Suid herpesvirus 1 (SuHV1, syn. Aujeszky's disease virus [ADV] or pseudorabies virus [PrV]), which belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus is the causative agent of Aujeszky's disease (AD, pseudorabies), a notifiable disease, that causes substantial economic losses to the swine industry in countries, where AD is present. Members of the family Suidae (true pigs) are the only natural hosts for PrV, although the virus can infect numerous other mammals including ruminants, carnivores and rodents. Despite the tremendous progress that has been made in controlling and eliminating PrV in domestic pigs, there is mounting evidence that PrV infections are more widespread in wild swine across the world than originally thought. Unfortunately, our understanding of the extent of PrV infections in these wild populations and of the threat to domestic swine is still fragmentary. This review aims at giving a global perspective on PrV infections in wild swine by scrutinizing the current state of knowledge concerning (i) the global occurrence of PrV infections in free-living populations of wild swine, e.g., wild boar and feral swine, (ii) the molecular characterization of wild swine PrV, (iii) infection characteristics of PrV in populations of wild swine, (iv) the risk of spillover infections to domestic pigs, (v) potential risk-mitigating measures, focusing on further research needs.

Pseudorabies virus (PrV) infections appear to be more widely distributed in the European wild boar (Sus scrofa) population than assumed. In Europe, attempts to isolate and characterize the causative agents have been limited so far. We therefore collected and examined a total of 35 PrV isolates obtained from wild boar or hunting dogs in Germany, France, Spain, Italy, Slovakia and Hungary between 1993 and 2008. Restriction enzyme analysis of genomic DNA using BamHI showed that all isolates, except one, belonged to genogroup I but different subtypes were evident. For further investigations of the phylogenetic relationships, a 732-bp fragment of the glycoprotein C (gC) gene was amplified by PCR. Sequence analysis revealed about 40 variant positions within this fragment. Comparison of the nucleotide sequences supported the separation into a clade containing isolates from North-Rhine Westphalia, Rhineland-Palatinate (Germany), France and Spain (clade B) and an apparently more variable clade comprising isolates from Brandenburg, Baden-Wurttemberg, Saxony, Saxony-Anhalt (Germany), Slovakia, Hungary, Italy and France (clade A).

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.

Between 1995 and 1998, we designed a series of studies in which we attempted to determine the main routes of transmission involved in the natural infection of pseudorabies virus (PRV) indigenous to free-ranging feral swine (Sus scrofa). Naturally infected feral sows transmitted the infection to uninfected feral boars, with which they had been commingled for a 6-wk period. Pseudorabies virus was isolated from boar preputial swabs, but not from nasal swabs. Three of the same PRV-infected feral sows did not transmit the infection to domestic boars during a 16 wk commingling period, despite the fact that they became pregnant. Feral boars, naturally infected with PRV, transmitted the virus to domestic gilts while penned together during 6 wk. Pseudorabies virus was isolated from vaginal swabs, but not from nasal swabs of gilts, after 2 and 3 wk of commingling. When the same infected boars were commingled with either feral or domestic boars for 13 wk, PRV transmission did not occur. None of the exposed boars developed neutralizing antibodies or yielded virus from their preputial or nasal swabs. Our results indicate that PRV indigenous to feral swine is preferentially transmitted to feral or domestic swine of the opposite sex by the venereal route. This mode of transmission differs from that seen in the natural transmission of PRV prevalent in domestic swine, where contaminated secretions, excretions and aerosols are responsible for the spread of the virus. Based on these results, we feel that as long as feral swine do not come into direct contact with domestic swine, PRV-infected feral swine probably pose only a limited risk to the success of the National Pseudorabies Eradication Program. The fact that PRV is usually transmitted from feral to domestic swine at the time of mating would indicate that the isolation of domestic herds by the use of a “double fence,” should be adequate protection against reinfection with PRV.

