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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Survival of Aujeszky's disease virus in infected pig slurry

It has been demonstrated that after experimental infection of pig slurry from the space under the slatted floor (infection dose of 10(6)PFU per ml), the Aujeszky's disease virus (ADV) survived for 72 hours at the temperature of 15 degrees C and at pH 6.5, but was inactivated after 96 hours. When technologically treated pig slurry from the storage tanks was saturated with water and infected with ADV at the dose of 10(5)PFU per ml, the virus survived for 23 days when kept at 15 degrees C and 4 degrees C and at pH 6.8, but was inactivated under the same conditions after 30 days. When the infective ADV dose in the technologically treated pig slurry in the storage tanks was reduced to 10(4)PFU per ml, the virus survived 16 days at +4 degrees C and pH 7.0 and 8.0 but was inactivated within 23 days after infection.     

Survival of Aujeszky's disease virus in frozen pig meat

The survival of Aujeszky's disease virus was studied in muscle, lymph node and bone marrow frozen at -18 degrees C, following infusion of a large dose of the virus into the hindquarter of a freshly killed pig. Previous attempts to induce an adequate viraemia for such studies, using intranasal and intravenous routes of inoculation of large doses of virus in live pigs, were unsuccessful. In frozen meat and marrow, the virus showed a biphasic inactivation curve with time, similar to that seen with cell-cultured virus. Most virus was rapidly inactivated initially but a small population of more stable virus persisted for a considerable period of time. In contrast, virus in lymph node showed a uniform inactivaton rate, like that of the more stable componet only. Virus was not detectable in any of the tissues after 35 days of storage at -18 degrees C.     

Studies of transplacental and perinatal infection with two clones of a single Aujeszky's disease (pseudorabies) virus isolate

The effect of Aujeszky's disease virus (ADV) infection in the last third of gestation was studied using two clones from an ADV isolate. Twelve sows were infected with one or other clone at 85 +/- 1 days of gestation. The dose of infection was 5 X 10(6) TCID50 per sow. The clinical and serological responses to the infection were different. One clone, Ls-1, produces a severe and acute illness. The course of the disease went from 9 to 14 days while the other clone, Ls-2, caused a mild or silent infection and for a shorter time. Transplacental infection occurred in only one sow which had been infected with Ls-2. The litter consisted of 8 mummified fetuses. Sows infected with Ls-1 produced piglets or mummified fetuses that were virus-negative. Perinatal infection was found in litters from both groups. Colostrum-deprived piglets that did not have postpartum contact with the sow, produced specific antibodies, 2 out of 6 born to sows infected with Ls-1 and 1 out of 4 born to sows infected with Ls-2. The antibody titers in colostrum-fed piglets were related to the extent of clinical response of the sows to the infection. Litters born to sows infected with Ls-1 had mean SNT titers from 1/12 to 1/112 while litters born to sows infected with Ls-2 showed titers of 1/2 or less than 2.     

Evaluation of field isolates of pseudorabies (Aujeszky's disease) virus as determined by restriction endonuclease analysis and hybridization

Sixty-seven isolates of pseudorabies virus (PRV) from 13 states were cleaved with 4 restriction endonucleases (RE), and after electrophoresis in agarose, their banding patterns were photographed and evaluated. The deoxyribonucleic acid (DNA) cleavage fragments were designated into regions specified by molecular weight ranges based on lambda phage DNA as a size marker. The 67 PRV isolates were evaluated according to the total number of cleavage fragments, by the number of fragments within designated molecular weight regions, and finally, by the migration of fragments within regions. Four of the 67 PRV isolates (all 4 from California) did not have a 4.1 to 4.6 megadalton HinfI band, but hybridization of the HinfI digests with a 32P probe made with the 4.4 megadalton band hybridized with 2 lighter fragments, 2.5 and 1.9 megadaltons, respectively. The BamHI digests of DNA from some PRV isolates with submolar fragments were hybridized with 32P probes made with fragments from the submolar region and the BamHI E fragment. Both probes hybridized to the submolar region of PRV with BamHI submolar fragments, but only to the trimolar (E, F, and G) band of PRV without submolar fragments in the 4.1 to 7.5 BamHI megadalton region. Epidemiologic evidence was obtained which indicated that a Missouri strain of PRV was transferred to an Illinois swine herd by importation of feeder pigs from Missouri. The results indicate that there are numerous genomically different PRV currently in circulation in the United States and that the combination of RE analysis and DNA hybridization offers useful epidemiologic information to evaluate the various strains.     

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.     

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.     

Prevalence of herds with young sows seropositive to pseudorabies (Aujeszky's disease) in northern Belgium

In Belgium, pseudorabies in swine has been the subject of a mandatory eradication programme since 1993. From December 1995 to February 1996, a survey was conducted in the five provinces of northern Belgium to estimate the provincial pseudorabies virus (PRV) herd seroprevalence. Seven hundred and twenty randomly selected herds were included in this survey. To detect recently infected animals, only young sows were sampled. The results show that 44% of these herds had an important number of PRV-seropositive young sows. The highest herd seroprevalence was observed in West Flanders (68%), followed by Antwerp (60%), East Flanders (43%), Limburg (18%), and Flemish Brabant (8%). Assuming a diagnostic test sensitivity and specificity of 95% and 99%, respectively, and a true PRV within-herd prevalence of 43%, the overall true PRV herd prevalence was estimated to be 35%. A logistic multiple-regression revealed that the presence of finishing pigs was associated with a two-fold increase in odds of a herd being seropositive (odds ratio (OR)=2.07, 95% confidence interval (CI)=1.31–3.26); a breeding herd size ≥70 sows was associated with a four-fold increase in odds of a herd being seropositive (OR=4.09, 95% CI=2.18–7.67); a pig density in the municipality of ≥455 pigs/km² was associated with a 10-fold increase in odds of a herd being seropositive (OR=9.68, 95% CI=5.17–18.12). No association was detected between the PRV herd seroprevalence and purchase policy of breeding pigs (purchased gilts, or use of homebred gilts only).