We studied the uptake and sequential transneuronal passage of pseudorabies virus (PRV) in rat CNS by use of a combination of immunohistochemistry and in situ hybridization. Protocols for rapid detection of PRV by immunohistochemistry and in situ hybridization in rats with PRV infection of the CNS after intranasal instillation of a wild-type strain of PRV were optimized in vitro, using porcine kidney-15 cells. Pseudorabies virus-specific hybridization signals appeared in the cytoplasm and nucleus of PRV-infected porcine kidney-15 cells by postinoculation (PI) hour 6. In tissue sections of PRV-infected rats, PRV nucleic acids were detected in areas of the rat brain in close proximity to the areas in which PRV antigens were evident. The PRV was initially found in the nucleus of trigeminal ganglion neurons at PI hour 24. At PI hour 72, PRV antigens were observed in the mid-brain, and 24 hours later, in the telencephalon. We also found evidence of specific progressive transsynaptic transmission of the virus, and, on the basis of that, we have constructed a map of the synaptic contacts and pathways in the brain. Therefore, combined use of immunohistochemistry and in situ hybridization was useful for characterizing the pathogenesis of PRV in the CNS of rats after intranasal inoculation, following a pattern that mimics PRV infection of the natural host.

A field trial was conducted on a commercial swine farm quarantined because of infection with pseudorabies virus. The purpose was to investigate, in growing pigs born to hyperimmunized sows, the immunogenicity of a vaccine with a glycoprotein I (gE) deletion. One hundred twenty pigs were assigned at random to 1 of 3 vaccination schedules at ages: 8 and 12 weeks; 8, 12, and 14 weeks; and 8, 12, and 16 weeks. Immune response was measured at 8, 12, 14, 16, and 18 weeks, using the serum neutralization test, a screening ELISA, and assays of IgG and IgA in serum and nasal secretions. Results of the serum neutralization test and the screening ELISA indicated that, for pigs vaccinated only at 8 and 12 weeks, the percentage of pigs with pseudorabies virus serum antibodies decreased substantially by 18 weeks; for pigs given a booster at 14 or 16 weeks, the prevalence of serum antibodies at 18 weeks was higher, with 16-week booster vaccination eliciting the best response. At each age, nasal IgA and IgG values were highly correlated (r greater than or equal to 0.70), as were serum IgA and IgG values; correlations of serum with nasal IgA and IgG values were somewhat lower (approx range, r = 0.40 to 0.70). Nevertheless, an increase in serum IgA or IgG values on vaccination was no guarantee of an increase in nasal IgA or IgG values. For serum and nasal mucosal antibodies, a poor immune response was associated with high quantities of maternally derived antibodies. Vaccination at 16 weeks was necessary to ensure eliciting of an immune response in almost all pigs.

Epidemiologic modeling of the likely herd-to-herd transmission of pseudorabies virus (PRV) was developed to assess the progress and potential for the PRV-eradication program in the United States. The herd-to-herd transmission of PRV over a 20-year period (1993 to 2012) in the United States was simulated under various scenarios, which included variable program-funding levels and variable prevalences. A transition model (Markov process model) was used to predict yearly changes in herd prevalence of PRV infection. Five mutually exclusive states of nature for herds were assumed: uninfected and not vaccinated; uninfected and vaccinated; known to be infected and not vaccinated; known to be infected and vaccinated; and infected, but not known to be infected. Three prevalences for states in the United States were assumed: higher prevalence, moderate prevalence, and lower prevalence. Three funding levels were assumed: no eradication program, continued funding at the current level, and increased funding of 25%. Estimates made by an expert panel for determining probabilities in the state-transition matrices were used. A model also was developed, and was considered to be the most optimistic scenario likely under increased funding of 25%. The most optimistic estimates of the probabilities that still lay within the range of estimates made by the expert panel were used for this model. Only the optimistic transmission matrices allowed for total eradication of PRV. Using the optimistic matrices, all states in the United States of America had moved into the moderate- or low-level risk status by the year 2000. The longest time taken to achieve eradication was for the state of Iowa, where eradication was not achieved until 2012.