Restriction fragment length polymorphism (RFLP) analysis and partial-genome DNA sequencing are commonly used to infer genetic relationships among pathogens. This study compares the application of both techniques to the analysis of 16 pseudorabies virus isolates collected during a 1989 outbreak. Genetic distances derived from RFLP and DNA sequence data were not significantly correlated with geographic distances between farms from which isolates were collected. RFLP-based genetic distance was, however, strongly correlated with temporal distance between isolates (days separating time of isolation). Sequence-based genetic distance was significantly correlated with temporal distance only when synonymous changes (nucleotide changes not leading to amino acid changes) were considered separately. Conversely, non-synonymous changes were correlated with the host species of origin of the viral isolate. These results indicate that selectively-neutral genetic changes most accurately reflect historical relationships, but that non-neutral changes most accurately reflect the biological environment of the viral isolate (e.g. host immune system).

Porcine reproductive and respiratory syndrome (PRRS) is a disease of domestic swine characterized by exceptionally high clinical variability. This study addresses the question of whether clinical variability in PRRS results from (a) genetic variation among viral isolates and/or (b) variation in management practices among farms on which isolates are found. Genetic data (open reading frame 5 gene sequences) and data on farm characteristics and associated clinical disease signs were collected for 62 PRRS virus (PRRSV) field isolates, representing 52 farms. Clinical disease signs were interrelated--confirming that a true reproductive syndrome exists (involving abortions, infertility in sows, deaths of sows and preweaning mortality). Pairs of farms experiencing deaths in their sow populations also tended to share viral isolates which were more similar to one another than expected by chance alone. This implies that sow death (one of the more-severe manifestations of PRRS) is under genetic influence. Large herd size was a significant risk factor for the death of sows and for respiratory disease in nursery pigs. All-in-all-out management practices in the nursery were protective against reproductive signs in the sow herd. All-in-all-out management practices in the finishing stages of production were protective against respiratory disease in nursery pigs--but were paradoxically associated with an increased risk of infertility in sows. These results suggest that farm-management practices can also influence which PRRS clinical signs are manifested during an outbreak. In general, signs associated with PRRS appear to result from a combination of genetic factors and herd-management characteristics. The relative contributions of these two influences differ depending on the specific clinical sign in question.

Porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene sequences were generated by RT-PCR from 55 field isolates collected in Illinois and eastern Iowa. Spatial and temporal patterns of genetic variation in the virus were examined on a local geographical scale in order to test the hypothesis that the genetic similarity of PRRSV isolates (measured as their percentage pairwise ORF5 nucleotide similarity) was positively correlated with their geographical proximity. Levels of genetic variability in the Illinois/eastern Iowa PRRSV sample were similar to levels of variability seen across broader geographical regions within North America. The genetic similarity of isolates did not correlate with their geographical distance. These results imply that the movement of PRRSV onto farms does not generally occur via distance-limited processes such as wind or wildlife vectors, but more typically occurs via the long-distance transport of animals or semen. Genetic distances between PRRSV isolates collected from the same farms at different times increased as the time separating the collection events increased. This result implies rapid movement of new genetic types of PRRSV into and out of farms. PRRSV ORF5 displayed a pattern of third-codon-position diversity bias that was not evident in a geographically comparable sample of pseudorabies virus (a swine alphaherpesvirus) gC gene sequences. This result provides evidence that PRRSV ORF5 is experiencing stabilizing selection against structural novelty. Despite high genetic variability at all geographical levels, PRRSV ORF5 nevertheless contained potentially antigenic regions that were invariant at the amino acid level. These regions should make effective vaccine targets if they prove to be immunogenic.

