Impact of equine herpesvirus-1 ORF15 (EUL45) on viral replication and neurovirulence.

Overview

• This research explores how the ORF15 gene (also known as EUL45) in Equine Herpesvirus 1 (EHV-1) affects the virus’s ability to replicate and cause neurological disease.
• The study provides insights into viral growth, gene regulation, and neurovirulence, highlighting EUL45’s potential as a target for vaccine development.

Background on Equine Herpesvirus 1 (EHV-1)

• EHV-1 is a virus that infects horses and is responsible for several health issues including respiratory illness, fetal loss, death around birth, and neurological disorders called myeloencephalopathy.
• The virus’s ability to cause neurological disease (neurovirulence) is linked to specific viral genes, and understanding these is critical for controlling infection.

Objective of the Study

• To investigate the role of the ORF15 gene (EUL45) in EHV-1 with regard to viral replication efficiency, gene expression regulation, and neurovirulence.
• To assess how deleting ORF15 affects virus growth and pathogenicity, which may inform vaccine development strategies.

Methods

• Used the Ab4p neurovirulent strain of EHV-1 as the experimental backbone.
• Generated three viral variants by manipulating bacterial artificial chromosome (BAC) DNA:
  • Ab4p attB (control with genetic insertion site)
  • Ab4p∆ORF15 (ORF15 gene/deletion mutant)
  • Ab4p∆ORF15R (revertant to confirm effects of ORF15 deletion)
• Transfected these viral DNA constructs into RK-13 cells (rabbit kidney cells) for virus production.
• Measured viral growth kinetics and plaque size in various cell types including:
  • Madin-Darby Bovine Kidney (MDBK) cells
  • Mouse neurons
  • Fetal equine brain cells
• Studied neurovirulence by intranasally infecting male CBA/N1 specific-pathogen-free (SPF) mice and monitoring:
  • Clinical signs of infection
  • Virus titers in tissues
  • Histopathological changes in the nervous system
• Analyzed gene expression of EUL45 and its neighboring genes EUL44 and EUL46 to assess regulatory effects.

Key Findings

• Impact of ORF15 (EUL45) Deletion on Viral Replication:
  • Deletion of EUL45 significantly reduced viral replication efficiency.
  • There was a noticeable decrease in virus release from infected cells.
  • Plaque sizes—zones of infected cells—were smaller in the deletion mutant, indicating impaired viral spread.
• Gene Regulation Effects:
  • Removing EUL45 led to upregulation (increase) of neighboring genes EUL46 and EUL44, suggesting that EUL45 normally represses these genes to some extent.
  • This points to a regulatory network within this region of the viral genome affecting viral gene expression and possibly replication dynamics.
• Reduced Neurovirulence:
  • The Ab4p-∆EUL45 virus displayed lower virulence in mice after intranasal infection, evidenced by reduced clinical signs and viral loads in neural tissues.
  • Growth of the mutant virus was poor in neural cell cultures compared to wild-type strains, confirming impaired neurotropism (ability to infect nerve cells).

Implications and Conclusions

• EUL45 is critical for efficient viral replication and contributes directly to the neurovirulence of EHV-1.
• The gene serves a regulatory role affecting the expression of nearby genes EUL44 and EUL46, which may also influence viral behavior and pathogenesis.
• Deleting EUL45 diminishes the virus’s ability to spread and cause neuronal disease, making the EUL45 deletion mutant a promising candidate for vaccine development.
• This research enhances understanding of molecular mechanisms controlling EHV-1 pathogenicity and suggests new targets for therapeutic intervention to control EHV-1 infections in horses.