Journal of Virological Methods 133 (2006) 70–75

Detection of equine herpesvirus type 1 by real time PCR

Gabriella Elia ∗, Nicola Decaro, Vito Martella, Marco Campolo, Costantina Desario,Eleonora Lorusso, Francesco Cirone, Canio Buonavoglia

Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari,S.p. per Casamassima km 3, 70010 Valenzano, Bari, Valenzano (BA), Italy

Received 5 August 2005; received in revised form 17 October 2005; accepted 20 October 2005Available online 22 November 2005

Abstract

A real-time PCR assay was developed for detection and quantitation of equid herpesvirus type 1 (EHV-1). The sensitivity of the assay wascompared with an established nested-PCR (n-PCR). The real-time PCR detected 1 copy of target DNA, with a sensitivity 1 log higher thangel-based n-PCR. The assay was able to detect specifically EHV-1 DNA in equine tissue samples and there was no cross-amplification of otherhorse herpesviruses. Real-time PCR was applied to determine EHV-1 load in tissue samples from equine aborted fetuses. The high sensitivity andrs©

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eproducibility of the EHV-1-specific fluorogenic PCR assay, combined with the wide dynamic range and the high throughput, make this methoduitable for diagnostic and research applications.

2005 Elsevier B.V. All rights reserved.

eywords: Equine herpesvirus 1; Quantitation; Fluorogenic PCR

. Introduction

Equid herpesvirus 1 (EHV-1), a member of Varicellovirusenus in the Alphaherpesvirinae subfamily, is one of the mostrevalent cause of disease in equine population. EHV-1 iselated genetically to bovine herpesvirus (BHV-1), herpes sim-lex viruses 1 and 2 (HSV-1, HSV-2) and pseudorabies virusPRV) (Heldens et al., 2001). EHV-1 is closely related to equiderpesvirus 4 (EHV-4) and the viruses were regarded initially asubtypes of the same virus (Sabine et al., 1981; Studdert et al.,981). Subsequently, restriction endonuclease and nucleotideequence analysis provided evidence that EHV-1 and EHV-4re two distinct viruses (Sabine et al., 1981; Studdert et al.,981; Turtinen et al., 1981). The genome of EHV-1 consists ofdouble-stranded DNA of about 145 kbp in length (Cullinane

t al., 1988). It is composed of a unique long (UL) region andunique short (US) region, which is flanked by two inverted

epeat sequences (TRS/IRS). EHV-1 genome was predicted toontain 80 genes with some of them present twice in the TRS/IRSegions, resulting in 76 unique genes (Henry et al., 1981; Telfordt al., 1992; Whalley et al., 1981). About 30 viral proteins have

been identified including at least 13 glycoproteins (Csellner etal., 2000).

EHV-1 is endemic in horses worldwide and it is respon-sible for respiratory infections, epizootic abortion and, moresporadically, neurological disorders. The infection has high mor-bidity rates and it is acquired easily by inhalation of saliva andnasal discharges, and by contact with aborted fetus and fetalmembranes (Allen and Bryans, 1986). Like other herpesviruses,EHV-1 may establish latent infection within its host (Allen andBryans, 1986). Intermittent viral shedding from asymptomaticanimal carriers may occur, contributing to spread of the infectionin equine population and likely accounting for unexpected out-breaks of EHV-1-related disease in closed populations (Welchet al., 1992).

Large-scale outbreaks of abortion and perinatal foal mortal-ity caused by EHV-1 are a significant cause of economic losses,while the burden of EHV-1-related respiratory diseases (losttraining time and poor race performance) is less quantifiable.The economic impact of abortions in horses stresses the needfor rapid and reliable diagnostic tools for detection of EHV-1infection, so that early sanitation measures, aimed at decreasingthe impact of virus spread, can be adopted.

∗ Corresponding author. Tel.: +390804679833; fax: +390804679843.E-mail address: g.elia@veterinaria.uniba.it (G. Elia).

Routine diagnosis of EHV-1 infection in live animals is usu-ally achieved by virus isolation in tissue culture cells fromnasopharyngeal secretions and blood, or from the tissues of

166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2005.10.024

G. Elia et al. / Journal of Virological Methods 133 (2006) 70–75 71

aborted fetuses. However, virus isolation is time consuming andnot very sensitive. Serological diagnosis is less conclusive andrequires both acute and convalescent serum samples.