Seventeen feral swine (FS) naturally infected with pseudorabies virus (PRV) and treated with dexamethasone (4 mg/kg body wt) on five consecutive days shed virus primarily from the genital tract and less frequently from the upper respiratory tract. The FS isolates were identified as PRV by virus neutralization with specific polyclonal antiserum and by direct immunofluorescence. Restriction endonuclease analysis with BamHI showed that representative samples from a total of 62 isolates were identical to each other, but differed in at least 5 DNA bands from the PRV Shope reference strain profile. DNA purified from FS isolates propagated in Vero cells or DNA extracted directly from genital swabs were amplified in the polymerase chain reaction using primers specific for the gpII (gB) gene of PRV. This amplification yielded a product of the expected size (200 bp), which specifically hybridized to a digoxigenin-labelled 30-mer probe complementary to an area within the region defined by the primers. In a transmission experiment, PRV was recovered from the vagina at 1 and 6 weeks after uninfected feral gilts were mixed with infected feral boars. PRV was not isolated from the upper respiratory tract of either gilts or boars. At eight weeks, 4 of the 5 gilts had developed low titer neutralizing antibodies to PRV. Our results indicate that PRV in FS is transmitted through sexual contact.

To understand the possible mechanisms of transmission of Aujeszky's disease virus (pseudorabies or PRV) from a feral pig reservoir, intranasal infections were initiated in domestic pigs and in pigs from a herd derived from captured feral pigs. Virus strains originating from feral pigs and from domestic pigs were compared. Similar shedding patterns were obtained in both feral-derived and domestic pigs, however, virus strains from feral pigs were markedly attenuated. Virus could be isolated after acute infection from nasal secretions, tonsils and occasionally from genital organs. In studies of transmission of PRV by cannibalism, either latently infected or acutely infected tissue was fed to both domestic and feral-derived pigs. In two similar experiments, latently infected tissue did not transmit virus, but tissues from acutely infected pigs did transmit infection, Cannibalism was observed typically in both types of pigs older than 6 weeks of age. It was concluded that transmission of PRV originating from feral pigs can occur by several mechanisms including the respiratory route and by cannibalism of pigs that die of acute infection, Transmission of PRV from feral swine may, however, result in sub-clinical infection. (C) 1997 Elsevier Science B.V.

The diagnostic performance of 2 enzyme-linked immunosorbent assays (gX-T, gX-H) for antibodies to pseudorabies virus (PRV) glycoprotein X (gX) were evaluated using 311 serum samples from a nonvaccinated quarantined herd. When the standardized virus neutralization (VN) test, which uses the Shope strain (VN Shope), was used as the comparative diagnostic standard, the gX-T test had a 7% false-negative rate and a 52% false-positive rate, and the gX-H test had a 19% false-negative rate and a 19% false-positive rate. When the VN test with a Bartha recombinant strain (VN Bartha gIII(Ka)) was used as the diagnostic standard, the gX-T test had a 9% false-negative rate and a 26% false-positive rate, and the gX-H test had a 24% false-negative rate and a 11% false-positive rate. Thus, the gX-T test was more sensitive and the gX-H test was more specific. Additional diagnostic tests on 79 serum samples from a noninfected herd did not produce false positives for the gX-H test, but there was an 8% false-positive rate for the gX-T test. Previous studies from our laboratory have demonstrated that VN Bartha gIII(Ka) has higher sensitivity than VN Shope, without losing specificity, and thus is a better comparative diagnostic standard. When adding a suspect range to the gX-T test, using the same criteria as the suspect range for the gX-H test, the false-positive rate of the gX-T test was reduced to 5% when evaluated versus VN Bartha gIII(Ka) in the infected herd and to 1% for the PRV-negative herd. However, 18% of the positive samples were classified as suspect (vs. 8% for the gX-H test). In PRV eradication programs, the cost of false negatives is greater than the cost of false positives; thus, the gX-T diagnostic used in this study is of greater diagnostic value.

