Multiplication and immunogenicity of a temperature-sensitive and thymidine kinase-deficient strain of Aujeszky's disease virus in pigs.

Hysterectomy-produced colostrum-deprived 5- and 27-day-old pigs were inoculated intramuscularly (IM) or intranasally (IN) with the temperature-sensitive and thymidine kinase-deficient ZHtsTK- strain of Aujeszky's disease virus (ADV), and the nasal swabs and organs of the pigs were periodically collected for virus isolation. No abnormal clinical signs were observed in these pigs, except for a mild febrile response. Viral shedding in the nasal swabs with low titers was detected in the pigs inoculated IN between postinoculation day (PID) 1 and 5, but not in those of the pigs inoculated IM. No contact infection, however, occurred in the cohabiting pigs. Viruses with low titers were isolated only from the muscles and lymph nodes at the site of inoculation in the pigs inoculated IM on PID 2 and 4, but not from any organs of the pigs inoculated IN. To investigate the ability of the ZHtsTK- strain to establish a latent infection in pigs, the pigs inoculated IM or IN with the ZHtsTK- strain were treated with prednisolone. No virus was detected in the trigeminal ganglia or the nasal swabs collected after prednisolone treatment by the cocultivation method. The immunological evaluation demonstrated that immunization of pigs with this strain was effective in preventing clinical signs caused by ADV infection. The duration of virus shedding was markedly shortened in immunized pigs, particularly in those immunized twice and the total quantity of virus recovered from immunized pigs was reduced in comparison with unimmunized pigs.     

Vaccination of swine with thymidine kinase-deficient mutants of pseudorabies virus

A mutant of pseudorabies virus (PRV) deficient in thymidine kinase (TK-) activity was isolated and characterized. The mutant grew well in cell culture and did not revert to the thymidine kinase-positive phenotype. The PRV-TK- was not virulent when inoculated intranasally into 3-to 4-week-old pigs and could not be reactivated from the ganglia of these pigs by explantation and cocultivation with susceptible cells several weeks after virus inoculation. Pigs that had been exposed to PRV-TK- were immune to challenge exposure with a virulent strain of PRV. Furthermore, the challenge virus was not recovered from the ganglia of most of these pigs, indicating that colonization of the ganglia by a super-infecting virulent PRV strain was considerably reduced by vaccination.     

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.     

Characterization of Japanese isolates of Aujeszky's disease virus by restriction endonuclease cleavage patterns, virulence in mice and thymidine kinase activity.

Twenty four cloned isolates of Aujeszky's disease virus collected from outbreaks of Aujeszky's disease from 1981 through 1989 in Japan were characterized by their restriction endonuclease (RE) cleavage patterns, virulence for mice and thymidine kinase (TK) activity. All of the isolates belonged to Type II of the four types classified by Herrmann et al. (1984). Based on the number and migration rate of the restriction fragments, the isolates were divided into 7 groups with Bam HI, 9 groups with Kpn I, 3 groups with BstE II and 2 groups with Sal I. The results indicate that the RE analysis, especially with Bam HI and Kpn I, provides useful epidemiological information about field isolates of Aujeszky's disease virus. All of the isolates showed virulence for mice ranging from 6.9 to 63.0 (PFU/LD50). It was interesting that the Nagano S87 strain, which had the highest virulence for the mouse, showed unique RE cleavage patterns with four enzymes. On the other hand, ara-T-resistant, TK-negative strain, was avirulent for mice (greater than 10(6.4) PFU/LD50). All of the isolates investigated in this study showed TK activity by the thymidine plaque autoradiography.     

Safety of an Aujeszky's disease vaccine based on deletion mutant strain 783 which does not express thymidine kinase and glycoprotein I

The safety of an Aujeszky's disease virus vaccine based on strain 783, a deletion mutant which does not express glycoprotein I and thymidine kinase, was assessed in pigs, calves and sheep. Four-day-old piglets which were inoculated intranasally and intramuscularly with 10(7) plaque forming units (PFU) developed only slight depression and fever. The virus was transmitted to a sentinel piglet. Six weeks after inoculation, the pigs were injected with high doses of corticosteroids in an attempt to reactivate the vaccine virus. The pigs did not shed Aujeszky's disease virus, did not develop a rise in virus neutralising antibody titres and sentinel pigs remained seronegative to Aujeszky's disease virus. Strain 783 was passaged in two series of three- to five-day old piglets, but after the third and fourth passages virus could no longer be recovered. Pregnant sows were inoculated with 10(7) PFU of virus strain 783 around day 35 or on day 85 of pregnancy, and their fetuses and piglets were assayed for Aujeszky's disease virus and antibodies against Aujeszky's disease virus. No evidence was found for transplacental transmission of the virus. Calves and sheep were given 10(7) PFU of virus strain 783 intranasally or intramuscularly; they survived and did not develop clinical signs of Aujeszky's disease. All the sheep and the calves inoculated intramuscularly developed neutralising antibodies to Aujeszky's disease virus.     

