Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction

Journal of Virological Methods 131 (2006) 92–98

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction Ibrahim S. Diallo ∗ , Glen Hewitson, Lucia Wright, Barry J. Rodwell, Bruce G. Corney Animal Research Institute, Yeerongpilly Veterinary Laboratory, Department of Primary Industries and Fisheries, Locked Mail Bag 5, Moorooka, Qld 4105, Australia Received 15 April 2005; received in revised form 22 July 2005; accepted 26 July 2005 Available online 30 August 2005

Abstract Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity. Crown Copyright © 2005 Published by Elsevier B.V. All rights reserved. Keywords: Equine herpesvirus; Taqman; MGB probe; Polymerase chain reaction; Diagnosis

1. Introduction Equid herpesvirus 1 (EHV1) is a member of the Alphaherpesvirinae, which affects horses (Bagust, 1972; Bagust et al., 1972). It is one of the most economically important diseases in the horse industry in Australia and other parts of the world (Dixon et al., 1977; Crabb and Studdert, 1995; Studdert et al., 1992). EHV1 causes abortion mainly in firstfoal mares and generally in late pregnancy (Hartley and Dixon, 1979). EHV1 is also associated with stillbirths, severe respiratory disease in young horses and perinatal foal mortality (Dixon et al., 1977; Studdert and Blackney, 1979; ∗

Corresponding author. Tel.: +61 7 3362 9531; fax: +61 7 3362 9457. E-mail address: [email protected] (I.S. Diallo).

Campbell and Studdert, 1983; Sabine et al., 1983). Occasionally EHV1 will cause outbreaks of neurological disease in horses with the predominant sign being myeloencephalitis (Studdert et al., 2003; O’Callaghan et al., 1983). A variety of techniques have been used for diagnosing EHV1. However, a number of these techniques cannot differentiate it from a closely related equid herpesvirus type 4 (EHV4), which also causes respiratory disease in young horses (Allen and Bryans, 1986). Over the years a variety of PCRs targeting the thymidine kinase (TK) gene (Carvalho et al., 2000) and glycoprotein genes such as B (O’Keefe et al., 1991; Wagner et al., 1992; Kirisawa et al., 1993; Borchers and Slater, 1993), C (Lawrence et al., 1994; Galosi et al., 2001), D (Galosi et al., 2001) and H (Varrasso et al., 2001) have been used widely in research laboratories. However, for diag-

0166-0934/$ – see front matter. Crown Copyright © 2005 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.07.010

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nostic laboratories these techniques have several drawbacks. They are cumbersome and they also have a high risk of crosscontamination due to downstream manipulation of amplified product. Some of these techniques are either nested or seminested PCRs or are used in combination with other molecular techniques such as southern blotting and REA (Borchers and Slater, 1993; O’Keefe et al., 1991; Welch et al., 1992; BallagiPordany et al., 1990; Sharma et al., 1992). Even though some of the more recent PCRs were designed to differentiate the two types (1 and 4) in one step, these techniques had a major drawback of using suspected carcinogens such as ethidium bromide and ultraviolet light (Kirisawa et al., 1993; Wagner et al., 1992; Lawrence et al., 1994; Carvalho et al., 2000). Real-time PCR is a very useful tool in diagnostic virology (Mackay et al., 2002). The technique combines the 5 exonuclease activity of Taq DNA polymerase and the specific hybridisation of a fluorogenic probe to a target gene. While amplifying a target DNA, Taq hydrolyses the bound dual-labelled probe, which in turn fluoresces. The detection of the resulting fluorescence is recorded by software and plotted as a graph (Livak et al., 1995; Heid et al., 1996). Real-time PCR has been used for the detection of herpesvirus in humans (Nicoll et al., 2001) and other viral pathogens such as West Nile virus (Lanciotti et al., 2000), Hendra virus (Smith et al., 2001) and Australian Bat Lyssavirus (Smith et al., 2002). The real-time PCR assay was shown to be specific, sensitive, fast, and effective in diagnosing viral diseases and has the advantage of low risk of cross-contamination. In this paper, we describe a fluorogenic probe assay for the diagnosis of EHV1. This is a minor groove-binding (MGB)

