Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation.

This research sought to develop markers to detect genetic variations in equine herpesvirus-1 (EHV-1) using PCR assays. The goal was to help group related viruses and better understand the dynamics of EHV-1 disease.

Research Methodology

• The experiment started by looking for variable restriction sites on equine herpesvirus-1 (EHV-1). The purpose of this was to develop markers, which could group epidemiologically related viruses.
• Crude viral DNA extracts of EHV-1 were prepared through a commonly used technique called Hirt extraction. These extracts were treated with restriction enzymes AluI, HaeIII, or RsaI. After this digestion, the resulting fragments were separated by size using electrophoresis and then Southern blotted, a method used to check for the presence of a specific DNA sequence in a DNA sample.

Findings and Conclusions

• The DNA ‘fingerprints’, obtained by probing the Southern blots with the EHV-1 specific fragment, EcoR1-I, could categorize 56 isolates of the virus into 16 groups. This demonstrates significant variation within EHV-1 species, which could potentially indicate different substrains or variations of the virus.
• The variable sites within the EcoR1-I fragment were mapped to estimate their positions, and the precise location was determined from the DNA sequence of this fragment.
• Special oligonucleotide primers, short strands of single-stranded DNA used to initiate PCR, were synthesized and used in PCR assays to detect these variable fragments.
• The investigated AluI variable fragment was a result of the presence or absence of a single AluI site, while the variable bands seen with HaeIII and RsaI resulted from variation in the copy number of two tandemly repeated sequences.
• In addition, an analysis of EHV-1 isolates probed with the glycoprotein B (gB) gene of EHV-1 revealed two distinct groups.

Implications of the Research

• The overall results of this research provide a foundation for rapid DNA fingerprinting on EHV-1 isolates.
• These new DNA markers can better group epidemiologically related viruses and offer important insights into the EHV-1 disease dynamics, ultimately aiding epidemiological investigations.
• Being able to distinguish between different EHV-1 strains can also have potential clinical relevance for treating affected horses, as disease severity and response to treatment may vary between different strains of the virus.