Influence of vaccination on Aujeszky's disease virus and disease transmission

Parenteral vaccination of fattening pigs with either modified live or inactivated Aujeszky's disease virus did not prevent infection with field strain virus or the development of clinical disease. The duration and severity of the clinical syndrome was, however, reduced and vaccinated pigs did not suffer the severe weight loss and high mortality experienced by non-vaccinated pigs in the acute phase of disease. The range of tissues in which challenge virus replication took place was more restricted in vaccinated animals and the concentration of virus in infected tissues was reduced. Vaccination shortened the duration of field virus excretion and carriage in the tonsil. Replication of modified live vaccine virus was restricted to the site of inoculation in the neck and associated lymph nodes for two days after vaccination and it was not excreted by vaccinated pigs. Attempts to infect pigs by feeding them tissues taken from non-vaccinated or vaccinated pigs soon after challenge infection were unsuccessful.     

Effects of swab materials and transport media on Aujeszky's disease virus.

The effect of swab materials on the recovery of Aujeszky's disease virus from various transport media held at 4 degrees C was investigated over a five day period. No significant loss in infectivity was found in control preparations, approximately 50 per cent of infectivity was recovered from fluids containing applicator wire and approximately 10 per cent from fluids containing polyester fibre or cotton wool. Virus recovery from fluids containing wooden applicator sticks ranged from no virus recovery in protein free media to from 1 to 10 per cent in protein containing media. Freezing followed by thawing was the most effective of the physical methods used for eluting virus from swab materials.     

Survival of Aujeszky's disease virus in slurry at various temperatures.

Survival of Aujeszky's disease virus in pig slurry was investigated during anaerobic storage at 5, 20, 35, 40, 45, 50 and 55 degrees C using 100-ml laboratory models simulating the conditions in slurry tanks during winter and summer seasons and during anaerobic digestion in batch reactors. The inactivation rate was found to increase with increasing temperature. Virus was inactivated at 5 and 20 degrees C in 15 weeks and 2 weeks, respectively. At 35 degrees C (mesophilic conditions) the virus was inactivated in 5 hours and at 55 degrees C (thermophilic conditions) no virus could be detected after 10 minutes.     

Vaccination of sheep against Aujeszky's disease with a gI-deleted mutant]

The use of gl deleted live vaccines against Aujeszky's disease (AD) facilitates to differentiate vaccinated from field-virus infected animals. In this study different modes of vaccination were tried to find out how sheep can be protected from a lethal infection with ADV. It could clearly be demonstrated that Aujeszky disease virus (ADV) is spread by horizontal transmission from infected pigs to sheep. The nasal discharges of infected pigs contained a maximum of 10(8.75)TCID50/g mucus at days 3 and 4 p.i. and those of the contact-pigs 10(8.5)TCID50/g mucus at days 6 and 7 after contact. Non-vaccinated contact sheep were infected horizontally by the pigs. The highest titres ranged from 10(6.25) to 10(7.5)TCID50/g mucus. These animals were sacrificed at day 5 p.i. exhibiting acute symptoms of AD. The nasal discharge of vaccinated sheep contained much lower amounts of ADV (maximum: 10(4.25)TCID50/g mucus). All surviving animals had developed antibodies. Following challenge with the ADV-strain NIA3, no febrile response or virus-shedding was observed in sheep vaccinated 2x s.c. or 2x i.m. with a gl deleted live vaccine, whereas sheep, vaccinated only 1x i.m. (4 out of 4 animals) or 1x i.m. (3 out of 4 animals) or 1x i.n. and 1x i.m. (1 out of 4 animals) had to be sacrificed after showing acute symptoms of AD. In conclusion it can be stated that a double parental vaccination with a gl deleted live vaccine protects sheep against a field-virus AD infection.     

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.     