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probe assay designed to detect the glycoprotein B gene of EHV1. The assay is able to differentiate EHV1 from EHV4. Based on the data presented the probe used in this assay was shown to be specific to EHV1; it did not react with other equine herpesviruses and it was very sensitive in the detection of EHV1 in a variety of samples including paraffin-embedded tissues. 2. Materials and methods 2.1. Samples and virus isolation Samples used in this study are listed in Table 1. The following reference viral cultures were obtained from the Centre for Equine Virology (Dr. C. Hartley, Centre for Equine Virology (CEV) University of Melbourne): EHV1 strain 438/77, EHV4 strain 405/76, equid herpesvirus type 2 (EHV2) strain 86/67 and equid herpesvirus type 3 (EHV3) strain 334/74. Other EHV1 strains were obtained from Primary Industries Research Victoria (Dr. J. Waddington, DPI, Victoria) and the Elizabeth Macarthur Agricultural Institute (Dr. P. Kirkland, DPI, NSW). These strains were used in these laboratories as positive controls in EHV1 serum neutralisation tests. Six EHV isolates from diagnostic samples (lung and liver from aborted fetuses) submitted to Yeerongpilly Veterinary Laboratory (YVL, DPI and F, Queensland) in 2002 were included in the study (Table 1). These isolates were identified as EHV1 based on clinical history, pathology, histology observations and virus isolation.

Table 1 Samples tested for validation Sample no.

Origin

Sample type

Clinical signs

Virus isolation

Suspected EHV type

TK PCR

GpB PCR

00-882 89-169752 02-23053 02-24594 02-26024 02-30610 02-30625 02-33546 A654 VIAS 438/77 405/76 86/67 334/74 94-21843 02-23053 02-24594 02-26024 02-30610 02-30625 02-33546 98-25342 03-190133

QLD QLD QLD QLD QLD QLD QLD QLD NSW VIC VIC VIC VIC VIC QLD QLD QLD QLD QLD QLD QLD NSW QLD

Culture Culture Culture Culture Culture Culture Culture Culture Culture Culture Culture Culture Culture Culture PE PE PE PE PE PE PE PE PE

Respiratory Venereal Abortion Abortion Abortion Abortion Abortion Abortion Abortion Abortion Abortion Respiratory Respiratory Venereal Abortion Abortion Abortion Abortion Abortion Abortion Abortion Abortion Diarrhoea

ED+ ED+ RK13+ RK13+ RK13+ RK13+ RK13+ RK13+ RK13+ RK13+ RK13+ ED+ ED+ ED+ RK13+ RK13+ RK13+ RK13+ RK13+ RK13+ RK13+ NC NC

2 3 1 1 1 1 1 1 1 1 1 4 2 3 1 1 1 1 1 1 1 1 None

−ve −ve +ve +ve +ve +ve +ve +ve +ve +ve +ve −ve −ve −ve +ve +ve +ve +ve +ve +ve +ve −ve −ve

−ve −ve +ve +ve +ve +ve +ve +ve +ve +ve +ve −ve −ve −ve +ve +ve +ve +ve +ve +ve +ve −ve −ve

PE tissue: paraffin-embedded tissue, NC: not cultured.

