[Development of PCR methods for detection of EAV infection]

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Epidemiology

Background:

• Equine Arteritis Virus (EAV) diagnosis traditionally relies on virus isolation, which is time-consuming.
• Molecular methods like RT-PCR offer potential for faster and more sensitive detection.
• Understanding EAV genetic diversity is crucial for effective disease management.

Purpose of the Study:

• To develop and validate real-time RT-PCR techniques for rapid and sensitive EAV diagnosis.
• To adapt conventional RT-PCR and real-time RT-PCR for broad-spectrum EAV strain detection.
• To perform molecular-epidemiological characterization of EAV strains.

Main Methods:

• Adaptation of two conventional RT-PCR methods and one real-time RT-PCR.
• Sensitivity testing using serial dilutions of the Bucyrus EAV strain.
• Validation using cell culture supernatants from 11 EAV isolates and semen samples from 7 stallions.
• Phylogenetic analysis of viral sequences.

Main Results:

• Real-time RT-PCR and heminested RT-PCR demonstrated a 100-fold higher sensitivity compared to simple RT-PCR.
• The developed techniques were successfully applied to diverse EAV isolates and clinical samples.
• Phylogenetic analysis revealed the existence of more EAV lineages than currently described.

Conclusions:

• Real-time RT-PCR provides a suitable alternative to virus isolation for fast and sensitive EAV diagnosis.
• The study identified novel EAV lineages, expanding our understanding of viral diversity.
• These molecular tools are valuable for both diagnostics and molecular epidemiology of EAV.