J. Millar Whalley

Detection and Quantification of Equine Herpesvirus-1 Viremia and Nasal Shedding by Real-Time Polymerase Chain Reaction

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% ± 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.

Comparison of the pathogenesis of acute equine herpesvirus 1 (EHV-1) infection in the horse and the mouse model: a review

The mouse models of the respiratory and abortion forms of equine herpesvirus 1 (EHV-1) infection have been used to investigate the vaccine potential of various EHV-1 immunogens, the effect of antiviral agents on EHV-1 infection and the pathogenicity of EHV-1 strain variants and deletion or insertional mutants. This review examines the similarities and differences in the pathogenesis of primary EHV-1 infection in the natural host, the horse, and in the mouse by comparing tissue tropism, clinical signs of infection, the effects of EHV-1 on pregnancy, haematological changes following infection, viral clearance, histopathology and latency. The evidence suggests that the mouse model provides a valid method for investigation of virological and histopathological aspects of EHV-1-induced disease in the horse. However, the extent to which useful and valid comparisons and extrapolations can be made of immunological parameters from mouse to horse is yet to be determined.

Immune responses and protective efficacy of recombinant baculovirus-expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and gD alone or in combinations in BALB/c mice

Baculovirus-expressed glycoproteins of EHV-1 gB, gC and gD alone or in combination evoked antibody responses and protected vaccinated mice against a challenge with EHV-1. gB, gD, gB + gC, gB + gD and gC + gD elicited very high levels of ELISA antibodies while gC and gC + gD elicited high levels of virus neutralising antibodies. Western blotting demonstrated that the antibodies produced were not only specific for the baculovirus-expressed glycoproteins gB, gC and gD, but also highly specific for each EHV-1 glycoprotein. Vaccination of mice with gB or gD prevented clinical signs of infection in mice challenged with EHV-1 and all vaccinated groups of mice except controls showed a rapid clearance of virus from the lungs and a reduction in lesions characteristic of herpesviruses in the lungs post-challenge. Notably, the lungs of mice vaccinated with gB, gD or gB + gD and challenged with EHV-1 showed prominent peribronchiolar and perivascular aggregations of mononuclear cells, predominantly lymphocytes. Immunocytochemical staining of these sections showed large numbers of T cells, suggesting an active role for these cells at the site of virus replication post-challenge.

Epidemiological investigation of equid herpesvirus-4 (EHV-4) excretion assessed by nasal swabs taken from thoroughbred foals

Equid herpesvirus-4 (EHV-4) was detected in nasal swabs taken from foals using a PCR based test and this information used to study the epidemiology of EHV-4 disease on three Australian Thoroughbred stud farms in NSW in 1992. There was a very high level of agreement (kappa value of 0.84) between the PCR results and virus isolation using cell culture techniques. There was a strong seasonal distribution of EHV-4 shedding. Twenty-five of 26 positive samples were collected in January and March with the remaining positive sample collected in February. Foals with clinical signs of upper respiratory tract infection per se were no more likely to be shedders of EHV-4 (odds ratio [OR] 1.4, 95% confidence limits [CL] 0.5-3.8). However, EHV-4 was more likely to be isolated from foals exhibiting copious serous or mucopurulent nasal discharge than those with no clinical signs (OR 4.6, 95% CL 1.1-19.0 and OR 2.5, 95% CL 0.8-8.0, respectively). The month of the year was more important than weaning or age as a risk factor for excretion of EHV-4. Male foals and those with a history of respiratory disease that had required veterinary treatment were more likely to shed EHV-4.

The Truncated Form of Glycoprotein gp2 of Equine Herpesvirus 1 (EHV-1) Vaccine Strain KyA Is Not Functionally Equivalent to Full-Length gp2 Encoded by EHV-1 Wild-Type Strain RacL11

ABSTRACT Most equine herpesvirus 1 (EHV-1) strains, including the naturally occurring virulent RacL11 isolate, encode a large glycoprotein, gp2 (250 kDa), which is expressed from gene 71. Besides other alterations in the viral genome, the avirulent strain KyA harbors an in-frame deletion of 1,242 nucleotides in gene 71. To examine the contributions of gp2 variation to virus growth and virulence, mutant RacL11 and KyA viruses expressing full-length or truncated gp2 were generated. Western blot analyses demonstrated expression of a 250-kDa gp2 in cells infected with RacL11 virus or a mutant KyA virus harboring full-length gene 71, whereas a 75- to 80-kDa gp2 was detected in cells infected with KyA or mutant RacL11 virus expressing KyA gp2. The RacL11 gp2 precursor of 250 kDa in size and its truncated KyA counterpart of 80 kDa, as well as the 42-kDa carboxy-terminal gp2 subunit, were incorporated into virus particles. Absence of gp2 in RacL11 resulted in a 6-fold reduction of extracellular virus titers and a 13% reduction of plaque diameters, whereas gp2-negative KyA exhibited a 55% reduction in plaque diameter and a 51-fold decrease in extracellular virus titers. The massive growth defects of gp2-negative KyA could be restored by reinsertion of the truncated but not the full-length gp2 gene. The virulence of the generated gp2 mutant viruses was compared to the virulence of KyA and RacL11 in a murine infection model. RacL11 lacking gp2 was apathogenic for BALB/c mice, and insertion of the truncated KyA gp2 gene into RacL11 was unable to restore virulence. Similarly, replacement in the KyA genome of the truncated with the full-length RacL11 gene 71 did not result in the generation of virulent virus. From the results we conclude that full-length and truncated EHV-1 gp2 are not functionally equivalent and cannot compensate for the action of their homologues in allogeneic virus backgrounds.

