Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR.
ABSTRACT Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Fifteen horses experimentally infected with EHV-1.
Experimental study: Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI (P< .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86-100) and 27% (95% CI: 20-35).
We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C(T) values are provided as well as justification of a minimum 10-day quarantine period.
Article: New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G2254 polymorphisms.[show abstract] [hide abstract]
ABSTRACT: A single-nucleotide polymorphism (A(2254) or G(2254)) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E(2)) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69-72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E(1)) was developed by redesigning primers and probes specific to ORF30. The E(1) and E(2) rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A(2254) or G(2254)) in all archived isolates plus 168 of the clinical samples. The E(1) assay was 10 times more sensitive than E(2), with a lower detection limit of 10 infectious virus particles. Furthermore, all A(2254) and G(2254) genotypes along with samples from three cases of dual infection (A(2254)+G(2254)) were correctly identified by E(1), whereas E(2) produced 20 false dual positive results with only one actual mixed A(2254)+G(2254) genotype confirmed. Based on these findings, E(1) offers greater sensitivity and accuracy for the detection and A/G(2254) genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.Journal of clinical microbiology 04/2012; 50(6):1981-8. · 4.16 Impact Factor
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ABSTRACT: Equine herpesvirus 1 (EHV-1) induces a variety of disease manifestations, including respiratory disease, abortions, and myeloencephalopathy. Several vaccines are commercially available but could not previously be distinguished by serologic testing from infection with EHV-1 (or the closely related EHV-4). Currently available vaccines are not reliably protective against the severe manifestations of the disease, including fatal myeloencephalopathy. We determined immunological parameters that can differentiate vaccinated from previously infected animals by comparing humoral and cellular EHV-1-specific responses in clinically healthy horses 10 months after vaccination. Forty-seven horses with known histories of vaccination and infection were studied, including a group of horses that survived a severe neurological outbreak 5 years prior to vaccination. Results of serum virus neutralization (SN), serum IgG isotyping, and cytokine profiling of lymphocyte subsets were compared. IgG4/7 levels strongly correlated with virus neutralization (P < 0.0001). IgG1/3 and SN values distinguished vaccinated/outbreak-exposed (vacc/outbreak) horses from vaccinated horses (P < 0.05). EHV-1-specific gamma interferon (IFN-γ)-producing CD4(+) (but not CD8(+)) T-cell numbers were also increased in vacc/outbreak horses, which distinguished them from vaccinated horses (P < 0.01). IFN-α secretion was similar between all groups and independent of previous exposure or vaccination. Our data suggest that IgG isotype responses to EHV-1 are more diverse under field conditions than is revealed by experimental studies and that the current modified-live virus (MLV) vaccine induces a more restricted IgG isotype response than does natural exposure to EHV-1. Since these parameters can be assessed in a high-throughput manner, they may prove useful in screening future vaccine candidates and assessing levels of protection.Clinical and vaccine immunology: CVI 12/2011; 19(2):235-41. · 2.37 Impact Factor