A major portion of the latent pseudorabies virus genome is transcribed in trigeminal ganglia of pigs.

Pseudorabies virus (PRV) is a porcine herpesvirus that establishes latent infections in trigeminal ganglia. To determine whether PRV expresses any transcripts that could play a role in latency, the trigeminal ganglia of 14 pigs previously inoculated through the nose and latently infected with PRV(Ka) were assayed by in situ nucleic acid hybridization for the presence of PRV-specific RNA. Hybridizations employing probes encompassing the entire viral genome revealed that an area extending from 0.64 to 0.82 map units was transcriptionally active. The DNA probe that most consistently detected transcripts was BamHI-8, a fragment which contains the gene for the immediate-early protein. With this probe, ganglia from 10 (71%) of 14 pigs scored positive for PRV RNA, although only 1 (8%) of 12 of the ganglia from the opposite side reactivated virus after explanation and culture of latently infected trigeminal ganglia. The RNA was transcribed from the strand opposite to that coding for the immediate-early protein; the signal was neuronally localized, with dense nuclear accumulation accompanied by variable numbers of grains over the cytoplasm. Northern RNA blot analysis showed that a discrete set of poly(A)- PRV transcripts were present in latently infected trigeminal ganglia. Additional in situ nucleic acid hybridization analysis revealed that the 3' limit of the transcriptionally active area was located in a 1.2-kilobase fragment upstream and adjacent to the 5' end of the immediate-early protein RNA, whereas the 5' limit was as much as 4.9 kilobases downstream from the 3' end of this RNA. PRV therefore expresses latent-phase transcripts that, although similar in many respects to latent-phase transcripts reported for other herpesviruses, have some unique properties.

Antiviral therapy.

Antiviral therapy is gradually coming of age. More agents including interferons as well as combinations of agents can be anticipated in the marketplace. Five antivirals are now approved for use, and there are at least seven that have a strong chance of becoming available.

Shedding patterns in swine of virulent and attenuated pseudorabies virus.

Shedding patterns of 2 virulent (P-2208 and KC-152-D) and 1 attenuated (BUK) strains of pseudorabies virus (PRV) were determined in groups of intranasally inoculated feeder pigs. Clinical signs observed following inoculation with the P-2208 and KC-152-D strains included increase in rectal temperatures up to 42.2 C, anorexia, severe respiratory disturbance, and fatal CNS signs in 2 cases. Clinical signs in pigs inoculated with 7.2 X 10(7) median tissue culture infective dose (TCID50) of the BUK strain were limited to depression and a rise in rectal temperatures to 40.5 C for 3 to 4 days. Evaluation of the efficacy of the virus isolation method used showed that the presence on swabs of only 12.5 TCID50 of the P-2208 strain or 8.4 TCID50 of the BUK strain resulted in a 50% chance of virus recovery. Intranasal inoculations with 500 TCID50 of the P-2208 or KC-152-D strain did not result in synchronous infection of the whole group. Intranasal inoculations with 5,000 TCID50 of the KC-152-D strain or 50,000 TCID50 of the P-2208 strain resulted in continuous virus shedding in all pigs between postinoculation days (PID) 4 and 13 (KC-152-D strain) or 14 (P-2208 strain). Some of the pigs in these 2 groups further shed the P-2208 or KC-152-D strain in a continuous or discontinuous pattern up to PID 19 (P-2208 strain) or 20 (KC-152-D strain). The time of onset or the level of virus neutralizing antibody production in individual pigs was not found to have an influence on their shedding patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo.

Porcine embryos (n = 93) were incubated on cell monolayers that had been previously inoculated with pseudorabies virus, porcine parvovirus (PPV), or each of 2 porcine enteroviruses. After 2, 24, or 48 hours of incubation, the embryos were fixed in glutaraldehyde and examined by electron microscopic procedures. It was found that pseudorabies virus adsorbed to the zona pellucida (ZP) and entered sperm tracks in the ZP. The PPV and both enteroviruses entered pores in the ZP and were associated with sperm that were at or near the outer surface of the ZP. In addition, PPV was seen enmeshed in cellular debris on the outer surface of the ZP. Evidence of a productive viral infection of the blastomeres of the embryos was not found.

Pseudorabies virus nucleocapsid antigen for skin testing in swine.

A procedure is described for the production of an effective pseudorabies virus skin test antigen consisting of nucleocapsids. The nucleocapsids were prepared by disruption of pseudorabies virus-infected pig kidney cells and nonionic detergent removal of the viral envelope. This preparation was noninfectious for pig kidney cell cultures and mice. Swine that had recovered from a pseudorabies virus infection and were injected intradermally with the nucleocapsid antigen developed a delayed type hypersensitivity response similar to that induced by inactivated virion antigen. The major advantage of the nucleocapsid antigen is that the occasional problem of seroconversion that is encountered with the virion antigen is avoided.

