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Passage of the basement membrane (BM), which forms a barrier between the epithelium and the underlying lamina propria, represents an important step in the early pathogenesis of different alphaherpesviruses. Rho GTPase signaling plays an important role in transmigration of cells across the BM during physiological and pathological processes. We reported earlier that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) interferes with Rho GTPase signaling and causes a reorganization of the host cell cytoskeleton, which as a consequence, enhances viral cell-to-cell spread in epithelial cell cultures. Here, using an ex vivo system of porcine nasal respiratory mucosa explants that allows to study PRV invasion through the BM, we found that a PRV strain that lacks US3 expression (ΔUS3 PRV) showed a reduced spread in mucosal epithelium and was virtually unable to breach the BM, in contrast to isogenic wild-type (WT) or US3 rescue PRV strains. Interestingly, addition of IPA3, an inhibitor of p21-activated kinases that blocks the effects of US3 on the cytoskeleton, suppressed the ability of WT PRV to spread across the BM. In addition, artificial suppression of RhoA signaling using CPC3 (cell-permeable C3 transferase) to mimic the effects of US3 on Rho GTPase signaling, significantly increased passage of ΔUS3 PRV through the BM, whereas it did not significantly affect BM passage of WT or US3 rescue PRV. In conclusion, these data indicate that US3 plays an important role in PRV mucosal invasion across the BM, which involves its interference with Rho GTPase signaling. This is the first report describing an alphaherpesvirus protein that drives viral BM passage. IMPORTANCE: Many viruses, including alphaherpesviruses, primarily replicate in epithelial cells of surface mucosae, such as the respiratory mucosa. Some of these viruses breach the basement membrane underlying these epithelial cells to reach underlying connective tissue and blood vessels and invade the host. Hence, epithelial spread and basement membrane passage represent crucial but still poorly understood early steps in (alphaherpes)virus pathogenesis. Here, using ex vivo porcine respiratory mucosa explants, we show that the conserved US3 protein of the porcine alphaherpesvirus pseudorabies virus (PRV) is critical for passage of PRV across the basement membrane and contributes to efficient viral epithelial spread. In addition, we show that US3-mediated viral epithelial spread and passage across the basement membrane depend at least in part on the ability of this viral protein to modulate cellular Rho GTPase signaling. This is the first report that identifies an alphaherpesvirus protein that drives viral basement membrane passage.

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Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process.

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The many faces of disease mapping include maps of disease case locations, regional counts of cases, and disease risk. Another approach is that of mapping the relative risk. Previous methods to map the relative risk were based on regression models of relative risk, given information about geographical locations and established risk factors. However, spatial epidemiological investigations are often exploratory with limited knowledge about the putative risk factors. Indeed, often the primary motivation for the analysis is to identify unknown geographically varying risk factors. An exploratory approach to mapping the spatial relative risk is to scale the risk map using the background risk in the unexposed (or less-exposed) population. Exposure to unknown spatial risk factors is defined via specific cluster analysis. Identification of spatial disease clusters separates the population into those inside and those outside high risk areas (the exposed and unexposed populations). This exploratory approach to relative risk mapping gives the investigator an impression about the importance and geographical distribution of the unknown spatial risk factors. Two examples illustrate the exploratory relative risk mapping approach using a spatial point data set on pseudorabies in pig-herds and a regional count data set on small fox tapeworm infections in red foxes.

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A Geographic Information System (VetEpiGIS) was used to analyze the ADV (Aujeszky's disease virus) sero-status in large-scale pig units regarding certain geographical features in a county of southern Hungary. The ADV sero-statuses were collected from all swine units in Csongrád county in 1998-2000. The units' coordinates were combined with a vector graphical digital map of the county, with a resolution of 1:100,000. Logistic regression tested the associations between sero-status of large-scale units and presence of topographical features, other units and villages in the neighborhood. "Neighborhood" was defined by circular zones with radius 1-10km around the unit (in 1km increments; one logistic regression for each radius). The following topographical features showed significant positive association with the ADV seropositivity: lake (3km OR: 5.7; 5km OR: 7.5; 6km OR: 6.1; 10km OR: 5.4) and highway (5km OR: 4.2; 6km OR: 5.3). Other features had negative association with ADV seropositivity: forest (3km OR: 0.13; 4km OR: 0.15; 5km OR: 0.15; 6km OR: 0.10; 7km OR: 0.10; 8km OR: 0.23) and uninfected large-scale unit (4km OR: 0.07; 5km OR: 0.27; 6km OR: 0.32; 7km OR: 0.31).