Because of its higher sensitivity and versatility, detection ofviral DNA/RNA by the polymerase chain reaction (PCR) hasbecome the diagnostic “gold standard” for a number of infec-tious diseases. Several PCR-based methods have been developedfor detection and identification of the DNA of EHV-1 and EHV-4in aborted fetuses or nasal swabs (Kirisawa et al., 1993; Borchersand Slater, 1993). Nevertheless, none of those PCR-based assayswas designated to be quantitative. In addition, PCR assays areexposed frequently to risks of carryover contamination, espe-cially when a large sample throughput is required.

Quantitation of virus DNA can be carried out accurately byreal-time PCR. Real-time PCR is less expensive and is amenableto automation, allowing higher throughputs and decreasingturnaround times. Furthermore, with respect to end-detectionby gel, the use of target-specific fluorescent probes in real-timePCR is able to increase sensitivity and specificity. Accordingly,the advantage of real-time PCR for detection of EHV-1 overother diagnostic techniques is the ability to differentiate EHV-1from EHV-4 and in the ability to quantify EHV-1 load in tis-sues, providing a powerful tool to improve investigation of theepidemiology and pathogenesis of EHV-1 infection.

This paper describes the development and validation of areal-time PCR assay based on TaqMan technology to detect andq

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DNeasy Tissue Kit (QIAGEN S.p.A.), according to the man-ufacturer’s instructions. Five to twenty-five milligrams tissuewere added to lysis buffer. DNA of each sample was eluted in200 �l of AE buffer (50 mM Na acetate, pH 5.3, 10 mM EDTA)and subjected to polymerase chain reaction (PCR) and nested-PCR (n-PCR) assay able to detect and to differentiate EHV-1and EHV-4 DNA (Kirisawa et al., 1993). Briefly, conventionalPCR was performed using AmpliTaq Gold (Applied Biosystem,Applera Italia, Monza, Italy) and primer pair FC2/RC (Kirisawaet al., 1993). The PCR products were then reamplified with apair of nested primers FC3/R1 to increase the sensitivity by 1000times (Kirisawa et al., 1993). The PCR products were detectedby electrophoresis through a 1.5% agarose gel and visualiza-tion under UV light after bromide ethidium staining. All DNAsamples were positive to EHV-1 and were stored at −70 ◦C.

Tissue samples from healthy horses, seronegative to EHV-1/-4, aged 8–9 months, were collected at slaughter house as negativecontrols and included in the study.

2.2. Design of primers and probe

In order to establish a real-time PCR specific for EHV-1, gB nucleotide sequences of EHV-1 and EHV-4 strainswere retrieved from GenBank and aligned using BioEdit soft-ware package (Hall, 1999). The aligned gB gene nucleotidessw

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uantify EHV-1 DNA in different samples.

. Materials and methods

.1. Samples collection and DNA preparation

During the winter of 2003, outbreaks of late-gestation abor-ions, suggestive of EHV-1 infection, were recognised in twoerds of half-breed horses in the South of Italy. Aborted fetusesere subjected to histopathological and virological investiga-

ions. Spleen, liver, lung, myocardium, and placenta were sam-led from a total of n = 15 fetuses. Samples were inoculated ontoabbit kidney cells (RK-13) for virus isolation, and the cultureere examined daily for herpesvirus-induced cytopathic effect.issue samples were processed further for DNA extraction with

ig. 1. Region of DNA-gB chosen to design a primer/probe set for specificaboratories Srl, Milan, Italy). The TaqMan probe was labeled with the fluoreye 6-carboxytetramethylrhodamine (Blackhole Quencher 1) at the 3′ end.

equences displayed 83% identity between the two EHVerotypes (Kirisawa et al., 1993). The primer/probe set (Fig. 1)as manufactured by MWG Biotech AG (Ebersberg, Germany).

.3. DNA standard for quantitation

A 1181-nucleotide region of the gB gene was amplified forhe standard plasmid DNA construction by using primer pairsC2/RC (Kirisawa et al., 1993).

