Blocking ELISA to distinguish pseudorabies virus-infected pigs from those vaccinated with a glycoprotein gIII deletion mutant

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV)-infected pigs from those immunized with a glycoprotein g92(gIII) deletion mutant, PRV(dlg92dltk). The blocking ELISA utilizes 96-well microtiter test plates coated with a cloned PRV g92(gIII) antigen, a mouse monoclonal antibody against gIII antigen (moMCAgIII): horseradish peroxidase (HRPO) conjugate, and undiluted test sera. Analyses can be completed in less than 3 hours with results printed out by an automated plate reader. Analyses on over 300 pig sera from PRV-free farms, on sera from other species, and on control sera containing antibodies to microorganisms other than PRV showed that the ratio of the optical density at 405 nm for the test sample to the optical density at 405 nm for the negative control (S/N value) was greater than 0.7 for all sera. No false positives were identified. Likewise, the S/N values were greater than 0.7 for over 400 sera obtained from pigs vaccinated twice with more than 1,000 times the standard PRV (dlg92dltk) dose or 1-4 times with the standard dose (2 x 10(5) TCID50/pig). Following challenge exposure to virulent PRV, the S/N values of the vaccinates were 0.1, showing that g92(gIII) antibodies in the sera of experimentally challenged pigs strongly blocked the binding of the moMCAgIII:HRPO conjugate to the antigen-coated wells. Sera of 233 pigs from PRV-infected herds with virus neutralization (VN) titers of 1:4 or greater were tested. All except 2 of these sera had S/N values less than 0.7 and more than 175 had S/N values less than 0.1. Sixteen sera from fetal pigs with VN titers of 1:4 or greater had S/N values of 0.24 or less, but 2 sera with VN titers of 1:4 when tested 5 years prior to the PRV g92(gIII) blocking ELISA test gave false negative S/N values. Twenty-four of 29 pig sera from PRV-infected herds with VN titers less than 1:4 were positive for g92(gIII) antibodies, illustrating the sensitivity of the PRV g92(gIII) blocking ELISA test. Analyses on 7 sera with VN titers of 1:4-1:64 showed that titers obtained with the PRV g92(gIII) blocking ELISA test were from 2- to 16-fold greater than the VN titers. The accuracy and sensitivity of the PRV g92(gIII) blocking ELISA test was further demonstrated by analyses of 40 unknown sera supplied in the National Veterinary Services Laboratories 1988 PRV check test kit.     

gIII antibody responses in pigs following vaccination and challenge to Aujeszky's disease.

Serological responses to a genetically engineered Aujeszky's disease "marker" vaccine (dl gIII + dl tk) were monitored using a blocking-ELISA (B-ELISA), a serum neutralisation test (SNT) and an indirect ELISA (I-ELISA). The B-ELISA is capable of differentiating pigs vaccinated with the above vaccine from natural infection. The SNT and the I-ELISA indicated that the pigs responded to vaccination and challenge. All three tests showed that the controls and the in-contact pigs always reacted negative for antibodies. The B-ELISA was able to detect pigs challenged with a field isolate 24 days post-challenge. These pigs remained positive until 110 days post-challenge when last tested. These findings indicate that the B-ELISA could be used successfully with this vaccine in a control eradication programme. This trial also shows that the vaccine virus did not spread to the in-contact pigs and also the vaccinated and challenged pigs did not transmit the disease to other susceptible pigs when they were introduced 14 days after challenge.     

Effect of experimental infection with pseudorabies (Aujeszky's disease) virus on pigs with maternal immunity from vaccinated sows.