Various procedures of vaccination for pseudorabies were compared for their effects on shedding, latency, and reactivation of attenuated and virulent pseudorabies virus. The study included 6 groups: group 1 (10 swine neither vaccinated nor challenge-exposed), group 2 (20 swine not vaccinated, but challenge-exposed), and groups 3 through 6 (10 swine/group, all vaccinated and challenge-exposed). Swine were vaccinated with killed virus IM (group 3) or intranasally (group 4), or with live virus IM (group 5) or intranasally (group 6). The chronologic order of treatments was as follows: vaccination (week 0), challenge of immunity by oronasal exposure to virulent virus (week 4), biopsy of tonsillar tissue (week 12), treatment with dexamethasone in an attempt to reactivate latent virus (week 15), and necropsy (week 21). Vaccination IM with killed or live virus and vaccination intranasally with live virus mitigated clinical signs and markedly reduced the magnitude and duration of virus shedding after challenge exposure. Abatement of signs and shedding was most pronounced for swine that had been vaccinated intranasally with live virus. All swine, except 4 from group 2 and 1 from group 4, survived challenge exposure. Only vaccination intranasally with live virus was effective in reducing the magnitude and duration of virus shedding after virus reactivation. Vaccination intranasally with killed virus was without measurable effect on immunity. Of the 55 swine that survived challenge exposure, 54 were shown subsequently to have latent infections by use of dexamethasone-induced virus reactivation, and 53 were shown to have latent infections by use of polymerase chain reaction (PCR) with trigeminal ganglia specimens collected at necropsy. Fewer swine were identified by PCR as having latent infections when other tissues were examined; 20 were identified by testing specimens of olfactory bulbs, 4 by testing tonsil specimens collected at necropsy, and 4 by testing tonsillar biopsy specimens. Eighteen of the 20 specimens of olfactory bulbs and 3 of the 4 tonsil specimens collected at necropsy in which virus was detected by PCR were from swine without detectable virus-neutralizing antibody at the time of challenge exposure. One pig that had been vaccinated intranasally with live virus shed vaccine virus from the nose and virulent virus from the pharynx concurrently after dexamethasone treatment. Evaluation of both viral populations for unique strain characteristics failed to provide evidence of virus recombination.

Subunit pseudorabies vaccines that contained only purified glycoproteins of either of 2 strains of pseudorabies virus (PRV) were prepared and subsequently tested for safety and efficacy. The strains of virus used for vaccine production differed in at least 2 properties. One strain (Kojnok) was virulent for pigs and was believed to code for the entire complement of viral glycoproteins. The other (Kaplan) was a deletion mutant that was unable to code for structural viral glycoproteins gI and gp63. Purified glycoproteins were dispersed in an oil-in-water emulsion and were administered IM to pigs. Both vaccines were found to be safe and effective immunogens. Neither caused any local or general reactions, as verified by examination of the injection site (local safety) and by vaccination of pregnant sows in PRV-infected and noninfected herds. Sows vaccinated with the gI+ or gI- vaccine protected their pigs at levels of 93 and 92%, respectively, against a severe challenge exposure that killed 98% of pigs born from nonvaccinated sows. Vaccinated pigs were tested for active immunity by intranasal challenge exposure with the NIA 3 strain. Protection was quantitated by measuring the relative daily weight difference, expressed in percent per day, between vaccinated and control pigs during the first week after challenge exposure (deltaG7); the estimated differences were 2.25 and 2.13% for gI+ and gI- vaccines, respectively. The absence of gI and gp63 did not affect the efficacy of this type of subunit glycoprotein vaccines. In pigs that were challenge-exposed intranasally 3 months after 1 injection with the gI- vaccine, the duration of viral excretion was highly reduced, compared with that in control pigs.

A deterministic mathematical model of the population biology of pseudorabies in swine was used to clarify some of the basic features of the host-virus relationship and to inquire into the circumstances that promote or impede virus persistence in a single herd. When the basic reproductive rate of the infection (ie, the number of secondary infections resulting from the introduction of a single infective animal into a wholly susceptible herd) is greater than unity, the model suggests that the number of infective individuals in the herd will undergo highly damped oscillations to a final equilibrium level. The most important determinants of virus persistence are herd size and the density at which sows are maintained. There is a threshold density of susceptible individuals below which the virus will eventually be eliminated from the herd, even when specific control measures are lacking. Test and removal strategies hasten virus elimination when herd density is already below threshold, but are otherwise likely to succeed only when the removal of latent infections reduces the basic reproductive rate of the infection below unity. Vaccination strategies may also result in virus elimination, but only in relatively small herds.

The ability of pseudorabies virus (PRV) to infect and establish latency in pigs with passively acquired (maternal) antibody for PRV was tested by exposing such pigs to the virus and subsequently attempting to reactivate latent virus by administering large doses of dexamethasone. Pigs of each of 4 litters that had nursed gilts with relatively high (512, gilts 1 and 2), moderate (32, gilt 3), and no (less than 2, gilt 4) serum titers of virus-neutralizing (VN) antibodies for PRV were allotted to 3 treatment groups (A, B, C) when they were 2 weeks old. Group-A pigs were separated from littermates and dam and thereafter kept in isolation; group-B pigs were experimentally exposed oronasally to PRV and 1 hour later returned to their dam; group-C pigs were kept with their dam and potentially exposed to PRV by contact with littermates of group B. Sera obtained from pigs at selected intervals until they were 17 weeks old were tested for VN activity and for precipitating activity for radiolabeled viral proteins. All group-A pigs remained clinically normal throughout the experiment. Depending on the initial amount of passively acquired antibody, little or no serum VN or precipitating activity remained by the time these pigs were 17 weeks old. Group-B and -C pigs, with relatively high amounts of passively acquired antibody when exposed to PRV, also remained clinically normal. However, most became latently infected as subsequently evidenced by either dexamethasone-induced or noninduced virus reactivation. Noninduced reactivation may have been initiated by weaning the pigs when they were about 8 weeks old. Group-B and -C pigs with no or moderate amounts of passively acquired antibody when exposed to PRV, had severe clinical signs. These pigs either died or recovered but remained stunted in growth. Virus was reactivated in all of the recovered pigs by treatment with dexamethasone. Quantitative and qualitative changes in serum precipitating activity, especially for viral proteins of relatively low molecular weight (less than 46,000), were a more consistent indication of virus reactivation than were either increased VN titers or virus isolation. Results with litters 1 and 2 clearly indicate that latent infection of young pigs with highly virulent PRV can develop in the absence of clinical signs.