Cytotoxic T lymphocytes (CTL) against mouse P815 cells were detected after stimulation of porcine peripheral blood mononuclear cells (PBMC) with irradiated Balb/c splenocytes. In vivo priming prior to in vitro stimulation slightly enhanced CTL activity, but lysis of targets was undetectable from lymphocytes from non-immune or immune animals that were not cultured with mouse splenocytes. After primary culture with Balb/c (H-2d) splenocytes, specific killing of P815 (H-2d) targets and not L929 (H-2k) targets indicated that recognition was specific for the H-2 locus. Similarly, CTL primed by mouse cells from either of two congenic strains recognized targets with alleles homologous to the stimulating cells. The anti-murine CTL was confirmed to be a CD8+ T cell based on studies using specific monoclonal antibodies to the porcine CD4 or CD8 cells. The cells responsible for the cytotoxicity of P815 targets lacked the characteristics of non-specific NK cells because (1) naive PBMC were unable to lyse NK targets (K562 cells) during the 4 h cytotoxic assay and (2) CTL killing of P815 targets increased with time after primary stimulation, whereas killing of K562 cells remained low at all times. These results suggest that porcine CTL can be readily generated against the xenogeneic mouse major histocompatability complex.

To examine effects of pseudorabies virus (PrV) on immune cells, we investigated the ability of PrV to infect and replicate in porcine peripheral blood leukocytes (PBLs). Flow cytometric analysis revealed a leukopenia after challenge, with loss of 40% of circulating monocytes and 50% of circulating lymphocytes. Virus was isolated from PBLs of challenged pigs by cocultivation with porcine kidney cells, indicating that PBLs were infected in vivo. Presence of virus in PBLs coincided with the appearance of neurological signs 1 to 2 days prior to death. Lymphocytes stimulated with mitogens and infected in vitro sustained a low-level infection (10(5) median tissue culture infective dose per 2 x 10(6) cells). In vivo challenge perturbed the CD4/CD8 ratio of circulating lymphocytes. Survival was associated with low CD4/CD8 ratios and high levels of CD8+ cells. Mortality was associated with low levels of CD8+ cells and CD4/CD8 ratios greater than one. A maturational deficiency of CD8+ cells was found in young pigs. Our results support a mechanism of PrV immunosuppression through direct infection of circulating lymphocytes, with CD8+ T lymphocytes being important for survival.

Antibody levels to pseudorabies virus (PRV) in cerebrospinal fluid (CSF) were compared to serum levels from immunized and infected pigs. Antibody was measured by single-dilution indirect solid-phase radioimmunoassay (IRIA). There was significantly higher CSF anti-PRV IgG relative to serum anti-PRV IgG (anti-PRV index, %) from infected pigs (1.390%, n = 14) than from vaccinated ones (0.141−0.149%, n = 5 and 7). The index from vaccinated and challenged pigs was intermediate (0.627%, n = 16), suggesting that vaccination cannot abrogate but can reduce the severity of encephalitis. Piglets with maternal antibody contained minimal CSF antibody similar to that of vaccinated animals. The CSF anti-PRV antibody was detected in piglets infected with as low as 102 TCID50 at 15 days postinfection. In infected pigs, the elevated CSF anti-PRV level was due to a leakage of serum antibody through a possible blood-brain barrier (BBB) impairment but not due to intrathecal antibody synthesis. Multiple regression analysis showed that the leakage was more time dependent than dose dependent. Leakage was detected until at least 4 weeks after disappearance of acute clinical symptoms. We have associated different levels of CSF anti-viral antibody with various infection or vaccination conditions.

