy (EHM), caused by equine herpesvirus type 1 (EHV-1), poses a major threat to the equine industry because of its devastating impact on animal welfare and athletic performance. A single-nucleotide polymorphism (SNP), G2254, in ORF30 (UL30), which encodes the viral DNA polymerase, has been used as a marker of neuropathogenic EHV-1 strains, although its contribution to EHM pathogenesis remains controversial. In addition, other ORF30 SNPs have been reported in association with EHM, but their functional significance is unclear. Clinical observations indicate that fever and high levels of viremia are closely associated with EHM onset. Here, we investigated EHV-1 replication under elevated-temperature conditions that mimic febrile states using a fetal horse kidney cell line, equine peripheral blood mononuclear cells (PBMCs), and equine vascular endothelial cells (EVECs). EHV-1 isolates derived from horses with EHM consistently retained the ability to replicate at elevated temperature in permissive cells and remained competent for infection despite restricted replication in PBMCs, whereas replication of many non-EHM-derived isolates was suppressed. Specific ORF30 SNPs were associated with replication at elevated temperatures, and a molecular epidemiological association analysis of these SNPs suggested an association with EHM. Together, these findings suggest that replication at febrile temperature is a viral property relevant to EHV-1 pathogenesis and EHM risk. IMPORTANCE: Equine herpesvirus myeloencephalopathy (EHM), caused by equine herpesvirus type 1 (EHV-1), represents a serious threat to the equine industry. Here, we show that EHV-1 field isolates and recombinant viruses harboring EHM-associated UL30 variants retain replication capacity at elevated temperatures in fetal horse kidney cells and equine vascular endothelial cells, and that peripheral blood mononuclear cells (PBMCs) infected with EHM-associated viruses can mediate cell-to-cell transfer under these conditions. In contrast, replication of many non-EHM-derived isolates and recombinant viruses in these cells, as well as PBMC-mediated cell-to-cell transfer of EHV-1, is suppressed at elevated temperatures. Notably, we show that single nucleotide polymorphism (SNPs) in ORF30, which encodes the viral DNA polymerase UL30, and which have been implicated in EHM, are linked to the ability of EHV-1 to replicate at elevated temperatures. These findings suggest that replication at febrile temperature is a viral property relevant to EHV-1 pathogenesis and EHM risk.