Real-time (Ct ) 0 0 18 21 18 17 19 19 15 18 15 0 0 0 19 35 34 28 26 26 20 0 0

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Additional samples of EHV3 (sample #89-169752) and EHV2 (sample #00-882) were obtained from the YVL culture collection. A total of 134 clinical samples (fresh lung and liver tissues from aborted fetuses) were also tested using the real-time PCR assay and virus isolation. The tissues were ground in virus transport medium using a mortar and pestle to a final concentration of 10%. The tissue homogenate was centrifuged at 10,000 × g for 6 min and 100 ␮l of the resulting supernatant was inoculated onto RK13 and ED cells. The cells were incubated at 37 ◦ C and checked for CPE every day. If no cytopathic effect (CPE) was observed after three 7-day passages the corresponding sample was considered to be free of equid herpesvirus. Archival tissues (lung and liver) were obtained from Toowoomba Veterinary Laboratory (courtesy of J. Gibson). These samples were paraffin-embedded tissues corresponding to viral cultures from YVL (Table 1) except for one sample, which was not cultured. All samples had been examined and characterised histologically. Sample #03-190133 was included as a negative control. It was a tissue sample from a horse with diarrhoea. All EHV1 and EHV2 isolates were grown in rabbit kidney cells (RK13). Virus was inoculated into 25 cm2 Roux Flasks and the cultures harvested after 48 h when the CPE reached 80%. Other viruses (EHV3 and EHV4) were grown on equine dermis cells (ED), as they did not grow on RK13. After harvest viral suspensions were examined by electron microscopy to confirm the presence of herpesvirus. 2.2. DNA extraction Two methods of extraction were used: • The heat lysis method described by Ballagi-Pordany et al. (1990) and Lawrence et al. (1994) was used in most of the cases. After three cycles of freeze thawing, the cell suspension was centrifuged at 1900 × g for 5 min to sediment cell debris. Two hundred microlitres of supernatant were transferred to a microfuge tube containing 20 ␮l of Proteinase K (Roche Diagnostics, Australia Pty Ltd.) (final concentration 50 ␮g/ml) and incubated at 56◦ C for 10 min. The tubes were then transferred to a block heater and heated at 95 ◦ C for 30 min and centrifuged very briefly to remove droplets from the lid. The heated suspension was used as the template in the PCR mix. • DNA was extracted from cell culture supernatant using the QIAamp® DNA Mini kit (QIAGEN Australia Pty Ltd.) according to manufacturer’s instructions. Fresh tissues from aborted fetuses (lung and liver) were ground using a mortar and pestle in virus transport medium to an approximate 10% suspension (w/v). The suspension was centrifuged at 1900 × g for 5 min and the supernate used for DNA extraction using the heat lysis as described above or using the QIAamp® DNA Mini kit (QIAGEN Australia Pty Ltd.) according to manufacturer’s instructions. The super-

nate was also used to inoculate RK13 and ED cells for virus isolation as described above. For paraffin-embedded tissues, the method used was previously described by Wright and Manos (1990). Briefly, 1 ml of xylene was added to paraffin-embedded tissue (approximately 1 mm3 thick) in a microfuge tube. The tube was centrifuged after 3 min. The tissue was then washed three times with absolute ethanol. After a brief centrifugation, the supernatant was removed and two drops of acetone were added and the tube was incubated at 56 ◦ C for 10 min (or until acetone has evaporated). Deparaffinised tissue was then digested in a digestion buffer (50 mM Tris pH 8.5, 1 mM EDTA and 5% Tween 20) containing Proteinase K (final concentration 150 ␮g/ml) by incubation at 56 ◦ C for 10 min. Proteinase K was inactivated by heating at 95 ◦ C for 10 min. After a brief centrifugation the supernatant was transferred to a new tube. Alternatively, DNA was extracted from deparaffinised tissue with QIAamp® DNA Mini Kit (QIAGEN Australia Pty Ltd.), according to manufacturer’s instructions. 2.3. Real-time PCR 2.3.1. Primers and probe The glycoprotein B of members of the Alphaherpesvirinae is well conserved, however it contains highly specific sequences that will allow the discrimination between the closely related equid herpesviruses, EHV1 and EHV4 (Wagner et al., 1992). Primers and probe targeting the glycoprotein B gene of EHV1 were designed using Primer ExpressTM software (PE Applied Biosystems Pty Ltd.). Primer and probe sequence specificity was confirmed by nucleotide–nucleotide blast search in the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/BLAST/). Primers were synthesised by Proligo Australia Pty Ltd. and had the following sequences: • EHV1 MGB F1: 5 -CAT GTC AAC GCA CTC CCA-3 • EHV1 MGB R1: 5 -GGG TCG GGC GTT TCT GT-3 The probe was an MGB probe with a fluorescent reporter 6-carboxy-fluorescein (FAM) at the 5 end of the probe and an MGB non-fluorescent quencher at the 3 end (Applied Biosystems Australia Pty Ltd.). EHV1 MGB PROBE 6 FAM-CCC TAC GCT GCT CCMGBNFQ The resulting amplicon was 63 bp long. Primers and probe were titrated and the working dilutions determined to be 0.4 ␮M for the primers and 0.1 ␮M for the probe. Standard reaction was a 25 ␮l reaction containing 2.5× Eppendorf RealMasterMixTM (Eppendorf, Australia) (dNTP, hot start Taq DNA polymerase and self-adjusting Mg2+ ), 0.4 ␮M of each primer, and 0.1 ␮M of probe and 2 ␮l of template.

