Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

An outbreak of paresis in mares and geldings associated with equid herpesvirus 1

An outbreak of paresis occurred on a small isolated stud farm in July 1980. Of the 42 horses on the stud, infection was confined to a group of nine in-foal mares and their foals and eight other horses which were either housed together at night or grazed adjacent pastures. Eight mares and two geldings developed ataxia or paresis and one mare died. Equid herpesvirus 1 was isolated from 17 animals and serological studies confirmed that 24 of 26 animals sampled had experienced infection.     

Serological detection of equid herpesvirus 1 infections of the respiratory tract.

An investigation was made of 3 serological tests (virus neutralization, complement fixation and indirect immunofluorescence), which are applicable to epidemiological studies of infections by Equid herpesvirus 1 (EHV-1). Sera from gnotobiotic foals inoculated intranasally with various strains of EHV-1 were unable in some cases to neutralize heterologous strains and these results were not consistent with the existence of clearly-defined subtypes of EHV-1, as previously proposed. The cross-reactions in complement-fixation tests paralleled those with neutralization but immunofluorescence tests were found to be both more sensitive and more broadly reactive than the other two. Complement-fixing antibodies declined more rapidly following experimental infection than did those measured by neutralization or immunofluorescence. The results are discussed in relation to the diagnosis of EHV-1 infection and the significance they may have for the epidemiology of this disease.     

Trials of an inactivated equid herpesvirus 1 vaccine: challenge with a subtype 1 virus

Vaccinated yearlings , two-year-old and in-foal pony mares with appropriate controls were exposed to aerosols of a subtype 1 virus one to two months after two or three vaccinations; all became infected. No obvious differences in the febrile responses, clinical signs and subsequent abortions were found between vaccinated and control mares. All vaccinated yearlings and two-year-old ponies developed a febrile respiratory disease but this was less severe than that suffered by the controls and the amounts and duration of virus shedding were reduced.     

Experimental reactivation of equid herpesvirus 1 (EHV 1) following the administration of corticosteroids

Eight ponies were experimentally infected with equid herpesvirus 1 (EHV 1) (subtype 1). All animals showed clinical and serological evidence of infection and virus was isolated from nasal swabs and leucocytes. These ponies were kept in isolation for a further three months during which time complement fixing antibody decreased at least four-fold. Following immunosuppression with dexamethasone and prednisolone subtype 1 virus was recovered from six of the eight animals within 14 days. Five of these six ponies were viraemic and three of them shed virus in nasal secretions; only four displayed significant rises in complement fixing antibody and only two in neutralising antibody. Clinical abnormalities were not detected during reactivation.     

Characterization of equid herpesvirus 1 (EHV-1) related viruses from captive Grevy's zebra and blackbuck

Equid herpes virus 1 (EHV-1) related isolates from a captive blackbuck (strain Ro-1) and Grevy's zebra (strain T965) behaved similarly to EHV-1 and EHV-9 in respect to their host cell range. Restriction enzyme analysis and a phylogenetic tree confirmed that Ro-1 and T965 were identical and more closely related to EHV-1 than to EHV-9. Differences from EHV-1 became obvious firstly, by amino acid alignments revealing two unique substitutions in the gB protein of Ro-1 and T965. Secondly, an EHV-1 type-specific monoclonal antibody did not detect its antigen on Ro-1, T965 or EHV-9 infected cells by immunohistochemistry. The results support the view that Ro-1 and T965 isolates represent a distinct, previously unrecognized species of equid herpesviruses.     

Serological study of an outbreak of paresis due to equid herpesvirus 1 (EHV-1).

Six cases of paresis occurred in a Swedish stud with 48 mares and a stallion. Complement-fixation tests revealed a recent infection with EHV-1 in most horses of the stud. Serumneutralisation tests showed rapid antibody-titre increases during the course of the disease. This type of antibody response was interpreted as induced by reinfection or, possibly, recurrent infection. Two diseased mares were sacrificed. No virus could be isolated from their central nervous system (CNS), liver or spleen, but there is a presumptive evidence for the presence of an antigen specific to EHV-1 in the CNS and liver. Neutralising antibodies to EHV-1 were demonstrated in the liver and kidneys following elution by acidification of the tissues. No such antibodies could be demonstrated in the brain and spinal cord. A possible reason for this failure is discussed.     