Live-virus and inactivated-virus vaccines were used to immunize sows against pseudorabies (Aujeszky's disease) virus. To test the efficacy of the vaccination, 53 pigs of different ages were taken from the 1st and the 2nd litters of vaccinated sows and placed separately in isolation units. The pigs were challenge exposed with virulent pseudorabies virus and examined for clinical signs, virus excretion, and serologic reaction. The challenge inoculum caused severe nervous or respiratory signs of disease in 12 of the 13 control pigs, with a mortality of 76%. The pigs from the 1st litters of sows vaccinated with the live-virus vaccine did not become sick, whereas 2 of the 9 pigs (22%) from the 2nd litters had clinical signs and died of pseudorabies. All pigs from sows vaccinated with the inactivated-virus vaccine remained healthy. The results of virus isolation from oronasal swabs, combined with the serotest results, indicated that challenge exposure of all except 1 of the pigs resulted in a subclinical infection with the formation of active immunity.     

Tonsillar changes in pigs given pseudorabies (Aujeszky's disease) virus

All of the eight 5-day-old pigs orally given pseudorabies (Aujeszky's disease) virus developed tonsillitis. The initial changes occurred in the subepithelial area between the lymphoid nodule and the crypt epithelium, showing a characteristic pattern of necrosis. The necrosis became more severe and gained access into the lymphoid nodule and crypt epithelium. Coincident with the histopathologic changes, numerous specific immunofluorescences were detected, first in the nucleus and in some parts of the cytoplasm of cells distributed in the subepithelial area. The fluorescence subsequently spread into adjacent lymphoid nodules and crypt epithelial cells. Ultrastructurally, many enveloped virus particles were detected in the center of the necrosis. Thereafter, the crypt epithelial cells also underwent degeneration, and a small number of virus particles were detected in the nucleus of the degenerating epithelial cells. In the more advanced stage, the enveloped virus particles were discharged into the crypt lumen.     

Air sampling procedure for evaluation of viral excretion level by vaccinated pigs infected with Aujeszky's disease (pseudorabies) virus.

Five groups of eight fattening pigs were vaccinated and then infected with Aujeszky's disease virus. Viral excretion was evaluated by two means: deep nasal swabbing and air sampling. It appeared that infectious airborne virus could be recovered from day 1 to day 6 after infection in the isolated units where control animals were raised. In vaccinated animals, airborne particles were also detected but the amount and duration varied in relation to their immune status at the day of virulent challenge: viral excretion was significantly lower in pigs presenting a high antibody level (1/16 to 1/64) just before infection. Results obtained with nasal swabs and with air samples were closely related. Despite its low sensitivity, the air sampling procedure could be considered as an efficient tool for reflecting infectious viral pressure in a confined atmosphere.     

Molecular biology of pseudorabies (Aujeszky's disease) virus.

In this review, some of the aspects concerning the molecular biology of pseudorabies virus (PrV), the causative agent of Aujeszky's disease, will be discussed. It will mainly focus on new findings concerning viral glycoproteins, factors determining PrV virulence, the problem of PrV latency and the development regarding genetically engineered vaccines.     

Aujeszky's disease (pseudorabies) virus detection in cerebrospinal fluid in experimentally infected pigs

The presence of Aujeszky's disease virus in cerebrospinal fluid of experimentally infected pigs was studied using the techniques of virus isolation and PCR. Pigs, some of which were previously vaccinated against Aujeszky's disease, were inoculated with different doses of the Aujeszky's disease NIA-3 strain. At the time of death or sacrifice, a sample of cerebrospinal fluid was taken and tested for the presence of virus using the mentioned techniques. Virus was isolated only from one sample, while it was detected by PCR in most of them. The higher sensitivity of the PCR technique and the possible presence of antiviral antibodies in the cerebrospinal fluid are reasons that can be argued to explain this fact. By PCR, the virus was detected more efficiently when digested cerebrospinal fluid cells were used as DNA source than when using whole cerebrospinal fluid, suggesting that the virus could be cell-associated. Aujeszky's disease virus could not be detected by PCR in pigs which survived the acute phase of the infection and were euthanased at 8 weeks post-inoculation, when they were latently infected. This indicated that the cerebrospinal fluid is not an adequate sample for the diagnosis of latency. Since Aujeszky's disease virus was detected from most of the tested samples, we believe that this could be an adequate procedure for the quick diagnosis of Aujeszky's disease.     

Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's disease vaccines.

Seven deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion. For each vaccine, the levels of clinical protection and viral excretion were compared. Groups of eight pigs were vaccinated twice with attenuated deleted Aujeszky's disease vaccines (which do not express certain glycoproteins: gI, gX or gp63). Pigs were vaccinated at the beginning of the fattening period and challenge took place at the end of it when the pigs were 18-19 weeks old. Live virus vaccines were suspended in water or in an oil-in-water emulsion. The experiment was performed in three successive assays of two groups of eight pigs (except three groups for the first assay). At each assay, a control unvaccinated group of eight pigs was added to compare the effects of challenge between vaccinated and unvaccinated animals. In total, 80 pigs were involved in this experiment. All the vaccinated pigs excreted virus from 3 to 9 d after challenge. However the level of viral excretion and the duration of the period of excretion were reduced after vaccination and especially, when oil-in-water emulsion was used. There were obvious differences between vaccines. With some vaccines, when the level of viral excretion was low, the level of clinical protection was high. However, in other cases, the level of clinical protection could be good despite a higher level of viral excretion. The seroneutralizing titres were significantly and inversely related to a low level of viral excretion but not to the level of clinical protection.     

Development of an ELISA to differentiate between animals either vaccinated with or infected by Aujeszky's disease virus

The use of two monoclonal antibodies specific for glycoproteins GI and GIII of the pseudorabies virus led to the development of a competitive ELISA which made it possible to differentiate animals infected with pseudorabies virus from animals vaccinated with the strains of the virus Bartha, NAI4 or Norden. A postvaccinal serological response could be detected from three to four weeks after vaccination. After the virulent challenge of these vaccinated pigs an infectious serological response became apparent two weeks after the challenge.     

Use of immunoenzyme (ELISA) diagnosis of Aujeszky's disease in pigs during the recovery period

Samples of blood and blood serums of pigs were examined for the presence of antibodies to the Aujeszky's disease virus. The virus-neutralizing (VN) test and the enzymoimmunologic (ELISA) method were used for this examination. As indicated by comparison of the average titres of antiviral antibodies determined by both methods, the ELISA method is 60 to 600 times more sensitive than the VN test. The high sensitivity of the ELISA method enabled to detect antiviral antibodies even in samples considered as negative after VN-testing. The method has been used with success for the sanitation of three swine stocks where the Aujeszky's disease was eradicated without interruption of operation.     

Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.     

Antigens involved in vaccination of swine against Aujeszky's disease (pseudorabies) virus.

The polypeptide and glycopolypeptide composition of a local virulent Aujeszky's disease virus (suid herpesvirus 1, SHV-1) strain (E-974) was determined in order to characterize the individual SHV-1 antigens inducing the serological responses in immunized and non-immunized animals. A commercially available inactivated vaccine of known efficacy and three experimental immunogen preparations (whole inactivated SHV-1 particles, lectin-purified glycoproteins from SHV-1 culture, and a combination of both) were used for immunization. Sera of two-month old immunized and non-immunized animals were analyzed by ELISA, seroneutralization and Western immunoblotting prior to and following challenge with E-974. Sera of 7- to 30-day-old piglets littered by immunized and non-immunized sows were likewise analyzed by immunoblotting. The following variables were determined: the total level of anti-SHV-1 antibodies, the level of neutralizing antibodies, the IgG responses to individual SHV-1 antigens, and the clinical parameters and degree of protection of the animals. The whole-particle experimental immunogen conferred greatest protection, but correlation between antibody levels and the degree of protection was imperfect. Serological responses seemed to be directed against certain structural polypeptides and viral envelope glycoproteins. The glycoprotein immunogen caused a selective response to bands which closely resemble the glycopolypeptides gII and gIII. A 71 kDa component of uncertain location within the viral structure appeared to be one of the main antigens involved in porcine serological response to SHV-1 and colostral protection of piglets.     