The resulting PCR product was cloned into pCR4-TOPOector (TOPO TA cloning, Invitrogen, Milan, Italy) and propa-ated in competent Escherichia coli TOP10F’ cells, accordingo the manufacturer’s instructions. Plasmid DNA was purifiedsing a commercial column (Fast Plasmid Mini, Eppendorf,amburg, Germany) and quantified by spectrophotometrical

nalysis. Ten-fold dilutions of standard plasmid, representing

tion of EHV-1, using the primer design software Beacon Designer (Bioradreporter dye 6-carboxyfluoroscein (FAM) at the 5′ end and with the quencher

72 G. Elia et al. / Journal of Virological Methods 133 (2006) 70–75

100 to 109 copies of DNA/10 �l of template, were made out inTE (10 mM Tris–HCl, pH 7.5, 1 mM EDTA) buffer. Aliquots ofeach dilution were frozen at −70 ◦C and used only once.

2.4. Real-time PCR

Duplicates of EHV-1 standards and DNA templates weresubjected simultaneously to real-time analysis with each runincluding one negative control (no template). Amplification wascarried out in a 25 �l reaction volume containing 12.5 �l of IQTM

Supermix (Bio-Rad Laboratories Srl), 900 nM of each primer(EHV-1For and EHV-1 Rev), 200 nM of probe EHV-1 Pr and10 �l of DNA. The thermal cycle protocol used was the follow-ing: activation of iTaq DNA polymerase at 95 ◦C for 10 min and45 cycles consisting of denaturation at 95 ◦C for 15 s, primerannealing and extension at 60 ◦C for 1 min.

2.5. Internal control

In order to verify the absence of DNA losses during theextraction step and of PCR inhibitors in the DNA templates,an internal control (IC), consisting of ovine herpesvirus type2 (Decaro et al., 2004), was added to the ATL buffer (tissuelysis buffer, QIAGEN S.p.A) at the concentration of 5000 DNAcopies/ml of buffer prior to tissue digestion. The fixed amountof the IC added to each sample had been calculated to give amorw(A(p2

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Fig. 2. Standard curve of the EHV-1 real-time PCR assay. Ten-fold dilutionsof standard DNA prior to amplification were used, as indicated on the x-axis,whereas the corresponding cycle threshold (CT) values are presented on the y-axis. Each dot represents the result of duplicate amplifications of each dilution.The coefficient of determination (R2) and the slope value (s) of the regressioncurve were calculated and are indicated. Real-time PCR was performed in ani-Cycler iQTM Real-Time Detection System (Bio-Rad Laboratories Srl) andthe data were analysed with the appropriate sequence detector software (version3.0).

3. Results

3.1. Reproducibility of real-time PCR

Ten-fold dilutions of standard DNA were tested and usedto construct the standard curve by plotting the plasmid copynumber logarithm against the measured CT values (Fig. 2). Thegenerated standard curve covered a linear range of 10 ordersof magnitude and showed linearity over the entire quantita-tion range (slope = −3.475), providing an accurate measurementover a very large variety of starting target amounts.

To determine the reproducibility of the assay, both within-run and between-run precision studies were undertaken (Fig. 3).Intra-assay CVs ranged from 7.37% (samples containing1.60 × 107 DNA copies) to 21.1% (8.64 × 103 DNA copies),whereas the interassay CVs were comprised between 20.39%(1.04 × 109 DNA copies) and 33% (1.66 × 107 DNA copies).

There was no detectable fluorescence signal in the tubes thatcontained DNA from the control (negative) samples or fromother equine herpesviruses and in the tubes without template,confirming that the assay was highly specific for the detectionof EHV-1 in clinical specimens.

To establish that the test had an acceptable sensitivity, itsperformance was compared with n-PCR (Kirisawa et al., 1993).Viral suspension of the EHV-1 strain Army 183 with a titre of105.00 TCID /50 �l was diluted 1:10 in 12 steps and analysediav

a

ean CT value in a real-time PCR assay (Hussy et al., 2001)f 32.21 with a S.D. of 1.02, as calculated by 100 separateuns (Decaro et al., 2005). Real-time PCR for IC detectionas carried out in a separate run, using primers oF-OvHV-2

5′-TGGTAGGAGCAGGCTACCGT-3′) and oR-OvHV-2 (5′-TCATGCTGACCCCTTGCAG-3′) and probe oP-OvHV-2

FAM-TCCACGCCGTCCGCACTGTAAGA-TAMRA). Sam-les in which the CT value for the IC was >34.25 (average plusS.D.) were excluded from analysis.

