Detection of Equine Herpesvirus-1 in Nasal Swabs of Horses by Quantitative Real-Time PCR

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Journal of Veterinary Internal Medicine (Impact Factor: 1.88). 08/2008; 22(5):1234-8. DOI: 10.1111/j.1939-1676.2008.0172.x
Source: PubMed


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.

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    • "But no study has been conducted to detect these viruses in horses settled in Shahrekord. Studies showed that molecular detection of EHV infection is more accurate and reliable owing to the limitations of serological test [10] [24]. Therefore, there is need to detect the presence of the viruses among equine population in Isfahan central and Shahrekord southwest regions, Iran. "
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    ABSTRACT: This study was undertaken to investigate molecularly the occurrence of EHV-1 and EHV-4 infection among equine population in regions, Iran. Blood samples from 53 and 37 randomly selected horses settled in Isfahan and Shahrekord, Iran, respectively, were collected. Detection of EHV-1 and EHV-4 genes in the blood samples was done using polymerase chain reaction (PCR). Out of 53 and 37 samples fromIsfahan and Shahrekord, 4 (18.18%) and 3 (8.10%) were positive for PCR of EHV-1, respectively. Nine (16.98%) and 6 (16.21%) were positive for PCR of EHV-4, while 6 (11.32%) and 3 (8.10%) were positive for PCR of both EHV-1 and EHV-4, in Isfahan and Shahrekord, respectively. Of the 7 blood samples positive for EHV-1, 4 (16.66%) and 3 (8.10%) were from horses >3 years old while 2 (18.18%) and 1 (16.66%) were from 2-3 years old horses, in Isfahan and Shahrekord, respectively. Out of the 7 and 3 samples positive for PCR of EHV-1 in Isfahan and Shahrekord, 4 (22.2%) and 1 (7.69%) were Standardbred, while 3 (14.28%) and 2 (13.33%) wereThoroughbreds, respectively. EHV-4 was detected in blood of 4 (22.22%) and 2 (15.83%) Standardbreds and from 4 (19.04%) and 4 (26.66%) Thoroughbred horses in Isfahan and Shahrekord, respectively.This study has shown that horses settled in Isfahan central and Shahrekord southwest regions, Iran, are infected by EHV-1 and EHV-4 and thus serve as potential reservoirs and disseminators of the viruses.
    BioMed Research International 09/2015; Volume 2015,(Article ID 917854,):7 pages. DOI:10.1155/2015/917854 · 2.71 Impact Factor
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    • "The extracted DNA was directly used for the detection by real-time PCR. There is no available comparative housekeeping gene in nasal swabs (Perkins et al. 2008); therefore, both loading a fixed amount of sample DNA and adding a known quantity of foreign DNA (Perkins et al. 2008) into the PCR reaction were considered good approaches for the comparative measure; however, the work, cost, and complexity of the assay were significantly increased, making it unsuitable for the large-scale analysis of clinical samples. "
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    ABSTRACT: The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15 pg/μl for EHV-1 and 5 copies/μl or 2.5 fg/μl for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.
    Applied Microbiology and Biotechnology 03/2014; 98(9). DOI:10.1007/s00253-014-5626-6 · 3.34 Impact Factor
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    • "The assay described in this report produces complete results within 2 hours and can be used as a rapid diagnostic tool. Although our test showed presence of EHV-4 in horses, many of them did not have any clinical symptom, which might be due to establishment of latency and lack of virus in peripheral blood mononuclear cells (PBMCs) (Perkins et al 2008). The observed clinical symptoms in some horses are general and similar to common respiratory infections. "
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    ABSTRACT: Objective: To detect the presence or absence of EHV-1 and EHV-4 in North-East equine population of Iran. Material and methods: Blood samples of 200 adult horses located in 80 different rural areas of North-East of Iran, were examined for Equine herpesvirus-1 and 4 presences. Absolute quantitation of EHV-4 target molecules was performed using standard curves and thedetection limit of the assay was shown to be six copies per reaction. Results: Our study showed a high prevalence of EHV-4 (88%) in these regions. EHV-1 DNA was not detected in any sample.Conclusion: In addition to previous serological study, our report is the first to detect the EHVs in blood samples of Iran's equine population by using a high sensitive real-time PCR diagnostic assay and it provides new information for the virus distribution map.
    Human and Veterinary Medicine 05/2013;
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