The effect of low-dose challenge of immunity with pseudorabies virus (PRV) on subunit-vaccinated pigs was studied in 2 experiments. In the first experiment, we studied the effect of challenge dose on the antibody response to an early excreted 98-kilodalton PRV-glycoprotein that was used as a diagnostic antigen in the ELISA. In the second experiment, we studied the effect of low doses of virus on the establishment of latent infections in subunit-vaccinated pigs. The relationship of virus exposure dose and vaccine dose to the response of pigs to diagnostic antigen was studied in 18 pigs. Two groups of 3 pigs were vaccinated with a total of 200 micrograms of a lectin-derived PRV subunit vaccine over a 5-week period. Two groups of 3pigs were similarly vaccinated with a total of 100 micrograms. Two groups of 3 pigs served as nonvaccinated controls. One group of pigs from each of the preceding categories was intranasally exposed to 10(6.0) and 10(2.7) plaque-forming units (PFU) of virus. Antibody to diagnostic antigen was detected by the ELISA and radioimmunoprecipitation 3 to 7 days earlier in pigs exposed to 10(6.0) PFU, demonstrating that the size of the virus challenge dose affects the antibody response to diagnostic antigen. The establishment of latent infections by low PRV doses and the ability to detect these infections was studied in 10 subunit-vaccinated pigs. Each pig was intranasally exposed to 10(2.3) PFU of virus (day 0). The serum virus-neutralizing antib ody titer of these pigs increased to their highest level 14 to 21 days after exposure and then steadily decreased through day 113, indicating absence of viral recrudescence. All pigs were treated with dexamethasone for 4 consecutive days, beginning on day 113. Latent infections were identified in 8 of 10 pigs on the basis of recovery of virus and/or 2 log2 or greater increases in serum virus-neutralizing titer. Antibody to diagnostic antigen was initially detected in the 8 latently infected pigs on day 14 or 21 and continued to be detected through days 21, 46, 53, 110, and 113 in 1, 2, 1, 1, and 3 pigs, respectively. The antibody titer to diagnostic antigen increased in 6 of the 8 latently infected pigs after dexamethasone treatment. However, antibody to diagnostic antigen was not detected by ELISA in the remaining 2 latently infected pigs, despite increases in serum virus-neutralizing titers in both pigs and the recovery of reactivated virus from one pig. The failure to consistently detect antibody to diagnostic antigen in latently infected pigs suggests that diagnostic tests using nonvaccine diagnostic antigen may be suitable only for detecting infections in vaccinated herds, but not in individual pigs.

Whereas the clinical efficacy of vaccination against pseudorabies has been studied extensively, methods to evaluate the influence of vaccination on pseudorabies virus (PRV) transmission have only recently become available. In this study, PRV transmission and growth performance in finishing pigs vaccinated either once or twice were compared. The incidence of PRV infections was significantly (P = 0.039) higher in the group vaccinated once (38%) than in the group vaccinated twice (10%). The reproduction ratio R, which is defined as the average number of new infections caused by 1 infectious individual, was estimated in both groups. This ratio was also significantly (P = 0.025) higher among single vaccinated pigs (R = 3.4) than among pigs that had received double vaccination (R = 1.5). In compartments where serologic evidence of PRV introduction was observed, the mean daily weight gain was significantly (P = 0.029) lower in pigs vaccinated once (698 g/d) than in pigs vaccinated twice (721 g/d). Results of this study document the possibility to objectively evaluate the effect of vaccination on PRV transmission under field conditions. From the results, we concluded that double vaccination is advantageous in populations of finishing pigs at risk for PRV introduction. However, even among pigs vaccinated twice, extensive spread of PRV can occur.

Tissue homogenates were obtained from swine co-infected with 2 vaccine strains of pseudorabies virus (PRV). Viral isolates derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures, were characterized as recombinant and parental PRV genotypes on the basis of thymidine kinase and glycoprotein X gene combinations. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant PRV. The thymidine kinase and glycoprotein X gene sequences were classified as wildtype or deleted, using a battery of polymerase chain reaction assays. Results substantiate the observation that PRV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection.