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.     

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.     

Evidence of long distance airborne transmission of Aujeszky's disease (pseudorabies) virus

Aujeszky's disease has been the subject of an eradication campaign in Denmark since 1980. A detailed knowledge of the virus strains present in the country was provided by restriction fragment analyses of older clinical isolates, and of isolates from all the virologically confirmed outbreaks since 1985. The introduction of foreign strains into southern border areas was demonstrated during the winters of 1984/85, 1986/87 and 1987/88. An epizootic during the winter of 1987/88 was shown to correlate with an unusual predominance of southerly winds. Both conventional and specific pathogen free herds became infected. A herd level case-control analysis of the outbreaks during the winter of 1987/88 revealed that there was a positive correlation between the risk of infection and the size of the herd. The observations support the hypothesis of airborne transmission of the disease.     

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.     

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.     

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.     

Study of the persistence of Aujeszky's disease (pseudorabies) virus in peripheral blood mononuclear cells and tissues of experimentally infected pigs

The presence of Aujeszky's disease virus (ADV) in peripheral blood mononuclear cells (PBMC) and tissues of experimentally infected pigs was studied. Vaccinated and unvaccinated pigs were inoculated with different doses of Aujeszky's disease NIA-3 strain. Pigs were periodically bled and PBMC were used for virus isolation and PCR detection of virus. Tissues were obtained at the time of death (8 weeks post-inoculation) and used for ADV genome detection by PCR. ADV genome was amplified from PBMC during the acute phase of infection and, in some experimental groups, up to 38 days post-inoculation (PI). The virus was sporadically detected by virus isolation performed from PBMC. In neural tissues, ADV was constantly amplified from the trigeminal ganglia and the olfactory bulb of persistently infected pigs (euthanised 8 weeks PI). In other tissues, the viral genome was rarely detected in lymph nodes and tonsils, and, occasionally, in the bone marrow. Our results indicated that PBMC are not an appropriate source for detecting ADV persistence, since inconsistent results were obtained throughout the experiments. In neural tissues, the olfactory bulb turned out to be as important a target for ADV persistence as the trigeminal ganglia. Viral genome detection in the bone marrow indicated that this tissue may play a role in the establishment of a persistent infection.     

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.     

Rapid detection of Aujeszky's disease (pseudorabies) virus infection of pigs by direct filter hybridisation of nasal and tonsillar specimens

A direct filter hybridisation method has been developed to diagnose acute Aujeszky's disease in live pigs. The advantages of the method are easy, fast sample processing; no DNA-purification is needed, and the hybridisation itself is simplified. The direct filter hybridisation method has been tested on pseudorabies virus infected cultured cells, experimentally infected pigs and on specimens from an outbreak of Aujeszky's disease. Virus isolation and filter hybridisation gave comparable results, indicating that the direct filter hybridisation method is a good tool for rapid diagnosis. It is independent of cell culture facilities and the disease can be diagnosed in live animals within 15 hours.     

Aujeszky's disease (pseudorabies) virus: the virus and molecular pathogenesis--state of the art, June 1999

Considerable progress has been made during the last years in understanding the molecular basis of protein function in pseudorabies virus (PrV), the causative agent of Aujeszky's disease (AD). Major topics have been the identification and functional characterisation of viral envelope glycoproteins and cellular virus receptors, elucidation of viral proteins involved in neurovirulence and neuropathogenesis, detection and characterisation of attenuating mutations present in and leading to successful attenuated live vaccines, and the near completion of the genomic sequence of PrV DNA. This review, which follows an article prepared for the 1993 AD symposium in Budapest, Hungary, will briefly summarise those recent developments and update the reader on the current state of the art in PrV research.     

Immunologic comparison of the proteins of pseudorabies (Aujeszky's disease) virus and bovine herpesvirus-1

The immunologic relationship between bovine herpesvirus-1 and pseudorabies virus was examined by 80% serum cross-neutralization test, enzyme-linked immunosorbent assays, and Western immunoblotting procedures. Immunogenic cross reactivity between the 2 viruses was observed with both the serum-neutralization test and the enzyme-linked immunosorbent assay. A probing of viral Western immunoblots with rabbit hyperimmune antiserum showed that there were a number of viral-specific cross-reactive proteins between bovine herpesvirus-1 and pseudorabies virus.     

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.     

Experimental infection of fattening pigs with pseudorabies (Aujeszky's disease) virus: efficacy of attenuated live- and inactivated-virus vaccines in pigs with or without passive immunity

The efficacies of attenuated live- and inactivated-virus vaccines against pseudorabies (PR) in fattening pigs were compared. Pigs born from vaccinated or nonvaccinated sows were vaccinated with one or the other vaccine and were challenge exposed at the end of the fattening period. The particular form of PR observed in fattening units in the field could be reproduced. A marked difference was seen between the control lot and the lots of the pigs vaccinated with the attenuated live- and inactivated-virus vaccines. The protection was real, but not absolute, in the vaccinated pigs. The inactivated-virus vaccine conferred a strong passive immunity to the young pigs of vaccinated dams which interfered with the development of an active immunity. The titer of the colostral antibodies in the sera of pigs born from the sows vaccinated with the attenuated-live virus vaccine was low and decreased rapidly. The active protection obtained with this vaccine was similar to that observed with the inactivated-virus vaccine. Thermal curves, weight losses, and time necessary for recovering the weight of pigs at the time of challenge exposure seemed to be good criteria for measuring the protection of fattening pigs against this particular form of PR. The conditions of the outcome of respiratory tract disorders in these pigs are discussed.     

Sensitive glycoprotein gIII blocking ELISA to distinguish between pseudorabies (Aujeszky's disease)-infected and vaccinated pigs.

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV) (Aujeszky's disease virus) -infected pigs from those immunized with a glycoprotein g92 (gIII) deletion mutant, PRV (dlg92dltk) [OMNIMARK-PRV]. This blocking ELISA test utilizes an anti-PRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate, TMB for color development and a cloned PRVg92 (gIII) antigen to coat wells of microtiter test plates. Undiluted sera are used to block the binding of the mAbgIII-HRPO conjugate to the antigen. The gIII blocking ELISA is specific and has a sensitivity comparable to screening ELISA and latex agglutination tests. PRV-negative sera and sera from pigs vaccinated once, twice, or four times with the gIII-negative vaccine all showed negative S/N values of greater than 0.70 (S/N defined as the optical density at 630 nm of test sera/optical density at 630 nm of negative control sera). Sera from PRV-infected herds, sera from pigs experimentally infected with virulent PRV, and sera from pigs vaccinated with modified-live or inactivated gIII+ vaccines were positive for gIII antibodies (S/N less than 0.7). Sera from pigs experimentally infected with 200 PFU virulent PRV seroconverted to gIII+ antibodies 7-10 days postinfection. Sera from pigs vaccinated with gpX- and gI- vaccines seroconverted to gIII+ antibodies 7-8 days after vaccination. The gIII antibodies persisted after gIII+ vaccinated for at least 376 days postvaccination. Sera from pigs protected by vaccination with PRV (dlg92dltk) and then challenge exposed to virulent PRV at 21 days postvaccination showed gIII+ antibodies by 14 days postchallenge. The specificity and sensitivity of the gIII blocking ELISA assay was further demonstrated on the United States Department of Agriculture-National Veterinary Services Laboratory (USDA-NVSL) sera from the 1988 PRV check set and the 1989 gIII PRV check set by comparing the gIII blocking ELISA assay with virus neutralization, screening/verification ELISA and latex agglutination assays.     

Mathematical modelling of pseudorabies virus (syn. Aujeszky's disease virus) outbreaks aids eradication programmes: a review

Pseudorabies virus will be eradicated from the Netherlands if a typical infectious pig (Rind) infects, on average, less than one other pig. In this review, we used a stochastic SIR model to estimate Rind using data from the field and from experiments. Rind in sow herds was estimated to be significantly less than 1 and in rearing and finishing pigs Rind was higher than 1. However, if Rind is higher than 1, PRV can still be eradicated if one infectious herd infects less than one other herd during the period that the herd is infectious(Rherd <1). Some future developments in Dutch pig husbandry (e.g. group-housing of sows) and possible risks after halting vaccination are also quantitatively evaluated.     

Factors affecting the geographic distribution of pseudorabies (Aujeszky's disease) virus infection among swine herds in Illinois

Data on the geographic distribution of swine herds tested for pseudorabies virus (PRV) in the state of Illinois (USA) were analyzed to determine whether the prevalence of PRV-infected herds was clustered geographically at the county level. Second-order analysis of spatial dependence indicated there was a spatial clustering of counties of high PRV prevalence rates and that this clustering was greater than the observed clustering of counties with a large number of swine herds. The clustering of county PRV prevalence rates was most apparent within a radius of 120 km (on the average, approximately two couties away). The association of county PRV prevalence rates with average herd size, geographic density of swine herds in the country and regional (within 120 km) density of PRV-infected herds was analyzed using multiple linear regression. The primary factor affecting county PRV prevalence rates was the regional PRV density, which interacted with the other model variables. For counties with a low regional density of PRV infection, county PRV prevalence rates charged little with a change in county herd density or average herd size. In contrast, for counties with a high regional density of PRV infection, PRV prevalence within a county increased with increasing average herd size and increasing geographic density of swine herds in the county. The results of the current and previous studies implicate an important role for the geographic proximity of infected herds in the spread of PRV among swine herds.