Equine herpesvirus 1 glycoprotein D expressed in Pichia pastoris is hyperglycosylated and elicits a protective immune response in the mouse model of EHV-1 disease.

Equine herpesvirus 1 glycoprotein D (EHV-1 gD) has been shown in mouse models and in the natural host to have potential as a subunit vaccine, using various expression systems that included Escherichia coli, baculovirus and plasmid DNA. With the aim of producing secreted recombinant protein, we have cloned and expressed EHV-1 gD, lacking its native signal sequence and C-terminal transmembrane region, into the methylotrophic yeast Pichia pastoris. The truncated glycoprotein D (gD) gene was placed under the control of the methanol inducible alcohol oxidase 1 promoter and directed for secretion with the Saccharomyces cerevisiae alpha-factor prepro secretion signal. SDS-PAGE and Western blot analysis of culture supernatant fluid 24 h after induction revealed gD-specific protein products between 40 and 200 kDa. After treatment with PNGase F and Endo H, three predominant bands of 34, 45 and 48 kDa were detected, confirming high mannose N-linked glycosylation of Pichia-expressed gD (Pic-gD). N-terminal sequence analysis of PNGase F-treated affinity-purified protein showed that the native signal cleavage site of gD was being recognised by P. pastoris and the 34 kDa band could be explained by internal proteolytic cleavage effected by a putative Kex2-like protease. Pic-gD, when used in a DNA prime/protein boost inoculation schedule, induced high EHV-1 ELISA and virus neutralizing antibodies and provided protection from challenge infection in BALB/c mice.

Equine herpesvirus 1 (EHV-1) glycoprotein D DNA inoculation in horses with pre-existing EHV-1/EHV-4 antibody

We have shown previously that equine herpesvirus 1 (EHV-1) glycoprotein D (gD) DNA elicited protective immune responses against EHV-1 challenge in murine respiratory and abortion models of EHV-1 disease. In this study, 20 horses, all with pre-existing antibody to EHV-4 and two with pre-existing antibody to EHV-1, were inoculated intramuscularly with three doses each of 50, 200 or 500microg EHV-1 gD DNA or with 500microg vector DNA. In 8 of 15 horses, inoculation with EHV-1 gD DNA led to elevated gD-specific antibody and nine horses exhibited increased virus neutralising (VN) antibody titres compared to those present when first inoculated. A lack of increase in gC-specific antibody during the 66 weeks of the experiment showed that the increase in gD-specific antibodies was not due to a natural infection with either EHV-1 or EHV-4. The increase in EHV-1 gD-specific antibodies was predominantly an IgGa and IgGb antibody response, similar to the isotype profile reported following natural EHV-1 infection.

Characterization of the glycoprotein D gene products of equine herpesvirus 1 using a prokaryotic cell expression vector.

The gene encoding equine herpesvirus 1 (equine abortion virus; EHV-1) glycoprotein D was engineered into the prokaryotic vector pEX, and expressed as a beta-galactosidase fusion product, which was recognized by pooled equine sera and anti-EHV-1 rabbit sera. Antibodies raised against the EHV-1 gD fusion product identified strong bands in infected cells at 66 and 68 K and at 138 K in purified virus, thus characterizing the several forms of this major envelope glycoprotein which is an important candidate for inclusion in subunit vaccines.

Characteristics of equine herpesvirus 1 glycoproteins expressed in insect cells.

A series of recombinant baculoviruses containing genes for glycoproteins C, D, H and L of equine herpesvirus 1 (EHV-1) have been constructed, and the EHV-1 products characterised by gel electrophoresis and immunoblotting. The EHV-1 glycoproteins expressed in insect cells were similar but not identical in apparent sizes to those expressed in EHV-1 infected mammalian cells. Each of the EHV-1 products was recognised by convalescent equine sera, indicating that they were all targets for an equine immune response. Mice immunised with baculovirus-expressed EHV-1 gD and gC acquired an enhanced ability to clear challenge EHV-1 from respiratory tissues, in association with both neutralising antibody and cell mediated immune responses.

Immunization of BALB/c mice with DNA encoding equine herpesvirus 1 (EHV-1) glycoprotein D affords partial protection in a model of EHV-1-induced abortion.

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) abortion. Whilst there are differences between the model and natural infection in the horse, literature suggests that EHV-1 infection of pregnant mice can be used to assess the potential ability of vaccine candidates to protect against abortion. Female BALB/c mice were inoculated twice, 4 weeks apart, with an expression vector encoding EHV-1 glycoprotein D (gD DNA). They were mated 15 days after the second inoculation, challenged at day 15 of pregnancy and killed 3 days later. The gD DNA-inoculated mice had fewer foetuses which were damaged or had died in utero (6% in gD DNA, 21% vector DNA and 28% in nil inoculated groups challenged with EHV-1), a reduction in the stunting effect of EHV-1 infection on foetuses (gD DNA: 0.40g+/-0.06, vector DNA: 0.34g+/-0.10), reduced placental and herpesvirus-specific lung histopathology and a lower titre of virus (TCID(50)+/-SEM/lung) in maternal lung than control groups (gD DNA 4.7+/-0.3, vector 5.3+/-0.2, nil 5.6+/-0.2). Maternal antibody to EHV-1 gD was demonstrated in pups born to a dam inoculated 123 days earlier with gD DNA. Although protection from abortion was incomplete, immunization of mice with gD DNA demonstrated encouragingly the potential of this vaccine strategy.