.6. Validation of the assay

The analytic specificity of EHV-1 DNA detection by real-timeCR was evaluated by testing DNA preparations of other equineerpesviruses, including EHV-4, EHV-2, EHV-5 and EHV-3.NA extracts from nasal swabs and lung samples of horses thatere negative for EHV-1 serum antibodies were included asegative controls.

To evaluate the detection limit of EHV-1 real-time PCR, 10-old dilutions of the standard DNA, ranging from 109 to 100

olecules, were tested. In addition, real-time PCR was car-ied out with DNA from EHV-1 strain Army 183 (Jones et al.,948) (kindly supplied by Dr. N. Cavaliere, Istituto Zoopro-lattico di Puglia e Basilicata, Foggia, Italy). The viral titreas 105.00 TCID50/50 �l on RK-13 cells. DNAs from 10-foldilutions of the viral suspension were extracted and tested byeal-time or n-PCR.

Reproducibility of the method was established by testingepeatedly field samples containing several concentrations ofHV-1 DNA, as previously described (Stelzl et al., 2004)

Fig. 3).

50n duplicate by both the TaqMan assay and n-PCR. Real-timenalysis predicted that the starting DNA copy number of theiral suspension was 1.86 × 108/10 �l.

The detection limit of the assay was shown to be 100 copiesnd 10−3.00 TCID50/50 �l for standard DNA and EHV-1 strain

G. Elia et al. / Journal of Virological Methods 133 (2006) 70–75 73

Fig. 3. Coefficients of variation intra-assay and interassay over the dynamicrange of the EHV-1 real-time PCR assay. DNA amounts were calculated for thesame samples in different assays (5 consecutive runs) and within an assay (5times in the same assay). In each run, 5 replicates/sample were analysed.

Army 183, respectively, with a sensitivity of 1 log higher thanthe gel-based n-PCR assay.

3.2. Samples

To validate the EHV-1 real-time PCR, the assay was appliedto detect and quantify viral load in tissue samples, comparingthe results with those obtained by the n-PCR assay used cur-rently in our laboratory. Table 1 summarizes the results of thisvalidation. We assayed a total of 39 samples tested positive byn-PCR, consisting of 5 different tissues (placenta, liver, lung,spleen and myocardium). As expected, the results obtained byreal-time PCR assay were consistent with the results obtainedby the standard n-PCR method. In all fetuses examined, the lungand the liver samples contained the highest EHV-1 loads with arange between 1.47 × 106 and 9.88 × 108 DNA copies/10 �l oftemplate. The spleen and myocardium ranged from 8.05 × 103

to 5.16 × 105 viral DNA copies/10 �l of template while theplacenta had a moderate viral load (about 3.60 × 103 DNAcopies/10 �l of template).

3.3. Internal control detection

The IC was detected in all the examined samples, with CTvalues below the threshold value of 34.25. Therefore, significantDDP

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Table 1Analysis of samples from horses naturally infected with EHV-1 by gel basedn-PCR and real-time analysis

