Edwin C. Hahn

Pseudorabies virus in wild swine: a global perspective.

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.

Genetic, geographical and temporal variation of porcine reproductive and respiratory syndrome virus in Illinois

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.

Mechanisms of transmission of Aujeszky's disease virus originating from feral swine in the USA.

To understand the possible mechanisms of transmission of Aujeszkys 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.

Associations between genetics, farm characteristics and clinical disease in field outbreaks of porcine reproductive and respiratory syndrome virus.

Abstract 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.

Variation of Aujeszky's disease viruses in wild swine in USA.

In the United States of America, Aujeszkys disease (pseudorabies) has been eradicated from all domestic swine. Some re-emergence of infection occurred as vaccine use diminished. Sporadic outbreaks have also occurred because of the reservoir of infection in feral swine that have spread across the southern two-thirds of the country and Hawaii. In order to be able to understand the origins of re-emerging virus, sequence analysis of variable genes in pseudorabies virus (PRV) has been used to differentiate strains. Most PRV from feral swine can be distinguished from virus circulating in domestic pigs during the national epizootic. However, several feral swine isolates of PRV from south central states are closely related or identical in sequence to strains from domestic pigs. Extensive study by PCR for the presence of virus in the oral cavity of feral pigs disclosed that the viral DNA is distributed widely in tonsils salivary glands, taste buds and even mucosa in the vicinity of tusks. Clearly the virus in feral swine has multiple mechanisms of transmission to insure persistent infection and the threat of re-emergence in domestic swine continues.

Characterization of pseudorabies virus of wild boar origin from Europe

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).

Genital infection and transmission of pseudorabies virus in feral swine in Florida, USA

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.

Comparative utility of restriction fragment length polymorphism analysis and gene sequencing to the molecular epidemiological investigation of a viral outbreak

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).

Interaction of pseudorabies virus with porcine peripheral blood lymphocytes.

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 (105 median tissue culture infective dose per 2 × 106 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/GD8 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.

Flow cytometric analysis of porcine peripheral blood leukocytes infected with pseudorabies virus.

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.