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

Overview

• This research investigates how specific mutations in the DNA polymerase gene (ORF30) of equine herpesvirus type 1 (EHV-1) allow the virus to replicate effectively at higher temperatures, such as those experienced during fever.
• The study links these mutations to the virus’s ability to cause equine herpesvirus myeloencephalopathy (EHM), a serious neurological disease in horses.

Background

• Equine herpesvirus type 1 (EHV-1): A virus that infects horses, causing respiratory disease, abortion, and neurological disorders.
• EHM (Equine herpesvirus myeloencephalopathy): A severe neurological complication of EHV-1 infection characterized by inflammation of the spinal cord and brain, leading to paralysis and sometimes death.
• Viral DNA Polymerase (UL30/ORF30): An enzyme critical for viral DNA replication. Genetic variations (single nucleotide polymorphisms or SNPs) in this gene may influence the virus’s behavior and pathogenicity.
• Previous controversy: The single-nucleotide polymorphism G2254 in ORF30 has been used as a marker for neuropathogenic strains but its exact role in causing EHM remains unclear.

Objectives and Rationale

• To determine if specific mutations in the viral polymerase gene (ORF30) influence the virus’s ability to replicate at elevated temperatures, mimicking fever conditions in horses.
• To explore the functional significance of various ORF30 SNPs beyond G2254 in the pathogenesis of EHM.
• To assess how elevated temperature affects viral replication in different equine cell types relevant to infection, including fetal horse kidney cells, equine peripheral blood mononuclear cells (PBMCs), and equine vascular endothelial cells (EVECs).

Methods

• Used multiple EHV-1 isolates, including those derived from horses with and without EHM, to study viral replication at normal and elevated temperatures.
• Tested replication efficiency in:
  • Fetal horse kidney cell lines (permissive cells for viral growth)
  • Equine peripheral blood mononuclear cells (PBMCs), which play a role in viral spread through the bloodstream
  • Equine vascular endothelial cells (EVECs), key targets during infection and vascular pathology
• Performed molecular epidemiological association studies analyzing SNPs in ORF30 to correlate genetic variants with replication at elevated temperatures and with EHM incidence.
• Created recombinant viruses containing EHM-associated UL30 variants to confirm the effect of specific mutations.

Key Findings

• Replication at Elevated Temperature: Viruses isolated from EHM-affected horses consistently replicated well at febrile temperatures in permissive cells, whereas many non-EHM isolates showed reduced replication.
• PBMC Infection and Cell-to-Cell Transfer: PBMCs infected with EHM-associated viral strains remained capable of transferring virus to other cells even at elevated temperatures, despite some replication restriction.
• ORF30 SNP Associations: Specific mutations in the ORF30 gene correlated strongly with the ability of EHV-1 to replicate at higher temperatures.
• Molecular Epidemiology: Analysis suggested an epidemiological link between these polymerase gene mutations and an increased risk of EHM in horses.

Interpretation and Implications

• The ability of EHV-1 to replicate efficiently during fever conditions (febrile temperature) may provide a selective advantage in vivo and contribute to the development of EHM.
• Mutations in the viral DNA polymerase that confer thermal stability or enhanced function at elevated temperatures could be critical determinants of neuropathogenic potential.
• These findings challenge or extend previous views that focused on single polymorphisms like G2254 alone, indicating that multiple mutations may contribute to virulence and disease risk.
• Understanding these genetic factors can aid in identifying potentially dangerous EHV-1 strains, improving disease management strategies, and developing targeted treatments or vaccines.

Summary

• The study identifies that mutations in the DNA polymerase gene of EHV-1 enable the virus to maintain replication efficiency at the higher temperatures found during fever, which is common during infection.
• This thermal replication capacity is associated with strains causing EHM, highlighting a viral property that may be critical for neuropathogenesis.
• The research supports the notion that viral genetic variation influences disease severity and opens pathways for improved diagnostic markers and therapeutic targets for equine herpesvirus infections.