Potential of DNA-mediated vaccination for equine herpesvirus 1.

The potential of DNA-mediated immunisation to protect against equine herpesvirus 1 (EHV-1) disease was assessed in a murine model of EHV-1 respiratory infection. Intramuscular injection with DNA encoding the EHV-1 envelope glycoprotein D (gD) in a mammalian expression vector induced a specific antibody response detectable by two weeks and maintained through 23 weeks post injection. Immune responses were proportional to the dose of DNA and a second injection markedly enhanced the antibody response. EHV-1 gD DNA-injected mice developed neutralising antibodies, and a predominance of IgG2a antibodies after the DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunised with 50 microg of EHV-1 gD DNA were able to clear virus more rapidly from lung tissue and showed reduced lung pathology in comparison with control mice. The data indicate that DNA-mediated immunisation may be a useful strategy for vaccination against EHV-1.     

Meningoencephalomyelitis in horses associated with equine herpesvirus 1 infection.

During an outbreak of abortion caused by equine herpesvirus 1, a neurologic disease characterized clinically by dullness and ataxia occurred in several mares. Equine herpesvirus 1 was isolated from brain and lung of two severely affected mares. Histologically, both mares had disseminated meningoencephalomyelitis characterized by necrotizing arteritis, focal malacia in grey and white matter of brain and spinal cord, and accumulation of lymphocytes and neutrophils in paravertebral ganglia. Eosinophilic intranuclear inclusion bodies occurred in foci of necrosis in thyroid adenomas of both mares.     

Clinical signs and humoral immune response in horses following equine herpesvirus type-1 infection and their susceptibility to equine herpesvirus type-4 challenge.

A group of three horses was experimentally infected with equine herpesvirus type 1 (EHV-1) and showed clinical signs characterised by a biphasic febrile response, leucopenia and cell associated viraemia accompanied by virus shedding from the nasopharynx. A second exposure to the virus 18 days later resulted in the isolation of virus from the nasopharynx of one horse. This and a further group of three EHV-1 seropositive horses were subsequently infected with equine herpesvirus type 4 (EHV-4) 147 days after the initial EHV-1 infection and virus was shed from the nasopharynx in the absence of clinical disease. Following the first EHV-1 infection, virus specific immunoglobulin M (IgM) was present by day 5 and remained high until the second exposure at day 18 at which point levels decreased. In contrast, EHV-1 specific IgG, detected at day 6 peaked at day 18, after which time levels remained high. Virus neutralising antibodies and antibodies able to mediate antibody-dependent cellular cytotoxicity were present by day 10. The immune response to EHV-1 is discussed with reference to the disease.     

Serologic and molecular comparisons of several equine herpesvirus type 1 strains

The molecular and serologic relatedness of 2 recent respiratory tract isolates of equine herpesvirus type 1, designated T1 and T2, were compared with the Army 183, Kentucky-A hamster-adapted (KyA-ha), and L-M cell-adapted (KyA-LM) strains. Electrophoresis in polyacrylamide gels revealed differences in virion structural proteins among 4 purified strains. Seven envelope glycoproteins (molecular weight of 93,000, 65,000, 62,000, 60,000, 36,000, 20,000, and 18,000) corresponding to virion proteins 13, 16, 17, 18, 23, 25, and 26a, respectively, found in both the Army 183 and KyA-ha strains had slightly different molecular weight counterparts in both the T1 and T2 isolates, which had identical structural protein profiles. virion protein 19 (58,000 daltons), a nonglycosylated protein, was present in reduced amounts in the respiratory tract isolates, whereas virion protein 8a (200,000 daltons) was absent. Virion protein 8a, an envelope glycoprotein, was only present in the KyA-ha strain. The T1 and T2 isolates were not neutralized by equine herpesvirus type 2 antiserum and revealed little cross-neutralizatio with the Army 183 and KyA-ha strains in plaque-reduction neutralization tests. Restriction endonuclease cleavage maps of viral DNA revealed a similar, but not identical, number and size of DNA fragments between T1 and T2 isolates. Likewise, DNa profiles of Army 183, KyA-ha, and KyA-LM were also similar to each other, but vastly different from the respiratory tract isolates.     

Serological evidence of equine herpesvirus type 1 (EHV-1) activity in polo horses in Nigeria.

Serological evidence of Equine Herpes virus type 1 (EHV-1) activity in Polo horses in Nigeria is reported for the first time. Eighty-two percent of horses tested with known antigen had precipitating antibodies to EHV-1 while 43% of sera tested against antigen prepared from nasal discharges were positive suggesting that the virus was being excreted in the nasal discharges and probably acting as a source of infection for incontact animals as occurs in on-going acute infections. The result of this study indicates a high prevalence of EHV-1 activity among Polo horses in Nigeria and demonstrates the ubiquitous distribution of the virus in a country that has not been previously investigated.     

Serological responses of mares and weanlings following vaccination with an inactivated whole virus equine herpesvirus 1 and equine herpesvirus 4 vaccine

Equine herpesvirus 1 (EHV-1) is a major cause of respiratory disease and abortion in horses worldwide. Although some vaccines have been shown experimentally to reduce disease, there are few reports of the responses to vaccination in the field. This study measured antibody responses to vaccination of 159 mares (aged 4-17 years) and 101 foals (aged 3-6 months) on a large stud farm with a killed whole virus EHV-1/4 vaccine used as per the manufacturer's recommendations. Using an EHV glycoprotein D (gD)-specific ELISA and a type-specific glycoprotein G (gG) ELISA, respectively 13.8 and 28.9% of mares, and 42.6 and 46.6% of foals were classed as responding to vaccination. Additionally, 16.4 and 17.6% of mares were classified as persistently seropositive mares. Using both assays, responder mares and foals had lower week 0 mean ELISA absorbances than non-responder mares and foals. Responder mares were ten times more likely to have responder foals, and non-responder mares were six times more likely to have non-responder foals than other mares using the gG ELISA. Mares aged 7 years or less and foals aged 4 months or more were more likely to respond to vaccination than animals in other age groups. There was no association between response of mares and the number of previous vaccinations received and persistently seropositive mares did not respond to vaccination. This study documents the responses of mares and foals to vaccination in a large scale commercial environment in 2000, and suggests that knowledge of antibody status may allow a more selective vaccination strategy, representing considerable savings to industry.     

Direct detection of equine herpesvirus type 1 DNA in nasal swabs by loop-mediated isothermal amplification (LAMP)

We evaluated loop-mediated isothermal amplification (LAMP) as a means of detecting equine herpesvirus type 1 (EHV-1) DNA directly from nasal swabs. To increase the sensitivity, we added a step in which the samples were heat-treated to the original LAMP procedure. The detection limit of the LAMP assay with heat treatment was 10 times more sensitive than the original LAMP assay even when the DNA extraction step was omitted. In addition, the LAMP assay with heat treatment was more sensitive than the original LAMP assay and the polymerase chain reaction using clinical samples. The LAMP assay with heat treatment is easy to perform and so should be applicable to the diagnosis of EHV-1 infections in clinical laboratories.     

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses

OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.     

Ataxia and paresis with equine herpesvirus type 1 infection in a herd of riding school horses

An outbreak of neurologic disease associated with serologic evidence of equine herpesvirus type 1 (EHV-1) infection occurred in a herd of 46 riding school horses. Ataxia and paresis were observed in 14 geldings and 5 barren mares. Eight affected horses had distal limb edema, 1 horse had a head tilt, and 3 others had urinary incontinence. Other clinical signs included fever, depression, and inappetance in 30 horses. Seven horses with neurologic signs were treated with acyclovir. Serum neutralizing antibody titers against EHV-1 increased 4-fold between acute and convalescent samples or exceeded 1: 256 in 19 of 44 horses, confirming recent infection. A significantly greater proportion of horses that seroconverted were mares ( P = .014). Of the 19 horses exhibiting ataxia and paresis, 17 made a complete recovery, 1 made a partial recovery, and 1 was euthanized.     

The protective antigens of equine herpesvirus type 1.

Equine herpesvirus type 1 was cultivated in swine testis cell cultures and partially purified by differential centrifugation and centrifugation in a linear sucrose density gradient. The viral envelope was separated from the nucleocapsid by treatment with Rexol 25J followed by sucrose gradient centrifugation. The envelope and nucleocapsid preparations were then electrophoresed in polyacrylamide gel after solubilization with sodium dodecyl sulphate. Hamsters were immunized with various preparations of the partially purified virus, including live or inactivated equine herpesvirus type 1 and viral envelope and nucleocapsid, all derived from the Kentucky D strain of the virus. Challenge of the immunized hamsters, with a hamster-adapted strain of equine herpesvirus type 1 demonstrated protection only in those animals which had been vaccinated with envelope-containing materials. When vaccination was carried out with fractions of electrophoresed envelope or nucleocapsid, protection was induced only by polypeptides of high molecular weight containing a glycoprotein component of the envelope of equine herpesvirus type 1.     

Impact of equine herpesvirus type 1 (EHV-1) infection on the migration of monocytic cells through equine nasal mucosa

The mucosal surfaces are important sites of entry for a majority of pathogens, and viruses in particular. The migration of antigen presenting cells (APCs) from the apical side of the mucosal epithelium to the lymph node is a key event in the development of mucosal immunity during viral infections. However, the mechanism by which viruses utilize the transmigration of these cells to invade the mucosa is largely unexplored. Here, we establish an ex vivo explant model of monocytic cell transmigration across the nasal mucosal epithelium and lamina propria. Equine nasal mucosal CD172a⁺ cells (nmCD172a⁺ cells), blood-derived monocytes and monocyte-derived DCs (moDCs) were labeled with a fluorescent dye and transferred to the apical part of a polarized mucosal explant. Confocal imaging was used to monitor the migration patterns of monocytic cells and the effect of equine herpesvirus type 1 (EHV-1) on their transmigration. We observed that 16–26% of mock-inoculated nmCD172a⁺ cells and moDCs moved into the nasal epithelia, and 1–7% moved further in the lamina propria. The migration of EHV-1 inoculated monocytic cells was not increased in these tissues compared to the mock-inoculated monocytic cells. Immediate early protein positive (IEP⁺) cells were observed beneath the basement membrane (BM) 48 hours post addition (hpa) of moDCs and nmCD172a⁺ cells, but not blood-derived monocytes. Together, our finding demonstrate that monocytic cells may become infected with EHV-1 in the respiratory mucosa and transport the virus from the apical side of the epithelium to the lamina propria en route to the lymph and blood circulation.     

Pathogenicity of a new neurotropic equine herpesvirus 9 (gazelle herpesvirus 1) in horses

Pathogenicity of equine herpesvirus 9 (EHV-9), a new type of equine herpesvirus isolated from Gazella thomsoni, in horses was investigated by intranasal inoculation of EHV-9 (10(7) pfu) to two conventionally reared 8-months old half-bred weanling horses. Fever higher than 39 degrees C was recorded. Virus was recovered from nasal swabs and peripheral blood mononuclear cells. Both horses developed neutralizing antibody to EHV-9. Perivascular infiltration of mononuclear cells and glial reaction were found in the olfactory and limbic systems. The results suggested that EHV-9 has a pathogenicity in horses.     

Induction of a Th-1-biased IgG subclass response against equine herpesvirus type 1 in horses previously infected with type 4 virus

An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were na?ve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses na?ve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.     

Responses of cats to nasal vaccination with a live, modified feline herpesvirus type 1

Intranasal vaccination with a cold-adapted strain of feline herpesvirus type 1 (FHV-1) two days before challenge gave partial protection, and four days before challenge gave complete protection, against feline viral rhinotracheitis. Protection at this time appeared to be specific since vaccination with FHV-1 did not affect the disease caused by the unrelated feline calicivirus. The time course of onset of protection also confirmed that the protective mechanism was likely to be specific. However, six days after vaccination only low levels of FHV-specific IgA and IgM antibody and of interferon were found in serum and nasal washings. In lymphocyte transformation assays neither peripheral blood lymphocytes nor tonsil lymphocytes gave a significant proliferative response in the presence of FHV antigen. Pathogenesis experiments demonstrated that the tonsil and nasal turbinates were the most important sites of virulent FHV-1 replication. Vaccination significantly reduced levels of infectious virus found in both sites. The results provide evidence that no one mechanism is responsible for protection following vaccination but local specific responses are more likely to be involved.     

Immunoglobulins in the serum and nasal secretions of lambs following vaccination and aerosol challenge with parainfluenza 3 virus.

Serial changes in the concentrations of IgM, IgG and IgA were compared in specific pathogen free (SPF) lambs which had been vaccinated with live or inactivated parainfluenza 3 virus (PI 3) by either intramuscular (IM) or intranasal (IN) routes followed by aerosol challenge with PI 3. In the serum, an increase in IgM was associated with the primary antibody response to the aerosol challenge, whereas increased IgG was associated with the secondary antibody response. No changes in immunoglobulin concentrations were observed in the nasal secretions of lambs administered live or inactivated virus IM or IN without adjuvant. Marked increases in IgG were found in the serum and nasal secretions of lambs vaccinated IM with inactivated virus in Freund's complete adjuvant (FCA) and fractionation by gel filtration confirmed that the antibody was associated with IgG in both these fluids.     

In vitro cytotoxicity of serum and peripheral blood leukocytes for equine herpesvirus type 1-infected target cells.

The immune response in horses following experimental infection with equine herpesvirus type 1 (EHV-1) was assessed by measuring cytotoxicity for EHV-1-infected target cells. A technique was developed, using [125I]5-iodo-2'-deoxyuridine ([125I]IUDR)-labeled equine fetal kidney cells infected with EHV-1 as the target cells. It was shown that peripheral blood leukocytes from a recovered horse were capable of lysing target cells, as measured by the loss of radio-active label. Following the experimental infection of specific-pathogen-free ponies with EHV-1, cytotoxicity was obtained with fresh autologous serum, peripheral blood leukocytes in autologous serum, and washed peripheral blood leukocytes. Cytotoxicity of the serum and peripheral blood leukocytes was detected as early as one day after infection. It is suggested that cytotoxic antibodies or cells could play an important part in restricting virus spread after infection of the horse with EHV-1.