J. M. Whalley

Epidemiological studies of equine herpesvirus 1 (EHV-1) in Thoroughbred foals: a review of studies conducted in the Hunter Valley of New South Wales between 1995 and 1997

Sero-epidemiological studies conducted between 1995 and 1997 on two large Thoroughbred stud farms in the Hunter Valley of NSW showed clear evidence of EHV-1 infection in foals as young as 30 days of age. Similarly, serological evidence suggested that these foals were infected with EHV-1 from their dams or from other lactating mares in the group, with subsequent foal to foal spread of infection prior to weaning. These studies also provided evidence of EHV-1 infection of foals at and subsequent to weaning, with foal to foal spread of EHV-1 amongst the weanlings. These data indicated that the mare and foal population was a reservoir of EHV-1, from which new cases of infection propagated through the foal population both before and after weaning. The results of these studies support the long standing management practices of separating pregnant mares from other groups of horses to reduce the incidence of EHV-1 abortion. Also, these results have important implications for currently recommended vaccination regimens, as the efficacy of vaccination in already latently infected horses is unknown.

Epidemiology of EHV-1 and EHV-4 in the mare and foal populations on a Hunter Valley stud farm: are mares the source of EHV-1 for unweaned foals.

The prevalence of EHV-1 and EHV-4 antibody-positive horses was determined using a type specific ELISA on serum samples collected from 229 mares and their foals resident on a large Thoroughbred stud farm in the Hunter Valley of New South Wales in February 1995. More than 99% of all mares and foals tested were EHV-4 antibody positive, while the prevalence of EHV-1 antibody positive mares and foals were 26.2 and 11.4%, respectively. Examination of the ELISA absorbance data for the individual mares and foals suggested that the EHV-1 antibody positive foals had been infected recently with EHV-1 and that a sub-group of the mare population was the likely source of infectious virus for the unweaned foals.

The detection of latency-associated transcripts of equine herpesvirus 1 in ganglionic neurons

Neural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the neuronal nuclei. EHV-1 nucleotide sequence data in the region reveals the presence of a putative EHV-1 LAT promoter that shares a similar motifs with the HSV-1 LAT promoter, including the LAT promoter-binding factor, and may have a role in EHV-1 LAT expression.

Characterization of immune responses to baculovirus-expressed equine herpesvirus type 1 glycoproteins D and H in a murine model

A murine intranasal infection model for equine herpesvirus type 1 (EHV-1) was used to evaluate immune responses following immunization with insect cells infected by baculoviruses that express EHV-1 glycoproteins. Baculovirus recombinant glycoprotein D (gD) and gH both induced serum antibodies to EHV-1 when measured by ELISA. The gD recombinant also produced a neutralizing antibody response. Protective immunity, determined by accelerated clearance of virus from the target organs in the respiratory tract, was demonstrated in mice immunized with a baculovirus recombinant expressing gD. In addition to the serological response, evidence is presented which shows that cell-mediated responses also play an important role in protection. Both recombinants induced delayed-type hypersensitivity and lymphoproliferation to EHV-1 antigen. The protective effects of T cells were confirmed by adoptive transfer of spleen cells from baculovirus gD-immunized donors to recipients that were challenged with live EHV-1. Depletion of either CD4- or CD8-bearing cells from the gD-immunized donors reduced the ability of the recipients to clear virus from the target organs, although depletion of CD4 cells had a more marked effect.

EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice

Summary. Insertional mutagenesis was used to construct an equine herpesvirus 1 (EHV-1) mutant in which the open reading frame for glycoprotein D was replaced by a lacZ cassette. This gD deletion mutant (ΔgD EHV-1) was unable to infect normally permissive RK cells in culture, but could be propagated in an EHV-1 gD-expressing cell line (RK/gD). Phenotypically complemented ΔgD EHV-1 was able to infect RK cells, but did not spread to form syncytial plaques as seen with wild type EHV-1 or with ΔgD EHV-1 infection of RK/gD cell cultures. Therefore EHV-1 gD is required for virus entry and for cell-cell fusion. The phenotypically complemented ΔgD EHV-1 had very low pathogenicity in a mouse model of EHV-1 respiratory disease, compared to a fully replication-competent EHV-1 reporter virus (lacZ62/63 EHV-1). Intranasal or intramuscular inoculation of mice with ΔgD EHV-1 induced protective immune responses that were similar to those elicited in mice inoculated with lacZ62/63 EHV-1 and greater than those following inoculation with UV-inactivated virus.