Photodynamic inactivation of pseudorabies virus with methylene blue dye, light, and electricity.

Pseudorabies virus was photoinactivated with a combination of methylene blue dye, light, and electricity. Viral suspensions were mixed with variable amounts of methylene blue dye and then were placed in a current source apparatus. Total inactivation of pseudorabies virus (1.7 X 10(6) 50% tissue culture infective doses per ml) was achieved with constant mixing, a methylene blue dye concentration of 10(-4) M, and an electrical current of 12 microA for 12 min.

Identification of immunologically distinct antigens in pseudorabies virus.

Antigenic proteins of pseudorabies viruses (PrV) are poorly understood. Proteins from purified PrV and membrane proteins from these viral infected cells, therefore, have been studied by antigenic analysis, using virus neutralization and agargel immunoelectrophoresis tests and by polyacrylamide gel electrophoresis, respectively. The study of crossed immunoelectrophoresis against specific antiviral serum antibodies revealed four immunologically distinct antigens involved in PrV. According to their electromobilities, these four immunologically distinct antigens were designated as Ag 1, Ag 2, Ag 3 and Ag 4. The study of dodecyl sulfate-polyacrylamine gel electrophoresis of a membrane-bound but detergent solubilized viral antigenic complex from PrV infected cells also demonstrated the involvement of four glycoprotein antigens. By interpolations of relative mobilities between known protein markers, the molecular weights of these four glycoproteins were estimated to be 61,500, 68,000, 75,000, and 88,000. Results from two dimentional immunoelectrophoresis seemed to be concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis. This report, therefore presents results, which strongly suggest antigenic similarities in the virion of PrV and cellular membrane glycoproteins of cells infected by this agent. The molecular weight of these four immunologically distinct antigens, Ag 1, Ag 2, Ag 3 and Ag 4, are presumed to have the following molecular weights of 88,000, 75,000, 68,000 and 61,500, respectively.

Detection of pseudorabies virus antibodies: time of appearance by two serotests.

This report describes a time-course comparison of detection of pseudorabies virus antibodies in experimentally infected swine by the virus-neutralization (VN) and indirect hemagglutination tests. Specific antibody titers were observed by the IHA test at 5 days after swine were inoculated, but not until 12 days by the VN test. The predominant immunoglobulin (Ig) class present in the serums of the swine at 5 and 7 days after inoculation was IgM, as determined by sulfhydryl reductions. The VN test lacked sensitivity to early Ig levels (IgM) in these experimentally infected swine, while the indirect hemagglutination test was highly sensitive to these same levels. On the basis of these results, it is possible that the VN test may read early infections as pseudorabies virus negative, due to the low presence of IgG in these samples.

Indirect hemagglutination test for pseudorabies antibody detection in swine.

An indirect hemagglutination test for the detection of antibodies in swine serum specific for pseudorabies virus is described. The indirect hemagglutination test was less time consuming than the standardized virus nertralization test while being highly sensitive and accurate. Serum samples that were toxic to virus neutralization indicator cells were readily tested in the indirect hemagglutination test. The indirect hemagglutination test may also be more sensitive than the virus neutralization test for determining early titers in pseudorabies virus infections. Complete methodology is described.

Comparison of pseudorabies virus inactivated by bromo-ethylene-imine, 60Co irradiation, and acridine dye in immune assay systems.

Pseudorabies virus infections among animals, especially swine, have become prevalent in the United States in the past few years. The disease in swine is now economically important. Test systems and antigens are being developed for use in control and disease suppression efforts. Pseudorabies virus was inactivated by three methods: chemically, with bromo-ethylene-imine; physically, with 60Co irradiation; and chemically and physically, with 3,9-diaminoacridine dye followed by exposure to white visible light. The antigenicities of the preparations were determined in the presence of specific antibody in immunodiffusion tests and through immunoelectrophoresis. The latter technique permitted quantitation of either antigen or antibody. In the electrophoretic patterns, the antigenic mass in bromo-ethylene-imine preparations was estimated to be 42 mg/ml, the same as in the untreated control material. After 60Co irradiation, 22 mg/ml was present, in comparison with 50 mg/ml in the untreated control antigen. In contrast, 67 mg/ml was present in the acridine dye-light-treated preparation, in comparison with 58 mg/ml in the untreated control material. A possible explanation for the acridine dye-light-treated preparation values is that photodynamic inactivation interferes with viral maturation during the replicative cycle within cells, with a resulting production of a greater amount of antigen, at least some of which is in the form of defective particles.