Sample Fetus no. n-PCR Real-time titrea

Lung 1 + 4.48 × 108

Liver 1 + 1.47 × 106

Lung 2 + 1.58 × 108

Liver 2 + 2.12 × 107

Liver 3 + 1.32 × 107

Liver 4 + 2.09 × 106

Liver 5 + 5.57 × 107

Lung 6 + 9.88 × 108

Lung 7 + 7.70 × 108

Liver 7 + 2.60 × 107

Spleen 7 + 3.31 × 105

Miocardyum 7 + 2.07 × 105

Liver 8 + 1.80 × 107

Spleen 8 + 8.05 × 103

Myocardium 8 + 8.52 × 103

Placenta 8 + 4.48 × 103

Liver 9 + 9.64 × 107

Lung 10 + 2.12 × 106

Liver 10 + 5.20 × 106

Spleen 10 + 8.64 × 104

Myocardium 10 + 1.04 × 104

Placenta 10 + 1.16 × 103

Lung 11 + 3.78 × 108

Liver 11 + 8.22 × 106

Spleen 11 + 4.46 × 104

Myocardium 11 + 8.92 × 103

Placenta 11 + 8.88 × 102

Lung 12 + 3.06 × 108

Liver 12 + 1.53 × 107

Placenta 12 + 2.56 × 103

Lung 13 + 7.34 × 108

Liver 13 + 1.59 × 108

Myocardium 13 + 8.64 × 103

Liver 14 + 1.98 × 106

Myocardium 14 + 5.16 × 105

Lung 15 + 8.19 × 108

Liver 15 + 6.40 × 106

Spleen 15 + 3.76 × 105

Placenta 15 + 5.01 × 103

a Real-time titres are expressed as number of EHV-1 DNA copies/10 �l oftemplate.

enabling simultaneous processing of several samples. The EHV-1 real-time PCR assay was shown to be efficient and more sen-sitive when compared to conventional gel-based n-PCR, beingable to detect as few as 100 copies of EHV-1 DNA. Such highsensitivity may be accounted for by the smaller size of the DNAfragment amplified in this real-time assay. Compared with n-PCR, the processing time is shorter, the contamination risks arelower because of the lack of post-PCR processing steps, and,finally, the specificity is enhanced by the probe hybridization.

The EHV-1 real-time assay was highly reproducible and lin-ear over a range of 10 orders of magnitude, from 100 to 109

copies, allowing a precise calculation of EHV-1 DNA load insamples containing a wide range of viral DNA amounts. Thereproducibility of the assay was high and CVs values were lowerthan other validated real-time PCR assays (Decaro et al., 2005;Paraskevis et al., 2002)

NA losses did not occur during nucleic acid extraction andNA polymerase inhibition was not observed during real-timeCR amplification.

. Discussion

The development of a TaqMan-based real-time PCR foretection and quantitation of EHV-1 from clinical specimens isescribed. The assay displays several advantages over conven-ional n-PCR assays, increasing the laboratory throughput and

74 G. Elia et al. / Journal of Virological Methods 133 (2006) 70–75

Conventionally, EHV-1 titration in clinical samples is under-taken in cells cultures, which is labour consuming and needs atleast 5 days for evaluation of virus-induced cytophatic effect. Todate, only conventional PCR methods have been applied for thestudy of viral shedding. Conventional n-PCR assay (Kirisawaet al., 1993) proved to have high sensitivity for detection ofEHV-1 DNA (Daly and Doyle, 2003). However, n-PCR is moretime-consuming and may lead to laboratory contamination withPCR-amplified cDNA, as handling of cDNA is required prior tothe second step of amplification. Recently, a competitive PCR-ELISA was developed for detection and quantitation of EHV-1DNA with the minimum detection level estimated as 63 viralgenome equivalents (Daly and Doyle, 2003). The detection limitof real-time PCR in this study was as low as 100 copies of EHV-1DNA. Accordingly, even a single viral particle in the specimencan be detected, theoretically, by a single-step procedure, thusproviding a highly sensitive diagnostic tool. The availability ofsuch highly sensitive assay will facilitate investigations on theepidemiology and pathogenesis of EHV-1 infection and will helpthe evaluation of candidate EHV-1 vaccine.

The control of EHV-1 infections is hampered by the phe-nomenon of virus latency and by re-activation of latent infec-tions, even in the presence of specific antibodies. Like otherherpesviruses, EHV-1 establishes and maintains a lifelong latentinfection in the host (Allen and Bryans, 1986). As latently-infected animals are the reservoir for new outbreaks of infection,ttAdsapcml2sDhdm

ntetsistEmshma

cines could be the assessment by real-time PCR of differencesin viral load and virus shedding in vaccinated and un-vaccinatedseronegative foals after virus challenge. In a similar manner, thedecrease in viral shedding after pharmacological re-activation invaccinated but latently infected horses could be another impor-tant target for the evaluation of vaccine efficacy.

In summary, a real-time PCR assay was developed for detec-tion of EHV-1 that proved to be sensitive and specific, and thatrepresents a new powerful tool for future investigations of EHV-1 pathogenesis and vaccine development.

Addendum

After the submission of this manuscript, a paper reporting areal-time PCR for EHV-1 was published as “ahead of print” onJournal of Virological Methods (Diallo et al., in press).

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