An outbreak of foal perinatal mortality due to equid herpesvirus type 1: pathological observations.

Twenty-nine cases of EHV1 infection occurred on a property, mainly in full term foals. Some foals were stillborn, some were born alive but weak and soon died and others were healthy at birth, became ill and died within 3 days of birth. Apart from voluminous, oedematous and atelectic lungs there were no gross lesions. Microscopically the lungs showed oedema, pneumonitis and bronchiolitis with intranuclear inclusions and, in many of the foals that survived over 6 hours, there was also hyaline membrane formation. Microscopic lesions were also seen in the liver, adrenal, thymus and spleen of some of these foals.     

In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1

The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at −1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine.     

Determination of equid herpesvirus 1-specific, CD8+, cytotoxic T lymphocyte precursor frequencies in ponies.

The frequency of antigen-specific, genetically restricted cytotoxic T lymphocyte precursors (CTLp) was measured in peripheral blood mononuclear cells (PBMC) of ponies before and after infection with equid herpesvirus 1 (EHV1). Split-well limiting dilution analysis (LDA) was developed to measure CTLp frequency using EHV1-infected 51Cr-labelled lymphoblasts as targets. Extensive characterisation showed that recombinant human interleukin-2, autologous antigen presenting cells and equine serum containing virus neutralising antibody were necessary for maturation of CTLp into effector CTL in vitro. CTLs were not induced when the equine serum (containing VN antibody) was replaced with either foetal calf serum or foetal equine serum (without VN antibody), or seronegative equine serum. CTLp frequency decreased significantly when CD8+ lymphocytes were depleted from the induction cultures. There was good inter- and intra-assay reproducibility using both fresh and recovered cryopreserved PBMC. Both EHV1 and EHV4 could be used to induce effector CTL which lysed EHV1-infected target cells. CTLp frequencies were measured in 2 groups of ponies: Group 1 consisted of two ponies (approx. 9 years old), which had multiple previous experimental infections with EHV1; Group 2 comprised five young (1-2 years) and two older (7 years) ponies which had presumed natural exposure to EHV1/EHV4 but no previous experimental infections. The results showed that CTLp frequencies were higher in the ponies of Group 1 compared with the others. Moreover, ponies with the higher CTLp frequencies were better protected against re-challenge infection with EHV1, showing reduced or absent clinical and virological signs. Consequently, measurement of EHV1-specific CTLp frequency is a potential in vitro correlate of immunity which may be useful for screening new vaccines in horses before embarking upon challenge protection studies to confirm efficacy.     

Abortion of virologically negative foetuses following experimental challenge of pregnant pony mares with equid herpesvirus 1.

From 1988 to 1991, 51 pregnant pony mares were challenged intranasally or by aerosol with an isolate of EHV-1 (AB4) originally recovered from a quadriplegic mare. This resulted in 32 abortions, occurring from 9 to 29 days after infection. In 14 of the early abortions (Days 9-14), EHV-1 was not demonstrated in the foetal tissues by virus isolation or immunostaining despite no other non-viral cause for the abortion being evident. Application of the polymerase chain reaction to foetal tissues from 9 of these cases also proved negative. One of the 14 mares was destroyed immediately after abortion, and post-mortem examination revealed severe and widespread vasculitis, thrombosis and secondary ischaemic damage in the endometrium with replication of EHV-1 in endothelial cells. These findings suggest that EHV-1 abortion can occur due to endometrial damage without the establishment of a foetal infection.     

In vitro assessment of the antiviral potential of trans-cinnamic acid,quercetin and morin against equid herpesvirus 1

The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at −1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine.