Molecular pathogenesis of equine coital exanthema: temperature-sensitive function(s) in cells infected with equine herpesviruses

Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 degrees C. The efficiencies of plating (EOP) observed were 10(-2) for both EHV 1 and 2, and 1 X 10(-6) for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34 degrees C and 39 degrees C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 degrees C, reflected those EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34 degrees C and 37 degrees C. Temperature shift experiments with EHV 3-infected cultures revealed that a temperature-sensitive function(s) responsible for the reduction in titer appeared to be a late function(s). All strains examined appeared to incorporate H3-thymidine into viral-density DNA at the non-permissive temperature of 39 degrees C. Electron microscopy of EHV 3-infected cell cultures, incubated continuously at the non-permissive temperature and examined at 18 h after infection, revealed structures consistent with the accumulation of nucleocapsids within the nucleus. The evidence presented is consistent with the hypothesis that in equine dermal cells infected with a plaque-purified wild-type strain of EHV 3 (1118LP), a function needed for the egress of nucleocapsids from the nucleus is absent at 39 degrees C. The significance of these findings relative to the pathogenicity of the disease (equine coital exanthema) caused by this virus is discussed.     

Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4)

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.     

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.     

Equine rhinopneumonitis virus (herpesvirus type 1): attenuation in stable monkey cell line.

An isolate of virulent equine herpesvirus (EHV) type 1 was adapted to Vero stable cell line by 13 serial passages at 37 C and 50 serial passages at 26 C. Characteristics of the attenuated EHV-1 were found to be avirulent, but immunogenic in horses if injected intramuscularly. The attenuated virus was regularly isolated from peripheral leukocytes in inoculated horses, but was not recovered from nasal turbinate tissues. A mild leukopenia was noticed. The attenuated virus produced characteristic large syncytia on primary isolation in rabbit kidney (RK13) or Vero cells at 37 C in contrast to cell rounding observed with virulent EHV-1. The syncytial marker was stable through 20 serial passages in Vero cells at 37 C. New application of double immunodiffusion test for distinguishing between EHV-1 and EHV-2 also is described.     

Immunologic relationships between equine herpesvirus type 1 (equine abortion virus) and type 4 (equine rhinopneumonitis virus)

The specificity of selected immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) was examined in 3 colostrum-deprived specific-pathogen-free foals. Single foals were vaccinated with inactivated EHV-1, inactivated EHV-4, or control cell lysate plus adjuvant followed by successive intranasal challenge exposures with EHV-1 and EHV-4 or with EHV-4 and EHV-1. Vaccination with inactivated virus preparations elicited cellular immune responses and antibody which were augmented by subsequent challenge exposures. Cellular immune responses, as measured by in vitro lymphocyte blastogenesis, were cross-reactive after foals were given either EHV-1 or EHV-4. Serum virus-neutralizing antibody responses were type-specific for foals given EHV-1, but were cross-reactive after EHV-4 administrations. It was concluded that diseases caused by EHV-1 and EHV-4 may be more effectively controlled with a bivalent vaccine containing both EHV-1 and EHV-4 than with the presently used monovalent vaccines based on EHV-1 alone.     

Surveillance of equine respiratory viruses in Ontario

The objective of this project was to develop and implement an active surveillance program for the early and rapid detection of equine influenza viruses in Ontario. For this purpose, from October 2003 to October 2005, nasopharyngeal swabs and acute and convalescent serum samples were collected from 115 client-owned horses in 23 outbreaks of respiratory disease in Ontario. Sera were paired and tested for antibody to equine influenza 1 (AE1-H7N7), equine influenza 2 (AE2-H3N8), equine herpesvirus 1 and 4 (EHV1 and EHV4), and equine rhinitis A and B (ERAV and ERBV). Overall, the cause-specific morbidity rate of equine influenza virus in the respiratory outbreaks was 56.5% as determined by the single radial hemolysis (SRH) test. The AE2-H3N8 was isolated from 15 horses in 5 outbreaks. A 4-fold increase in antibody levels or the presence of a high titer against ERAV or ERBV was observed in 10 out of 13 outbreaks in which AE2-H3N8 was diagnosed as the primary cause of disease. In conclusion, AE2-H3N8 was found to be an important contributor to equine respiratory viral disease. Equine rhinitis A and B (ERAV and ERBV) represented an important component in the equine respiratory disease of performing horses.     

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.     

Absolute monocyte counts as an aid to early diagnosis of equine herpesvirus 1 (EHV1) infection in Hong Kong thoroughbred horses

Two isolates of the abortion strain (subtype 1) of equine herpes virus 1 (EHV1) were recovered from nasopharyngeal swabs of 3 Thoroughbreds in training in Hong Kong taken during an outbreak of clinical respiratory disease. There are 2 subtypes of EHV1, the abortion (1) and respiratory strains (2) and serologically it is not possible to differentiate between the 2 due to antigen cross-reactivity.⁴Monocyte counts undertaken by the same experienced technologist on blood films made by a modification of the traditional ‘wedge’ smear taken from blood samples collected from 58 Thoroughbred horses during the outbreak revealed a significant correlation to EHV1 serum titres.This may allow for earlier identification of affected animals during an outbreak especially in situations where virus confirmation facilities are not readily available.     

The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989

The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Bam HI fingerprints the commonest virus identified in our study was EHV1.IP (P is for prototype strain). There was a single notable exception in that the Bam HI fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.IB that was identified as a cause of abortion in Central Kentucky in 1970 to 1974. We present evidence that EHV1.IB caused abortion in California in 1964 and has remained unaltered in its Bam HI restriction pattern. No antigenic differences were found among 4 distantly related EHV1 isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHV1 isolates is further evidence for a recent origin for EHV1 and may help to explain the natural history of this virus in the horse in which it seems to be a cause of serious epidemics of abortion and perinatal mortality, and less commonly of encephalitis.     

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.     

The polymerase chain reaction (PCR) for the detection of DNA of equine herpesviruses 1 and 4]

Formalin-fixed and Paraplast-embedded tissue samples of 42 aborted equine fetuses were examined by polymerase chain reaction for the presence of equine herpesvirus DNA. The used set of primers was located in the glycoprotein 13 open reading frame and allowed the amplification of both EHV 1 und EHV 4. By cleaving pattern analysis after Hinf I digestion EHV 1 could be distinguished from EHV 4. In 9 of the cases investigated EHV 1-DNA was detected. This finding is in absolute context with the results of the virological investigations.     

Duration of immunity induced by an adjuvanted and inactivated equine influenza, tetanus and equine herpesvirus 1 and 4 combination vaccine

An adjuvanted vaccine containing inactivated equine influenza, herpesvirus antigens, and tetanus toxoid was administered to young seronegative foals of 8 months of age by deep intramuscular injection in the neck (Group A). The first two vaccinations were given 4 weeks apart. The third was administered 6 months later. Another group of foals (Group B) was vaccinated according to the same scheme at the same time with monovalent equine herpes virus (EHV) vaccine (EHV1.4) vaccine. Antibody responses to the equine influenza (single radial haemolysis; SRH) and tetanus (ToBi ELISA) components of the vaccines were examined from first vaccination until 1 year after the third vaccination. The influenza components of the combination vaccine induced high antibody titres at two weeks after the second vaccination whereafter titres declined until the time of the third vaccination. After the third vaccination, the titres rose rapidly again to remain high for at least 1 year. Antibody titres against tetanus peaked only after the third vaccination but remained high enough to offer protective immunity for at least 1 year. Foals vaccinated with monovalent EHV1.4 remained seronegative for influenza and tetanus throughout the study. Four and a half months after the third vaccination of groups A and B, a third group of animals was vaccinated twice with monovalent EHV1.4 vaccine 4 weeks apart (Group C). Two weeks after the administration of the second dose in the later group, all groups (A, B, C and an unvaccinated control group D) were challenged with EHV-4. Vaccinated foals (Group A, B, C) showed a clear reduction of clinical symptoms and virus excretion after EHV-4 challenge compared with the unvaccinated control foals. No difference could be demonstrated among the vaccinated groups, suggesting that the combination vaccine protects as well as the monovalent vaccine. In EHV1.4-vaccinated foals both antigenic fractions induced clear protection up to 6 months after vaccination (9). It can therefore be anticipated that the efficacy of the combination vaccine against EHV-1 challenge is similar to the efficacy against EHV-1 induced by EHV1.4 vaccination.     

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.     

Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals

The envelope glycoprotein D of equine herpesvirus 1 (EHV-1 gD) has been shown in laboratory animal models to elicit protective immune responses against EHV-1 challenge, and hence is a potential vaccine antigen. Here we report that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant Iscomatrix elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses. The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/4 whole virus vaccine. Intramuscular inoculation of EHV-1 gD DNA encoded in a mammalian expression vector was less effective in inducing antibody responses when administered as the sole immunogen, but inoculation with EHV-1 gD DNA followed by recombinant EHV-1 gD induced increased gD ELISA and virus-neutralizing antibody titres in six out of seven horses. However, these titres were not higher than those induced by either EHV-1 gD or the whole virus vaccine. Isotype analysis revealed elevated gD-specific equine IgGa and IgGb relative to IgGc, IgG(T) and IgA in horses inoculated with EHV-1 gD or with the whole virus vaccine. Following inoculation of pregnant mares with EHV-1 gD, their foals had significantly higher levels of colostrally derived anti-gD antibody than foals out of uninoculated mares. The EHV-1 gD preparation did not induce a significant mean antibody response in neonatal foals following inoculation at 12 h post-partum and at 30 days of age, irrespective of the antibody status of the mare. The ability of EHV-1 gD to evoke comparable neutralizing antibody responses in horses to those of a whole virus vaccine confirms EHV-1 gD as a promising candidate for inclusion in subunit vaccines against EHV-1.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination

In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. Two to 3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system.     

Application of a type-specific enzyme-linked immunosorbent assay for equine herpesvirus types 1 and 4 (EHV-1 and -4) to horse populations inoculated with inactivated EHV-1 vaccine

A type-specific enzyme-linked immunosorbent assay (ELISA) using equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) glycoprotein G was applied for sero-epizootiology of EHV infections in Japan. Recently, an inactivated EHV-1 vaccine has been administered to racehorses for prevention of upper respiratory disease. To examine the effect of the vaccination on the result of the ELISA, 6 horses were experimentally inoculated three times intramuscularly or intranasally with inactivated EHV-1 vaccine. Sera collected from these horses were used to the type-specific ELISA and complement-fixation (CF) test. Although the CF test detected a significant increase of antibody elicited by vaccination, the ELISA did not detect any antibody response. Next, sera collected from thirty-eight horses, which were intramuscularly inoculated with inactivated EHV-1 twice at an interval of four weeks, were used in the ELISA and CF test. The results also indicated that CF titers increased by vaccine inoculation, but ELISA titers did not. To examine epizootiology of EHVs serologically in racehorse populations at two Training Centers of the Japan Racing Association, the type-specific ELISA and CF test were carried out using paired sera collected from racehorses before and after the winter season. The results showed that the ELISA could distinguish EHV-1 and EHV-4 infections in vaccinated horses serologically. In conclusion, the type-specific ELISA is considered to be useful for sero-diagnosis and sero-epizootiological research on EHV-1 and EHV-4 infections not only in unvaccinated horses, but also in vaccinated horses in Japan.