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2.3.2. Cycling parameters The real-time PCR was performed on a Rotor-GeneTM model 3000 from Corbett Research. The cycling parameters were as follows: 95 ◦ C

• Initial denaturation at for 2 min • Cycling consisted of 50 cycles at 95 ◦ C for 15 and 60 s at 64 ◦ C. Data collection was performed at the annealing/ extension phase. Specific amplified products generate fluorescence, which is plotted as an exponential curve. The fluorescence is proportional to the amount of amplified product. A threshold line is established to account for non-specific background fluorescence. The cycle at which the curve crosses the threshold line is the Ct value. The results were interpreted based on Ct values as follows: • Ct values between 5 and 35 were considered positive. • Ct values between 36 and 40 were considered suspect and retested by real-time PCR, virus isolation and gel-based PCR targeting TK and glycoprotein B genes. • Ct values over 40 and values of zero were considered negative. 2.4. Other PCR used in this study The samples were also tested using the EHV1 PCR of Carvalho et al. (2000), which targets the thymidine kinase (TK) gene and the EHV1 PCR of Wagner et al. (1992), which targets the glycoprotein B gene. Reaction setting and cycling parameters were slightly modified. Briefly, the PCR was performed in 25 ␮l reaction containing 1.5 mM MgCl2 , 200 ␮M each dNTP, 0.4 ␮M each primer and 0.5 U AmpliTaq Gold (Applied Biosystems). The cycling parameters included an initial denaturation at 95 ◦ C for 5 min, followed by five cycles of 95 ◦ C for 2 min, 55 ◦ C for 1 min and 72 ◦ C for 1 min, 40 cycles of 95 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 45 s and a final elongation at 72 ◦ C for 10 min. The PCR was performed on a PCR Express Thermocycler (Thermo Hybaid). All samples were also tested for EHV4 using a published TK gene PCR (Carvalho et al., 2000). 2.5. Sensitivity Analytical sensitivity of the assay was tested using a suspension of EHV1 grown in cell culture (RK13). The viral suspension was diluted 10-fold (10−1 to 10−5 ) and titrated on RK13 cells. Simultaneously, DNA was extracted from these dilutions and used for detection of virus in real-time PCR. The results were compared to check the minimum virus quantity, expressed in TCID50 , detectable by the test. Similarly, to evaluate the minimum amount of DNA detectable by the test, DNA was extracted using QIAamp® DNA Mini Kit (QIAGEN) and then quantified using a spectrophotometer (Eppendorf) and subsequently 10-fold diluted and dilutions used for real-time PCR.