The susceptibility of fractionated porcine peripheral blood leukocytes (PBL) to pseudorabies virus (PRV) was studied by flow cytometry and defined by viral antigen expression. Viral antigens on the surface of infected cells and cell viability were evaluated by forward angle light scatter (FALS), 90-degree light scatter (90LS), green fluorescence (FITC-anti-PRV), and red fluorescence (propidium iodide). Approximately 10% of infected mononuclear cells from healthy pigs expressed cell-surface PRV antigen. Cell-surface fluorescence and cell type were confirmed by sorting live positive cells for microscopy. In sorted positive samples, the lymphocyte versus monocyte ratio was approximately 50%:50%, defined by morphology. Positive lymphocytes represent 5.75% of total mononuclear cells. When cells were stimulated with phytohemagglutinin (PHA) and lipopolysaccharide (LPS) before infection, mitogen-stimulated T-lymphoblasts showed increased susceptibility to PRV (40.7% positive) and died of infection. Monocytes, particularly adherent monocytes, were highly susceptible (40% to 71.4% positive). Granulocytes appeared to be refractory. The relative susceptibility of various PBL populations was compared by normalizing lymphocyte susceptibility to 1 as follows: resting total lymphocytes (1); B-lymphocytes (0.67); T-lymphoblasts (7.08); total monocytes (4.27); adherent cells (4.03 to 10.88); adherent monocytes (6.95 to 12.42); granulocytes (0.24). These findings suggest a possible mechanism by which PRV could have an immunosuppressive effect as well as a pathway for dissemination of PRV.

In pseudorabies virus (PRV) infection of pigs, antibody-dependent cellular cytotoxicity (ADCC) may be an early defense mechanism. Peripheral blood leukocytes (PBL) and pulmonary macrophages mediate ADCC activity. Antibody-dependent cellular cytotoxicity against PRV-infected target cells was assessed, and the effect of infection of cells having an ADCC-effector function was determined. Although pulmonary lavage cells (PLC) had ADCC activity, in vitro infection of PLC led to PRV replication, loss of cell viability, and loss of ADCC activity. In contrast, infection of PBL did not lead to replication, decreased cell viability, or reduced ADCC activity, compared with those in non-infected controls. Measuring ADCC activity in a longitudinal study revealed that PBL from neonates had lower ADCC activity than did PBL from pigs greater than 3.5 months old. Peripheral blood leukocytes and not PLC may have a greater role in control of PRV dissemination in the pig. The difference in activity between cells from neonates and older pigs might explain, in part, the age dependency in the severity of the disease.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.

New proteins appearing after infection of cultured L 929 cells with pseudorabies virus (PRV) were analyzed by SDS polyacrylamide gel electrophoresis. Analysis was facilitated by using a virus-cell system with marked inhibition of host protein synthesis after infection. Infected cells were pulsed during successive two hour periods through the infectious cycle with35S-methionine. Proteins were extracted with detergent and analyzed on high resolution reducing gels. Thirty-four protein bands were resolved on gels of different concentrations that varied from 7 to 15 percent. Calculated apparent molecular weights of the protein peaks were not dependent on gel concentration except for very large or small sized proteins. Eight glycoproteins were resolved after labeling with14C-glucosamine. The time course of incorporation of label was used as a measure of protein synthesis allowing the grouping of proteins according to the time of maximal synthesis. Several proteins shifted in MW during the course of infection, indicating possible post-translational cleavage or other minor modification.

A single-dilution indirect solid-phase radioimmunoassay (IRIA) was developed for the detection of low levels of anti-pseudorabies immunoglobulin G in swine sera. The assay derived increased sensitivity from the use of a second amplifying antibody. The IRIA was examined for its stoichiometry, amplification by secondary antibody, advantage of a single-dilution assay vs an end-point titration, sensitivity, and specificity. The assay had a near linear dose-response relationship with positive sera (serum-neutralization titer less than or equal to 1:16) and lacked a dose response with negative sera. With addition of the secondary antibody, the IRIA was enhanced 8.5-fold in net specific binding, and the end-point titer was amplified 32-fold. The single-dilution assay was proved to be a feasible test, compared with end-point titration. Anti-pseudorabies virus titers were at least 128-fold higher by IRIA than those by serum-neutralization test. Evidence indicated that there may be minimal or no cross-reactivity of IRIA antigen with anti-infectious bovine rhinotracheitis sera. The single-dilution IRIA was a rapid and sensitive test for anti-pseudorabies virus immunoglobulin G in swine sera.