Comparative analysis of 3 qPCR primer-probe sets for the detection of equid alphaherpesvirus 1.

Equine alphaherpesvirus 1 (EqAHV1), a virus that affects horses, can now be detected using three different qPCR primer-probe sets recommended by the World Organisation for Animal Health. This study compared these sets to identify which provides the most accurate and sensitive detection.

Background and Purpose

• Equid alphaherpesvirus 1 (EqAHV1), previously named equine herpesvirus 1, is a significant equine pathogen causing respiratory disease, abortion, and neurological disorders.
• The World Organisation for Animal Health (WOAH) revised its Terrestrial Manual in 2024 to recommend three qPCR primer-probe sets for detecting EqAHV1.
• The goal of this research was to compare the sensitivity (ability to detect low copy numbers of the virus) and specificity (ensuring detection is specific to EqAHV1 and not other viruses) of these three qPCR sets to find the most reliable diagnostic tool.

The Three Primer-Probe Sets

• gB1H and gB1P: Both target the glycoprotein B (gB) gene of EqAHV1.
• gC1: Targets the glycoprotein C (gC) gene and is ISO 17025-accredited, currently used by the WOAH reference laboratory.

Methodology

• Assessed sensitivity through detection ability at very low viral copy numbers (down to and below 10 copies).
• Evaluated specificity to confirm that primers detect only EqAHV1, without cross-reacting with related equine herpesviruses like EqAHV4.
• Compared gB1H with a previously recommended set, gB1D, using 120 nasal swabs collected from febrile horses.
• Tested whether adjusting primer and probe concentrations improved gC1 sensitivity.

Key Findings

• Sensitivity:
  • gB1H and gB1P were able to detect 10 copies or fewer of EqAHV1, demonstrating high sensitivity.
  • gC1 failed to detect viral copies at or below 10, indicating lower sensitivity.
  • Adjusting primer and probe concentrations did not improve gC1 sensitivity.
• Specificity:
  • gB1P detected not only EqAHV1 but also equid alphaherpesvirus 4 (EqAHV4), likely due to amplification of the homologous gB gene in EqAHV4.
  • This cross-reactivity means gB1P is not dependable for highly specific detection of EqAHV1.
• Comparison of gB1H and gB1D:
  • Using clinical samples, gB1H showed slightly higher sensitivity than the older gB1D set.
  • This suggests that gB1H is an improved detection method for clinical use.

Conclusions and Recommendations

• The gB1H primer-probe set offers the best combination of high sensitivity and specificity for detecting EqAHV1 among the three evaluated sets.
• Although gB1H is recommended as the most reliable qPCR method, laboratories should still validate these primer-probe sets under their own conditions before routine implementation.
• Using gB1P may risk false positives due to detection of related herpesviruses, and gC1 may miss low viral loads due to poor sensitivity.

Implications for Veterinary Diagnostics

• Implementing the gB1H qPCR assay can improve early and accurate diagnosis of EqAHV1 infections in horses.
• Better detection helps with timely disease management and biosecurity measures, potentially reducing outbreaks related to equine abortion virus.
• Validated and reliable qPCR assays support epidemiological studies and monitoring of EqAHV1 prevalence.