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Table 2 Comparison of virus isolation versus real-time PCR Virus isolation

Real-time PCR Positive

Negative

Total

Positive Negative

1 1

0 132

1 133

Total

2

132

134

To assess the relative sensitivity of the assay, virus isolation results for 134 clinical samples were compared to real-time PCR results (Table 2). The test was also performed on DNA extracted from archival tissues to check if the PCR would be effective in detecting EHV1 sequences in paraffin-embedded tissues. 2.6. Specificity The following equine herpesviruses were used to assess the analytical specificity of the real-time PCR: EHV2 strain 00-882 (YVL), EHV3 strain 89-169752 (YVL), EHV4 strain 405/76 (CEV), EHV2 strain 86/67 (CEV) and EHV3 strain 334/74 (CEV). The relative specificity of the assay was assessed by comparing the results of virus isolation and real-time PCR. One hundred and thirty four clinical samples were tested in both systems and the results compared. A variety of bacterial strains, which could act as specific pathogens and possible causative agents for equine abortion (Streptococcus zooepidemicus, Streptococcus equi, Streptococcus equisimilis, Rhodococcus equi, Taylorella equigenitalis) and potential bacterial contaminants such as Escherichia coli, Enterobacter agglomerans, Pseudomonas aeruginosa and Klebsiella oxytoca were tested to check if they would react with the real-time PCR. All bacterial strains were obtained from YVL culture collection (Table 3). 2.7. Housekeeping gene All DNA extracted from paraffin-embedded tissues and clinical samples were tested for PCR competency using primers targeting the Equus caballus Endothelin-B receptor gene. This ensures that samples giving a negative EHV1 PCR result are negative due to the absence of EHV1, rather Table 3 Bacterial strains from YVL culture collection Bacterial spp.

Strain

Isolated from

Escherichia coli Klebsiella oxytoca Pseudomonas aeruginosa Enterobacter agglomerans Streptococcus equi Streptococcus equisimilis Streptococcus zooepidemicus Taylorella equigenitalis Rhodococcus equi

ATCC-25922 J495/1 ATCC-27853 J3658/8A Unknown I1901/6 J1968/2 UQVS-CMO 4050

– Equine urine – Equine fetus (spleen) Unknown Equine (abscess) Equine uterus Equine metritis Bovine BAL

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than due to template degradation or the presence of PCR inhibitors. The primer sequences used were kindly provided by the Australian Equine Genetics Research Centre (Dr. I. Biros, University of Queensland). This method was modified from Metallinos et al. (1998). The primer sequences were as follows: • Forward: 5 -GGA GAC TTT CAA GTA CAT CAA CAC A-3 • Reverse: 5 -CAA GTC TCC CAG AGC CAG G-3 Cycling parameters were as follows: 95 ◦ C for 5 min followed by 10 cycles of 95 ◦ C for 2 min, 55 ◦ C for 1 min, 72 ◦ C for 1 min, followed by 35 cycles of 95 ◦ C for 15 s, 55 ◦ C for 30 s and 72 ◦ C for 30 s. The cycling was done on a PCR Express Thermocycler (Thermo Hybaid). The resulting amplicon was 154 bp. All PCR competent samples should be positive for this reaction.

3. Results The six EHV1 isolates were positive for EHV1 real-time PCR with Ct values ranging from 17 to 21 (Table 1). All isolates were identified as herpesviruses by electron microscopy (data not shown). All samples tested with real-time PCR were also tested with gel-based PCRs targeting the TK and glycoprotein B genes of EHV1. The results of EHV1 real-time PCR were found to agree with gel-based PCR results (Table 1). All suspect EHV1 samples were negative when tested for EHV4 using PCR targeting the TK gene as described by Carvalho et al. (2000). Primer and probe sequences were specific to EHV1. They matched the glycoprotein B sequence of EHV1, while they did not match any other sequence in NCBI database (data not shown). When the methods of DNA extraction were compared, it was found that the DNA extraction method using the commercial kit performed similarly to the heat lysis method, with Ct value difference of between 1 and 2 cycles in most cases (Table 4). Of 134 clinical samples tested in RK13 and ED cells only one lung sample yielded a virus after the first passage in RK13, but not in ED. The CPE was characteristic of herpesvirus with formation of syncytia; the presence of Table 4 Difference of Ct between DNA extractions Isolates

Heat lysis: 95◦ 30 min (Ct )a

Extracted DNA (Ct )b

02-23053 02-24594 02-26024 02-30610 02-33546

17 20 19 19 16

16 18 18 18 14

a b

Ballagi-Pordany et al. (1990) and Lawrence et al. (1994). QIAamp® DNA Mini Kit (QIAGEN).

Fig. 1. Amplification plots of EHV1 reference strain (from CEV), EHV1 from EMAI and sample 02-33546 using Rotor-Gene, plot 1: EHV1 (EMAI) neat: Ct = 15.02 (total DNA detected is a mixture of viral and cellular DNA), plot 2: EHV1 (CEV) neat: Ct = 15.69 (approximately 15 ng of total DNA 108 ge) (the cycle at which the plot crosses the threshold line is the Ct for a given sample, ge: genome equivalent), plot 3: sample 02-33546 neat: Ct = 16.25, plot 4: EHV1 (CEV) 10−1 : Ct = 19.11 (approximately 1.5 ng of total DNA or 107 ge), plot 5: EHV1 (CEV) 10−2 : Ct = 22.54 (approximately 150 pg of total DNA106 ge), plot 6: EHV1 (CEV) 10−3 : Ct = 25.82 (approximately 15 pg of total DNA or 105 ge), plot 7: EHV1 (CEV) 10−4 : Ct = 29.22 (approximately 1.5 pg of total DNA or 104 ge), plots 8 and 9: EHV1 (CEV) 10−5 : Ct = 32.88 and 33.9 (approximately 0.15 pg of total DNA or 103 ge). Note: All plots are in duplicate, negative control had a Ct of zero.

herpesvirus was confirmed by EM. The results were in agreement with histopathology observations (data not shown). 3.1. Sensitivity The real-time PCR assay can detect as little as 1 TCID50 of EHV1 virus and approximately 0.15 pg of DNA (Fig. 1). When virus isolation was compared to real-time PCR, 2 clinical samples (lung and liver from the same fetus) out of 134 samples tested, were positive for real-time PCR with Ct values of 30.5 and 22.4, respectively; while virus was isolated only from one of the two samples in RK13 (Table 2), but not in ED. There was no viral growth for the remaining 133 samples in both cell lines. Real-time PCR results agreed with histopathology results (data not shown). The test detected EHV1 in paraffin-embedded tissues, except for one tissue (#98-25342), which was thought to be EHV positive based on histological observations, clinical signs and pathology. There were no virus isolation data, as the sample was not cultured. When real-time PCR results were compared to virus isolation results, there was a good agreement between cell-culture grown virus from a sample and corresponding fixed tissue. However, the Ct values were higher in paraffin-embedded tissues. 3.2. Specificity Equine herpes viruses other than type 1 such as EHV4 (405/76), EHV2 (86/67, #00-882), EHV3 (334/74, #89169752) did not react in this test. Similarly, none of the bacterial strains reacted with the real-time PCR. Their Ct

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values were equal to zero when tested in real-time PCR. Furthermore, a number of clinical samples that were culture positive for Actinobacillus equuli, S. zooepidemicus, R. equi, Corynebacterium spp. and hemorrhagic E. coli were negative for real-time PCR (data not shown). The results of virus isolation and real-time PCR agreed for all clinical samples tested except one. Of the 134 samples tested 132 and 133 were negative in real-time PCR and virus isolation, respectively. These results were in agreement with histopathology observations. 3.3. Housekeeping gene All paraffin-embedded tissues tested in the real-time PCR, except one (#98-25342) were positive for the housekeeping gene (endothelin B receptor), confirming their PCR competency. Sample #98-25342 was also negative for EHV1 real-time PCR. Similarly, all tested clinical samples were confirmed to be PCR competent as they all reacted in the housekeeping PCR.

