A novel PCR protocol for detection and differentiation of neuropathogenic and non-neuropathogenic equid alphaherpesvirus 1

Abstract

Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A2254→G2254) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of the genotype by sequencing and restriction enzyme analysis (REA). Thirty samples from horses with signs typical for EHV-1 infection were tested by rtPCR and our new conventional PCR. The results showed that compared to rtPCR, the conventional PCR protocol combined with sequencing and REA was more reliable concerning unambiguous determination of the EHV-1 genotype. Results of our new assay confirmed previous findings, according to which the non-neuropathogenic genotype A2254 is predominantly found in animals with fever, respiratory signs, and abortions or perinatal mortality, whereas the neuropathogenic genotype G2254 is primarily detected in animals suffering from neurologic disease. In some samples, results pointed towards coinfection with both genotypes. Further studies are required in order to elucidate the significance of infections with genotype A2254 and G2254 in neurologic and non-neurologic cases, respectively.

Molecular Surveillance of EHV-1 Strains Circulating in France during and after the Major 2009 Outbreak in Normandy Involving Respiratory Infection, Neurological Disorder, and Abortion

“…A2254G typing was subsequently used on a regular basis for strain discrimination [40,41]. In our study, 137 strains collected from 2009 to 2018 were typed, and A 2254 strains were more significantly associated with abortion cases than to neurological cases ( p = 0.0002), as recently described by Lechmann et al (2019) [42]. However, no significant correlation between G 2254 strains and the neurological form of disease was measured.…”

Multiplex real-time PCR for the detection and differentiation of equid gammaherpesvirus 2 and 5

“…DNA had been extracted from the nasal swabs shortly after their collection using the QIAamp DNA Mini Kit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturer's instructions. Following multiplex rtPCR for EHV-1 and EHV-4 (Diallo et al, 2006, Diallo et al, 2007Lechmann et al, 2019) as well as rtPCR for the equine 12 S rRNA gene as extraction control, the DNA was stored at − 20 • C (data not shown).…”

Development of multiplex gold nanoparticles biosensors for ultrasensitive detection and genotyping of equine herpes viruses

“…Traditional diagnostic methods like virus isolation and serology are often inadequate in detecting the virus in animals with low viral loads or those who are infected but asymptomatic. Therefore, it is essential to develop highly sensitive detection and genotyping techniques to enable early identification, disease monitoring, and control measures to prevent the spread of EHV infections 7 .…”