Detection of EHV‐1 and EHV‐4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm

REASONS FOR PERFORMING STUDY A silent cycle of equine herpesvirus 1 infection has been described following epidemiological studies in unvaccinated mares and foals. In 1997, an inactivated whole virus EHV-1 and EHV-4 vaccine was released commercially in Australia and used on many stud farms. However, it was not known what effect vaccination might have on the cycle of infection of EHV-1. OBJECTIVE To investigate whether EHV-1 and EHV-4 could be detected in young foals from vaccinated mares. METHODS Nasal and blood samples were tested by PCR and ELISA after collection from 237 unvaccinated, unweaned foals and vaccinated and nonvaccinated mares during the breeding season of 2000. RESULTS EHV-1 and EHV-4 DNA was detected in nasal swab samples from foals as young as age 11 days. CONCLUSIONS These results confirm that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned, unvaccinated foals. POTENTIAL RELEVANCE The evidence that the cycle of EHV-1 and EHV-4 infection is continuing and that very young foals are becoming infected should assist stud farms in their management of the threat posed by these viruses.

A prime-boost immunization strategy with DNA and recombinant baculovirus-expressed protein enhances protective immunogenicity of glycoprotein D of equine herpesvirus 1 in naïve and infection-primed mice

The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding equine herpesvirus 1 (EHV-1) glycoprotein D (gD) followed by EHV-1 gD expressed by a recombinant baculovirus was assessed in a murine model of EHV-1 respiratory infection. Compared with mice inoculated with DNA or protein only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA and neutralizing antibody titres to EHV-1 and had accelerated clearance of virus from lungs following challenge infection. The enhanced protective effects of this consecutive immunization were also evident in mice which had a previous infection with EHV-1 and had pre-existing antibodies. The T-helper 1 (Th1) type of immune response induced by EHV-1 gD DNA was maintained after the protein boost, despite the gD protein alone appearing to direct a Th2 response.

DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) respiratory infection. A single intramuscular injection with plasmid DNA encoding EHV-1 glycoprotein D (EHV-1 gD), including its predicted C-terminal membrane anchor sequence, induced a specific antibody response detectable by 2 weeks and maintained through 23 weeks post injection. A second injection at 4 weeks markedly enhanced the antibody response and all EHV-1 gD-injected mice developed neutralizing antibodies. A lymphocyte proliferative response to whole EHV-1 was observed and a predominance of IgG2a antibodies after DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunized with EHV-1 gD DNA were able to clear virus significantly more rapidly from lung tissue and showed reduced lung pathology, in comparison to control mice.

N-terminal sequence analysis of equine herpesvirus 1 glycoproteins D and B and evidence for internal cleavage of the gene 71 product

Signal cleavage sites of equine herpesvirus 1 (EHV-1) glycoproteins D and B (gD and gB) and an endoproteolytic cleavage site of EHV-1 gB were determined by N-terminal amino acid sequencing and compared with known cleavage sites of homologues in other herpesvirus. Signal cleavage of EHV-1 gD occurred between Arg35 and Ala36 in a region of basic amino acids resembling the endoproteolytic cleavage sites of viral glycoproteins, nine amino acids downstream of the predicted site, while EHV-1 gB was cleaved as predicted between Ala85 and Val86. Endoproteolytic cleavage of EHV-1 gB occurred between Arg548 and Ala549, 28 amino acids downstream of the cleavage site predicted from conserved sequences of other herpesvirus gB homologous. One interpretation of these data is that EHV-1 gB is cleaved internally at both sites, a possibility which was supported by the apparent molecular masses of the unglycosylated gB subunits produced in the presence of tunicamycin. This double cleavage would release a stretch of amino acids which is not present in sequenced gB molecules of other herpesviruses. Experiments with glycosylation inhibitors indicated that cleavage of EHV-1 gB can occur in the absence of glycosylation. N-terminal sequencing also determined that a 42 kDa EHV-1 glycoprotein was a product of internal cleavage of the protein encoded by gene 71. Staggered endoproteolytic cleavage after adjacent arginine residues 506 and 507 separates the 42 kDa C-terminal subunit containing all the cysteine residues from the serine and threonine rich N-terminal region.

Evidence for involvement of equine herpesvirus 1 glycoprotein B in cell-cell fusion

SummaryMonoclonal antibodies specific for equine herpesvirus 1 (EHV-1) glycoproteins (gB, gD, gp2 and a cleaved translation product of gene 71) were tested for ability to inhibit cell-cell fusion as measured by syncytium formation in EHV-1 infected cell cultures. Syncytium formation was inhibited by a complement-dependent neutralising antibody (7B10) which recognised the large subunit of EHV-1 gB. This indicated that EHV-1 gB, in common with gB homologues of herpes simplex virus and other herpesviruses, plays a role in the cell-cell fusion process.