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Arquivo Brasileiro de Medicina Veterinária e Zootecnia

Print version ISSN 0102-0935On-line version ISSN 1678-4162

Arq. Bras. Med. Vet. Zootec. vol.52 n.4 Belo Horizonte Aug. 2000 

Detection of equine herpesvirus 1 DNA in a single embryo and in horse semen by polymerase chain reaction

[Detecção de DNA de herpesvírus eqüino-1 em embrião e em sêmen eqüino por meio de PCR]


R. Carvalho1, L.M.F. Passos2*, A.M. Oliveira1, M. Henry2, A.S. Martins3


1Ministério da Agricultura e do Abastecimento
2Escola de Veterinária da Universidade Federal de Minas Gerais
Caixa Postal 567
30123-970 - Belo Horizonte, MG
3Instituto de Ciências Biológicas - UFMG


Recebido para publicação, após modificações, em 9 de março de 2000.
*Autor para correspondência




The genome of one equine embryo and three equine semen specimens collected from a Brazilian farm were tested by polymerase chain reaction (PCR) for the presence of EHV-1 and EHV-4-specific timidine kinase (TK) sequences. The PCR detected specific EHV-1 TK gene sequences in all samples tested. The peripheral blood leukocytes (PBL) of the embryo donor mare also was amplified by EHV-1 TK primers. Infectious virus was not recovered from any specimens. The animals did not show any clinical signal of EHV-1 or EHV-4 infections. EHV-4 was not detected in the studied specimens. The results indicate that PCR was more sensitive than virus isolation in cell culture for detecting EHV-1 in semen of carrier horses.

Keywords: Equine, semen, embryo, EHV-1, EHV-4, PCR



O genoma das amostras de um embrião e semen de três eqüinos, coletados em uma fazenda brasileira, foram testados para a presença de seqüências específicas dos genes da timidina kinase (TK) de herpesvírus eqüino-1 (HVE-1) e herpesvírus eqüino-4 (HVE-4) por meio de reação em cadeia da polimerase (PCR). A PCR detectou seqüências específicas do gene de TK de HVE-1 em todas as amostras testadas. O DNA template extraído de leucócitos periféricos da égua doadora do embrião também foi amplificado pelos pares de primers designados para TK do HVE-1. Vírus infeccioso não foi isolado desses espécimes. Os resultados indicaram que o PCR foi mais sensível do que o método de isolamento em cultura de células para a detecção do HVE-1 em sêmen de cavalos portadores.

Palavras-chave: Eqüino, sêmen, embrião, HVE-1, HVE-4, PCR




Equine herpesvirus (EHV) types 1, 2, 3, 4, and 5 are widespread in equine populations throughout the world. EHV-1 is known as the major cause of abortion in horses and EHV-4 primarily causes respiratory infections and it is rarely implicated in cases of abortion (Crabb & Studdert, 1996). EHV-1, like many others a -herpesviruses, may establish a latent infection and persist in the host, following a primary infection. The reactivation of latent EHV-1 and the re-shedding of infectious virus has been demonstrated under natural and experimental conditions (Burrows & Goodridge, 1984; Edington & Bridges, 1985; Gibson et al., 1992).

The use of the polymerase chain reaction (PCR) has contributed to answer questions about the sites and prevalence of latent EHV-1 (Welch et al., 1992; Edington et al., 1994; Baxi et al., 1995; Chesters et al., 1997). According to many authors, the detection of a -herpesviruses in semen and other specimens by PCR is more sensitive than conventional virus isolation (Sharma et al., 1992; Lawrence et al., 1994; Xia et al., 1995b; Rocha et al., 1998; Wald et al., 1999). The most important EHV-1 transmission route is the respiratory tract and young horses are usually infected in the first year of life (Gilkerson et al., 1997). The role of semen in the transmission of EHV-1 infection has not been evaluated. This paper describes the detection of EHV-1 DNA using PCR in a single embryo and three semen specimens from which the virus had not been isolated by cell culture infection.



The specimens examined in this study were obtained during an experimental horse embryo transfer research program carried out at the Veterinary School, Universidade Federal de Minas Gerais (Brazil), where the horse population is not vaccinated against EHV-1 and EHV-4 infections. The donor mare and stallions were clinically healthy during the whole observation period between insemination and embryo collection, and no abortion, during late-pregnancy had been reported during the previous two breeding seasons.