4. Discussion Real-time PCR main advantage is its ability to provide reliable results within 3–4 h including DNA preparation time. The real-time PCR described in this paper was shown to be fast and easy to perform. When used in combination with the rapid sample preparation method described by BallagiPordany et al. (1990) and Lawrence et al. (1994) results were obtained within 2 h including DNA extraction time. This method is especially suitable for high throughput laboratories. However, it would also be suitable for routine diagnostic as it is less cumbersome and faster than currently used techniques. Based on the results presented in Table 2, the real-time PCR was more sensitive than virus isolation. The real-time PCR was positive for two samples while only one of these samples yielded a herpesvirus. However, even though the liver sample had a higher Ct value (22.4) compared to the lung sample (Ct = 30.5), it did not yield any virus in cell culture even after three passages, while a herpesvirus was isolated from the lung sample. This could be due to the fact that real-time PCR can detect viral DNA even when viral particles have been inactivated and unable to grow in cell culture and the liver sample may have contained very little viable virus, but enough viral DNA to be detected by the assay. Furthermore, the liver suspension showed some cell toxicity when virus isolation was attempted. Unfortunately, only two positive samples were available at the time of this study. Further testing maybe required as fresh tissues become available. The real-time PCR described in this paper was also shown to be sensitive. It was capable of detecting low levels of virus (1 TCID50) and 0.15 pg of DNA. This was estimated to be equal to approximately 103 genome-equivalents. This

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figure is an over-estimate of the minimum amount of viral DNA detected by the real-time PCR, as no attempt was made to purify the virus prior to DNA extraction. Therefore, an undetermined amount of host DNA would also have been present. The test was also sensitive enough to detect viral DNA in paraffin-embedded tissues. However, it was negative for sample #98-25342, which presented histology suggestive of equine herpesvirus abortion. The negative result in real-time PCR and housekeeping gene PCR suggested that the DNA was sufficiently damaged to be unsuitable for PCR. Based on the sensitivity of the assay for fixed tissues, it can be extrapolated that the test is sensitive enough to detect DNA in fresh tissues. DNA in fixed tissues is more likely to be damaged than in diagnostic samples. Therefore, it can be assumed that the assay would be suitable for diagnostic purpose. The real-time PCR was specific in the detection of EHV1 and had the ability to discriminate EHV1 from EHV4. The assay was also shown to be specific, as it detected EHV1 only in samples with characteristic herpesvirus inclusion bodies as seen in histopathology. When samples free of EHV1 were tested using real-time PCR and in cell culture, the results were in agreement for 133 out of 134 samples tested. Moreover, when bacterial cultures known to cause abortion (Timoney, 1996; Fitzgerald and Yamini, 1995; Hong et al., 1993) were tested, they were negative and clinical samples that were negative for real-time PCR were culture positive for S. zooepidemicus, Actinobacillus equuli, R. equi, Corynebacterium spp. and hemorrhagic E. coli. Thus, bacteria likely to be present in diagnostic samples will not cause false positive reactions in the real-time assay. The use of this test would provide diagnostic laboratories with a good tool for detection of EHV1 and its differentiation from EHV4, a closely related virus. There was a 100% agreement between real-time PCR results and other published EHV1 PCR methods such as TK gene (Carvalho et al., 2000) and glycoprotein B (Wagner et al., 1992) (Table 1). However, this method has an added advantage compared to other published methods; there were no post-amplification manipulations involved, thereby reducing the risk of carry-over contamination. In conclusion, the real-time PCR designed to target the glycoprotein B of EHV1 is a specific, sensitive and reliable test for the detection of EHV1 in diagnostic samples based on the data presented. As such, the test should be particularly suited to high throughput diagnostic laboratories when coupled with the rapid DNA extraction method.

Acknowledgements We wish to thank J. Gibson, J. Taylor and W. Townsend (Toowoomba Veterinary Laboratory) for providing the samples. We also would like to thank Dr. C. Hartley (CEV, University of Melbourne), Dr. P. Kirkland (EMAI, DPI, NSW), and Dr. J. Waddington (PIRVC, VIC) for providing us with EHV reference strains.

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