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This paper describes a new approach for spatial relative risk mapping as a tool for geographical risk assessment. The spatial epidemiological analysis is based on geographically referenced data about pseudorabies (Aujeszky's disease) virus infections at farm level in a region of high animal density in Germany at the beginning of the national eradication project. On the basis of serological findings 186 farms were classified as positive out of a total of 482 investigated farms listed in veterinary administrative registers. Geographical cluster analysis was used to identify two areas of high risk (RR = 2.4 and 3.3). Non-parametric density estimation was used to estimate the proportion of infected farms per square kilometre. Furthermore, the spatial relative risk function was approximated through the prevalence ratio defined by the ratio of the local prevalence and the overall prevalence of the farms outside the cluster regions. The corresponding approximated relative risk map indicates and quantifies a clear spatial pattern of disease frequency.

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Aujeszky's disease (AD) manifested itself in both German states in 1960. Owing to the historical development, in the subsequent two decades, the development of the disease and of its control in the Western and Eastern parts of Germany went different ways. This article describes differences and particularities in the development of AD in Germany leading to the establishment of a national AD eradication programme after re-unification of the two German states at the beginning of the last decade. The basic principles of the German AD eradication programme are described, and the results of 10 years of efforts to control the disease are presented and discussed. Without any doubt, as in other European countries, implementation of the national eradication programme resulted in a considerable progress in the eradication of AD. Since the eradication programme has been established in 1989, particularly in West Germany, the number of AD outbreaks has decreased steadily from about 2000 cases in 1987 to 0 cases recorded in 2001. Recently, Germany has been declared as officially AD-free by the European Commission.

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Two epidemiological studies were conducted from August 1997 to May 1998: a case-control study to identify herd level risk factors for antibodies to Aujeszky's disease virus (ADV) in sows in the state of Yucatan, Mexico and a cross-sectional study to determine the prevalence of antibodies against ADV in fattening pigs. In the case-control study, data on herd management and biosecurity were obtained from all the 27 ADV known field-virus-seropositive farms (cases) and 62 randomly selected seronegative farms (controls) by questionnaire. Breeding animals of these seropositive farms had received a gE-deletion vaccine. In the cross-sectional study, 26 farrow-to-finish farms of the 27 seropositive farms were used and blood samples taken from 60 fattening pigs per herd (15 pigs for each stage of production). Serum samples were analyzed by the screening-ELISA and gE-ELISA tests. In the case-control study, three of the 15 risk factors were significant. Odds ratios for distance to the nearest farm (< or = 2.5km), not sampling for the detection of ADV and herds with origin of breeding animals within the state were 9.5, 18.1 and 8.7. In the cross-sectional study, 11 (42.3%) of the 26 sampled farms were seropositive to vaccine antibodies. None of the piglets were positive to antibodies against field virus risk--suggesting that the strategy of vaccinating only the breeding animals reduced the ADV infection of the piglets.

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An epizootic of Aujeszky's disease (pseudorabies) in four captive European brown bears (Ursus arctos) in November 1994, in the Val di Non, Trentino Region, Italy, was linked to consumption of raw pork. Affected animals had severe pruritus resulting in self-mutilation, and all four died within 24 hr after onset of clinical signs. Aujeszky's disease virus was isolated on first passage from the brain and was characterized by means of restriction endonuclease analysis. Based on these data, we believe that bears are extremely susceptible to the disease, and that wildlife managers should consider pseudorabies as a potential risk for the captive and wild bear populations.

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Pancreatic B cells are known to be damaged by a wide range of viruses, causing diabetes. Though these viruses belong to different taxonomic groups, their single shared characteristic is neurotropism. In the present study, pseudorabies viral infection was modelled on fetal porcine islets cultivated in vitro. It was demonstrated that the endocrine cells of the pancreas, especially B cells, were infected in vitro and so served as a medium for the replication of the virus. All stages of the morphogenesis of the virus were observed ultrastructurally within the cells. The exocrine cells located close to the endocrine ones were free from attachment and invasion of the virus. The potential of the pseudorabies virus to develop within pancreatic endocrine cells is regarded as evidence of the paraneuronal nature of these cells.