The three semen ampoules were thawed and frozen twice, briefly sonicated in an ultrasonic water bath, diluted 1:10 in cell culture medium containing antibiotics, centrifuged at 800´ g for 15min, and the supernatant collected and stored at –70ºC. The frozen embryo cells were disrupted manually by grinding with a pestle and mortar in a dry ice bath. Dilutions of 1:50 and 1:10 were used, respectively, for the semen and the embryo inoculum, and these were inoculated in parallel onto RK13 and ED monolayers. The tissue supernatant diluted 1:10 was used for phenol/chloroform DNA extraction and the DNA concentration was checked out by absorbency at 260nm and an aliquot stored at –20° C as described elsewhere (Sambrook et al., 1989). A suspension of PBL from the donor mare buffy coat was cultivated with RK13 (Welch et al., 1992). The infected cell cultures were daily inspected for cytopathic effect (CPE) during seven days. All passages showing no CPE were repeated twice after the freezing/thawing procedure. In order to avoid contamination, the extraction of DNA, the manipulation of tissues, the cell inoculation and PCR reactions were processed in different laminar flow cabinets located in different rooms.

Primers for PCR were designed based on sequence analysis of EHV-1 and EHV- 4 DNA (Nilcolson et al., 1990; Telford et al., 1992). The forward primer TkF1 (5’- CGG GAC CGC AGC TGG AAA T -3’), the reverse primer TkR2 (5’- CTG GCG AGA ACG CTA CCC -3’), and the inner forward TkF3 (5'- CCT TGG TTC CTT TGG CGA CGC AC-3') were combined in pairs as TkF1/TkR2 and TkF3/TkR2, to amplify fragments of 333bp (57.3% of GC) and 226bp, respectively, from the TK coding region of the EHV-1 gene. The forward primer TkF5 (5’-TTG GGC CGT GGC CGA AAA C-3’) and the reverse primer TkR6 (5’- CTA GCC AAA ACC TTG CCT -3’) were designed to differentiate EHV-4 from EHV-1. The latter primers spans a 333bp sequence within the EHV-4 TK coding region. The best cycling condition for the 333bp of EHV-1 and EHV- 4 TK PCR reaction was: DNA denaturation at 95°C for 5min, amplified by 5 cycles at 94ºC for 1min, 56ºC for 1min, 72ºC for 1min; followed by 35 cycles of denaturation at 92ºC for 1min; annealing at 56oC for 1min, synthesis at 72oC for 1min, and a final extension cycle of 72oC for 1min. For the inner EHV-1 TK primer pair, the denaturation temperature was dropped to 90oC and the annealing temperature increased to 58oC during the 35 cycles that followed the initial cycle. To determine the specificity of EHV-1 and -4 TK sequence amplification, genomic DNA extracted from RK13 and ED cells, as well as a bovine semen sample, were used as negative controls. The EHV-2/LK (ATCC) and PRV/ VDA 936 (Brazilian isolate) were used as virus negative control DNA templates. EHV-1/Ab1 (kindly provided by Dr. R.A. Killington) and EHV-4 405/75 (ATCC) DNA extracted from infected monolayers showing 100% of CPE were used as positive control templates. All the negative cell culture passages were scraped, centrifuged at 14,000rpm in a microcentrifuge (Eppendorf), ressuspended in 1ml of TE, thawed-frozen twice, briefly sonicated and centrifuged again at 14,000rpm. The supernatant was used for DNA extraction. The DNA from mare PBL sample was obtained using the same procedure. DNA amplification was performed using 1m l of each DNA (approximately 0.5-1.0 mg/ml) in 50ml containing 200mM of each dNTP; 5ml of 10X reaction buffer [50mM KCl, 10mM Tris-HCl (pH 8.4), 2.5mM MgCl2, 0.01% (w/v) gelatin, 1% Triton]; 25pmol of each virus specific primer; plus 0.25ml (5 U/mL) Taq DNA polymerase (Promega). Re-amplifications used 0.5mL of the PCR product. Blank tubes containing the same primer mix with water but no DNA, and positive DNA template but no primer mixes were included in each group of reaction. All PCR reactions were set up in a laminar flow cabinet fully separated from the room where the amplification products were manipulated and equipped with exclusive positive displacement automatic micropipette set, aerosol-resistant tip racks and disposable gloves. Ten ml of each PCR reaction were fractionated by electrophoresis using a 8% polyacrylamide gel at 200V for 2 hours in TBE pH 8.0. The gels were stained by ethidium bromide (0.5 mg/ml) and subsequently by silver nitrate as previously described (Herring et al., 1982).



Virus was not recovered from RK13 and ED cells inoculated with semen or embryo samples. The negative control, bovine semen DNA, amplified with this primer, did not yield neither EHV-1 nor EHV-4 TK sequences. By using EHV-1 and EHV-4 primers, no amplification products were obtained with EHV-2, PRV and genomic ED and RK13 cell DNA templates. The positive control EHV-1 and EHV-4 DNA templates extracted from infected monolayer cell cultures were consistently amplified with their specific primer pairs. The specificity of these amplifications was confirmed by obtaining the predicted restriction digest of the PCR product with TaqI and HindIII, respectively to EHV-1 and EHV-4 (data not shown).

DNA extracted from the negative passages in cell cultures did not show any positive PCR product using TkF1/TkR2, TkF3/TkR2 and TkF5/TkR6 in two amplification rounds. The PBL from donor mare failed to produce virus by cultivation on RK13 monolayers, but amplified EHV-1 TK sequences after the first and second amplification rounds, using TkF1/TkR2 and TkF3/TkR2, respectively. The serum from the mare donor had previously been positive by virus neutralization against EHV-1, but only at its lowest serum dilution (1:4). The embryo tissue DNA was not amplified during the first amplification round with TkF1/TkR2 primer pair for EHV-1, but a positive 226pb fragment was detected after a second amplification round using the TkF3/TkR2 pair. Weak bands of 333pb were visualized for each semen DNA sample after the first amplification round in silver stained gel, using 1mg DNA template (Fig 1a). These results were confirmed by a re-amplification round using the nested set TkF3/TkR2, showing clearly bands of 226bp for each semen samples (Fig 1b). These results were visualized in both ethidium bromide and silver stained gels. No positive product was detected for EHV-4 in two different amplification/re-amplification rounds using TkF5/TkR6.


a02fig01.tif (257944 bytes)




The results presented herein describe the detection of EHV-1 DNA in a single equine embryo and in three horse semen samples. Previous studies in BHV-1 and herpes simplex virus have shown that virus recover from semen is difficult by conventional isolation procedures in cell cultures, either under experimental or natural conditions (Xia et al., 1995a; Wald et al., 1999).

The PBL from the embryo donor mare was positive by PCR and negative for infectious virus by cultivation, suggesting a low level of acute infections or the presence of the latent form. This is in agreement with other authors (Slater et al., 1994), who failed to recover virus from latent infected horses. No specific or spurious PCR product was produced by EHV-4 TK primer pair in two amplification rounds.

A previous study carried out in horses at the Veterinary School using PCR, serum neutralization and an indirect-ELISA (using the EHV-1 Ab1 as antigen), demonstrated that 100% of non-symptomatic mares and young horses were EHV-1 carriers (Carvalho et al., unpublished data).

Our results show that the use of diluted semen in tissue culture medium for virus isolation seems to be a practical alternative to reduce its cytotoxic effect, although it could have diminished the chance of virus isolation (van Oirschot, 1995). In the present study, the failure of virus isolation was probably due to the low level of infectious virus or to the presence of latent forms in these specimens.

According to previous studies on BHV-1 (van Engelenburg et al., 1995; Rocha et al., 1998), our results show that the PCR is more sensitive than the virus isolation method to detect virus in semen specimens. These findings highlight for the first time the importance of applying PCR to study the pathogenesis of EHV-1 infection in a carrier stallion.

Due to the high sensitivity of the PCR used in this study, we do not exclude the possibility of embryo sample contamination with endothelial or trophoblastic cells aggregated in the uterine flushing. However, the three semen DNA samples showed specific amplification and re-amplification for EHV-1 TK gene sequences in two different amplification reaction sets. The high specificity of the amplicons produced for the TK gene region of EHV-1 was confirmed by the lack of PCR amplification for the homologue EHV-4 TK gene in these specimens. The PCR amplification of EHV-1 DNA was not interfered by components of the seminal plasma fraction, which are frequently reported as inhibitory for PCR (Guerin et al., 1995; Xia et al., 1995a).

The importance of the venereal route for infection has been investigated in BHV-1 (Afshar & Eaglesome, 1990; Weiblen et al., 1991; van Oirschot, 1995) and PRV infections (Guerin et al., 1995). However, the role of persistently infected stallion that may shed virus through the semen and the consequent implication of the venereal route in the transmission of EHV-1 have not been investigated. These results suggest that new investigations are needed to understand the role of semen in the transmission of EHV-1 infection.

Further studies should be carried out to test new approaches for neutralizing the toxic activity of the semen in cell culture, aiming to improve the diagnosis by virus isolation. This would be useful to detect the virus, viral antigen or nucleic acid, and to identify if parental cells are involved in the EHV-1 mechanism of infection. Such studies are important for ascertaining the potential of the venereal route transmission of EHV-1 for abortive infection.



This work was supported by research grants from Ministério da Agricultura e do Abastecimento da República Federativa do Brasil. We wish to thank Dr. M. Resende and Dr. R.C. Leite for providing the laboratory facilities and Dr. D.A. Leib for helpful comments on an early version of this paper.



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BHV-1, bovine herpesvirus 1; CPE, cytopathic effect; EHV-1, equine herpesvirus 1; EHV-2, equine herpesvirus 2; EHV-3, equine herpesvirus 3; EHV-4, equine herpesvirus 4; EHV-5 equine herpesvirus 5; PCR, polymerase chain reaction; RK13 , rabbit kidney cell line; ED, equine dermal cell line; TK, thymidine kinase; PBL, peripheral blood leukocytes; PRV, pseudorabies virus; ATCC American type cell collection; TBE, tris-borate buffer; TE, tris-HCl, EDTA buffer

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