Attenuated properties of thymidine kinase-negative deletion mutant of pseudorabies virus

A thymidine kinase (TK)-negative (TK-) deletion mutant of the Bucharest (BUK) strain of pseudorabies virus (PRV) was isolated. The mutant, designated as PRV (BUK d13), did not revert to TK-positive (TK+), even when propagated in medium that selected for TK+ viruses. The mutant also replicated equally well at 39.1 C and 34.5 C, and was easily distinguished from other PRV strains by molecular hybridization experiments, restriction nuclease fingerprints, and plaque autoradiography or other assays for the TK phenotype. The PRV (BUK d13) had greatly reduced virulence for mice and rabbits, compared with parental TK+ strains, PRV (BUK-5) and PRV (BUK-5A-R1), and provided mice with solid protection against the TK+ BUK and Aujeszky strains of PRV. Experiments were done in 5- to 6-week-old pigs to assess the safety and efficacy of PRV (BUK d13) in the natural host. In one experiment, pigs were vaccinated IM with 7.5 X 10(8) plaque-forming units of TK- PRV (BUK d13), and were then challenge exposed intranasally (IN) with 4.3 X 10(8) TCID50 of virulent PRV [Indiana-Funkhauser (IND-F)]. Vaccinated pigs did not have clinical signs of illness after vaccination or after challenge exposure. One nonvaccinated control pig died on postchallenge day 4; a 2nd nonvaccinated control pig became moribund, but eventually recovered. Pigs developed virus-neutralizing antibodies after vaccination, and had a secondary immunologic response after challenge exposure; however, PRV was not isolated from the tonsils or trigeminal ganglia of vaccinated pigs at postchallenge exposure day 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of different genes in the virulence and pathogenesis of Aujeszky's disease virus.

In this study the role of different genes located in the unique short region of the genome of Aujeszky's disease virus was examined. Inactivation of the genes encoding the protein kinase (PK), gp63, and gI reduced virulence of the virus for pigs, in contrast to inactivation of the genes encoding the 28 kDa protein, and gX. There was no correlation between virulence and virus multiplication in vitro or in the oropharynx in vivo. The morphogenesis of the PK mutant was altered. The gI mutant replicated to normal titres in the oropharynx and could be recovered from the trigeminal ganglia but not from other parts of the central nervous system, suggesting that gI facilitates the spread of the virus from neuron to neuron. All mutants induced neutralizing antibody and complete or partial protection against a challenge infection. PK and gp63 were required for the induction of complete protection, although these proteins are reportedly not targets for neutralizing antibody or cytotoxic T cells.     

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.     

Serological response of pigs infected with Aujeszky's disease virus

The temporal development of antibody in four groups of pigs infected with different Aujeszky's disease virus isolates was examined. The enzyme-linked immunosorbent assay detected antibody by five to six days after infection and the antibody-dependent cell-mediated cytotoxicity assay detected antibody seven to nine days after infection. Neutralising antibody was first detected nine to 10 days after infection, whereas assays measuring complement mediated antibody lysis did not detect antibody until 10 days after infection. These results are discussed in terms of their importance to the diagnosis of and recovery from Aujeszky's disease.     

Aujeszky's disease virus infection patterns in European wild boar

Evidence of exposure (i.e. seroprevalence) to Aujeszky's disease virus (ADV) is high among wild boars from south-central Spain. This research aims to determine the presence of ADV by molecular detection, and to describe the patterns of ADV infection in wild boars. Tonsils (TN) and trigeminal ganglia (TG) for ADV molecular detection, and sera were collected from wild boars (n = 192) in 39 hunting estates from south-central Spain (2004/2005). A nested polymerase chain reaction (PCR) for a fragment of the ADV surface glycoprotein B was performed on collected tissues. Individual status of presence of viral DNA was tested against explanatory variables by means of a Generalized Linear Mixed Model (GLIMMIX) analysis. Viral detection prevalence was 30.6 ± 6.7%. Although there was an increasing pattern with age and females presented higher prevalences, no statistically significant influence of sex and age was found for viral presence. Molecular testing in TN and TG allowed classifying infection status into (i) ADV negative (in both TN and TG), (ii) only positive in TN, (iii) only positive in TG and (iv) positive in both TN and TG. ADV DNA was statistically more frequently evidenced in TN in females than in males. With the exception of one individual, all wild boars with presence of ADV DNA in TN and TG or only in TG reacted positive in the ELISA. In contrast, animals with only ADV DNA in TN serorreacted positively and negatively. Interestingly, 45% of the PCR positive wild boars (n = 59) were seronegative in the serological test, all of them with viral DNA only in TN. Our results provide evidence for latency of ADV in wild boars and stress the fact that antibody detection based tests may fail to detect a proportion of recently infected animals. This is of great concern since current management schemes in our study promote animal translocation for hunting purposes, with the associated risk of under-detecting ADV infected individuals when using serology to screen for ADV infection.