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Pigs exposed to fluctuating temperatures (high, 30 +/- 2 degrees C; low, 4 +/- 1 degrees C) were intranasally inoculated with Aujeszky's disease virus (ADV). ADV-infected pigs, exposed to the fluctuating temperatures, showed severe clinical signs and ADV in the nasal secretions persisted longer than in the ADV-infected control pigs kept at the normal temperature (20 +/- 2 degrees C). High concentrations of ADV were isolated from nasal secretions on the 1st day after inoculation of the virus. Pathologically, all ADV-infected pigs had non-suppurative encephalitis and trigeminal ganglionitis. The lesions were more widely distributed in pigs exposed to fluctuating temperatures than in infected control pigs. Two infected pigs given the stress had severe malacic foci in the frontal lobe and four of them had prominent interstitial pneumonia. In the pigs exposed to fluctuating temperatures, a significant number of immunoglobulin-containing cells, especially IgM-containing cells, did not respond to ADV infection. A significant (P < 0.01) difference in the number of IgG- and IgM-containing cells was observed between the ADV-infected pigs exposed to the fluctuating temperature and ADV-infected control pigs, respectively. These results demonstrated that the stress of fluctuating temperatures enhanced the susceptibility to ADV infection.

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Pigs (3 and 10 weeks old) were infected intranasally with Aujeszky's disease virus (ADV) mutants that functionally lacked one of the non-essential genes in the unique short region of the genome (except the gene encoding the 11K protein). Virus excretion in oropharyngeal fluid and disease symptoms were monitored. Some pigs were killed to study pathogenesis, whereas others were challenged with virulent ADV 8 weeks after the primary infection. Mutants lacking protein kinase, or glycoproteins gp63 or gI showed reduced virulence, but mutants lacking gX or the 28K protein showed normal virulence. Glycoprotein gI appears to affect the tissue tropism of ADV in pigs, presumably by facilitating the spread of the virus through the central nervous system. In this study, there was no correlation between virulence and virus multiplication in either cultured cells or in the oropharynx in vivo. All mutants induced neutralizing antibody and complete or partial protection against challenge infection. Complete protection was obtained by inoculation with the gI and gX mutants, whereas incomplete protection was obtained using gp63 and protein kinase mutants. Complete clinical and virological protection was associated with the absence of secondary antibody responses in the serum.

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Latent viral DNA was detected by the polymerase chain reaction in trigeminal ganglia of all of 10 pigs that were necropsied 81 or more days after they had been infected intranasally with a thymidine kinase-negative (TK-) vaccine strain of pseudorabies virus (PRV). Failure to reactivate virus from any of the same pigs by earlier treatment with dexamethasone suggested that even though latency can be established with TK- PRV, subsequent reactivation may be a relatively rare event.

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Insertion of reporter genes into complex viral genomes and monitoring virus replication by detecting the corresponding protein products is increasingly used in pathogenesis studies. We present here the isolation and characterization of a recombinant neurotropic alpha-herpesvirus, pseudorabies virus (PrV), which stably carries the gene encoding firefly luciferase. To express the enzyme the complete open reading frame for luciferase was fused to the promoter and first seven codons of the non-essential glycoprotein gX gene of PrV. A recombinant PrV carrying the luciferase gene inserted into the gX gene and exhibiting strong luciferase activity after infection of cultured cells was further characterized. Kinetic analyses showed that luciferase activity was detectable as early as 90 min after infection. Luciferase expression could be monitored in cell extracts in a luminometer. For facilitating plaque isolation of luciferase recombinant viruses it was also visualized in situ on sensitive film. Kinetic experiments in mice proved the suitability of luciferase as an excellent marker for following herpesvirus spread in the animal. By way of luciferase detection we show that PrV invasion of the central nervous system after intranasal infection of mice occurred independently of replication in non-neural tissues such as lung or thymus. Furthermore, comparison of isogenic luciferase recombinant PrV strains carrying intact or deleted glycoprotein gI genes showed differences in the organotropism between these two viruses.

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Knowledge of the factors that place susceptible gilts at highest risk of pseudorabies virus (PRV) infection in a quarantined herd is crucial to reduce spread of PRV within the herd. Cohorts of PRV seronegative gilts were monitored in 17 herds that were endemically infected with PRV to determine the location of breeding females at the time of infection with PRV and identify herd characteristics and management and housing factors that may influence spread of PRV in the breeding section of swine herds endemically infected with PRV. Blood samples were collected every 1 to 2 months for an average of 13.6 months. In addition, blood was collected from a representative sample of finishing pigs (greater than or equal to 20 weeks old) 3 times per year to determine their serologic PRV status. Incidence rates and relative risks of PRV infection were estimated for 4 areas of the breeding section: gestation barn, gilt pool, farrowing room, and breeding area. Overall, 28, 11, 8, and 2 females became infected with PRV in each of these areas, respectively. The greater number of females infected in the gestation barns, compared with the number of females infected in other locations, is probably a consequence of being at risk for a longer period rather than of a higher incidence rate. Herd size, common housing for gilts in the gilt pool and sows, and serologic pattern of PRV infection in finishing pigs were associated with the detection of spread of PRV in the breeding section of the 17 herds.(ABSTRACT TRUNCATED AT 250 WORDS)

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Attenuated, gene-deletion mutants of pseudorabies virus (PRV) were tested for their ability to establish a reactivatable latent infection in pigs. The viruses (designated A, B, and C) were from each of three vaccines commercially available in the United States. Viruses A and C were similar in that they had genetically engineered gene deletions for thymidine kinase (TK) and glycoprotein X (gX); however, they had been prepared from genetically different parental strains. Virus B was TK positive, but had a naturally occurring gene deletion for glycoprotein I (gI). Four pigs were exposed oronasally to each of the viruses, and 10 weeks later they were treated with dexamethasone in an attempt to induce virus reactivation. All of the viruses replicated after initial exposure as evidenced by virus isolation from nasal swabs and the pigs' immune responses. Virus reactivation was subsequently induced by dexamethasone treatment in two of four pigs exposed to virus A. Notably, both pigs remained free of serum antibody for gX. Restriction endonuclease analysis and tests for TK activity and the presence of gX indicated that reactivated virus was similar, if not identical, to virus A used to establish latent infection. Virus shedding after dexamethasone treatment was not identified for either of the other pigs exposed to virus A nor for any of the pigs exposed to viruses B or C. The results indicated that attenuated, TK-negative PRV can establish a reactivatable, latent infection in pigs.

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To determine the sites of replication and the evolution of pseudorabies virus infection in boar genital organs, 5 Belgian Landrace boars were inoculated with pseudorabies virus unilaterally in the cavum vaginale of the testis. Virus replication took place only in cells of the tunica vaginalis of both cava vaginalia. Infection of the serosa led to exudative periorchitis and increased scrotal fluid, resulting in a severely swollen scrotal region. These experimental findings were similar to findings in boars with naturally acquired pseudorabies virus infection. Scrotal fluid contained large amounts of virus, making it a useful specimen for diagnosis of the disease in affected boars.

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Pseudorabies virus was inoculated intratracheally into sheep to investigate the pathogenesis of pseudorabies virus infection. Clinical signs of pyrexia, depression, frequent swallowing, facial fasciculations, chorea, excessive salivation, mild tympanites, labored breathing and focal pruritus were followed by death Macroscopic lesions were severe focal facial trauma, petechiae in cervicothoracic ganglia and dilated esophaguses. The medulla oblongata and the trigeminal, cranial cervical, cervicothoracic and parabronchial ganglia contained pseudorabies virus and pronounced nonsuppurative inflammatory changes. The neural distribution of lesions and virus suggests that the virus travelled from the respiratory mucosa to the central and sympathetic nervous system by two routes: 1) in the vagus and glossopharyngeal nerves to the medulla oblongata and 2) in the postganglionic fibers to the sympathetic ganglia. The presence of virus in the nasal mucus indicated that horizontal transmission of pseudorabies virus may occur among sheep.

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In HPCD pigs inoculated with PRV, latent PRV could be reactivated in-vivo by the administration of large doses of prednisolone 3 months after the primary infection. In two pigs, virus shedding was without clinical signs of disease, whereas depression of circulating lymphocytes was prominent. Reactivation of PRV was also demonstrated by cultivation of the brain cortex on the 7th day and the mandibular lymph node on the 9th day after the prednisolone began treatment. Coincident with the virus isolation, characteristic lesions were observed in 2 pigs in the central nervous tissues and mandibular lymph nodes and these were composed of cell necrosis and eosinophilic intranuclear inclusion bodies. Cells containing the intranuclear inclusion bodies had immature and mature PRV particles. Results of the present study with HPCD pigs indicated that the lesions in the brain and lymph node accompanied by eosinophilic intranuclear inclusion bodies were pathogonomonic lesions induced by reactivation of PRV.

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