Functional antibody responses in pigs vaccinated with live and inactivated Aujeszky's disease virus

Functional antibody tests, including virus neutralising activity of serum, antibody dependent cellular cytotoxicity and complement mediated lysis, were used to measure the response of pigs given either live or inactivated Aujeszky's disease virus vaccines. Pigs were then challenged with virulent Aujeszky's disease virus and antibody responses were analysed and found not to correlate with protection. Reasons for this lack of correlation are discussed and it is suggested that these results indicate that more emphasis should be placed on measuring the local immune response.     

The porcine humoral response to detergent extracted Aujeszky's disease (pseudorabies) virus antigens

Twenty Aujeszky's disease (AD) virus antigens were demonstrated by crossed immunoelectrophoresis in a Triton-X-100 detergent extract of virus-infected PK-1a cells. Eight of these antigens were shown to be glycosylated based on their ability to be specifically bound by the lectin Ricinus communis agglutinin II. Pigs nasally infected with AD virus showed a significant serum antibody titer to seven of the known glycosylated antigens and to four additional antigens. The antibody titer to these antigens persisted for at least 116 days. Pigs which were vaccinated parenterally with the whole detergent extract survived a nasal challenge of 10(8 . 5) PFU of virulent AD virus. The antibody response of these vaccinated pigs on the day of challenge was essentially identical to the recovery response previously observed in non-vaccinated nasally infected pigs. These results indicate that the optimum components of future AD virus subunit vaccines and their complementary diagnostic reagents should be selected from these 11 antigens.     

Immune response in pigs to Aujeszky's disease viruses defective in glycoprotein g1 or gX.

Two Aujeszky's disease virus glycoprotein genes, gX and g1, have been used to produce deletion mutants which have then been developed into vaccines. These deletions then allow differentiation between pigs infected with wild type virus and those given the vaccine. It is not clear whether the glycoproteins encoded for by these genes are needed to induce a full protective immune response, in which case deletion mutants would suffer from lack of potency. To test this, commercially available Aujeszky's virus vaccines which lacked either gX or g1 were compared and isogenic constructs were made which differed only in the absence or presence of gX and, or, g1. These constructs and vaccines were used to vaccinate the natural host of Aujeszky's disease, the pig, and potency was measured using challenge with wild type virus. In all cases vaccines which lacked g1 performed significantly less well than those in which g1 was present, whereas deletions of gX had no significant effect on vaccine performance.     

Survival and immunization of raccoons after exposure to pseudorabies (Aujeszky's disease) virus gene-deleted vaccines

In a controlled experiment, 16 wild-trapped raccoons were exposed to 1 of 2 genetically modified live pseudorabies virus (PRV) vaccines used in swine. One vaccine had genes deleted for thymidine kinase (TK(-)) and glycoprotein G (gG(-)); the other had an additional deletion for glycoprotein E (gE(-)). These vaccines were administered orally and intranasally at four dose levels: 10(3), 10(4), 10(5), and 10(6) TCID(50). The 21 days survival rate was 37.5% for the gG(-)TK(-) vaccine; all of the survivors developed antibodies to PRV. All animals receiving the gG(-)gE(-)TK(-) vaccine survived; 75% (all except the lowest dose) developed anti-PRV antibodies. Survivors were challenged intranasally with a 3.2x10(3) TCID(50) dose of the virulent wildtype PRV Shope strain. Two of the remaining three gG(-)TK(-) vaccinated raccoons survived the challenge; for the gG(-)gE(-)TK(-) vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody titers were more likely to survive the challenge with the virulent PRV; there was a 100% mortality rate for raccoons lacking detectable anti-PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent virus.