Skin virulence of attenuated pseudorabies virus (PRV) strains.

After intradermal inoculation of the attenuated pseudorabies virus (PRV) strains, Bartha, MK35 (gI+) and MK35-T-2 (gI-) to rabbits, they caused local inflammatory nodules with oedema reddiness and necrosis. The specifity of these reactions was confirmed by their inhibition with anti-PRV serum. The minimal dose that caused visible skin reaction was about 10(2) TCID50/ml for Bartha and MK35-T-2 (gI-) strains and over 10(4) TCID50/ml for the MK35 (gI+) strain. It is assumed that the established residual skin virulence can serve as an additional distinction marker for attenuated PRV strains.     

Virus reactivation in pigs latently infected with a thymidine kinase negative vaccine strain of pseudorabies virus

Summary 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.     

A radioimmunoassay for herpes simplex virus (HSV) thymidine kinase

A sensitive RIA for HSV-1 thymidine kinase (TK) has been developed. This assay is based on competition for the binding site of a rabbit antibody against purified HSV-1 TK, between a purified ³H-labeled HSV-1 TK and a sample containing an unknown amount of viral TK. The assay is capable of detecting 8 ng or more of the HSV enzyme. Purified HSV-1 TK denatured to <1% of its original kinase activity is as effective in binding to the antibody as is native HSV-1 TK. Viral TK is detectable at ranges of 150–460 ng/mg protein of cell extract from infected cells or cells transformed by HSV or HSV genetic material. HSV-2 TK appears highly cross-reactive, VZV TK is slightly less so, and the vaccinia TK shows little or no cross-reactivity. This RIA may serve as a tool for monitoring the expression of the HSV TK during an active herpes virus infection, a latent ganglionic infection, or in neoplastic cells which may have arisen by viral transformation.     

Comparison of Aujeszky's disease (pseudorabies) virus strains by SDS-PAGE of the virus proteins

Summary Comparison of Aujeszky's disease virus strains by SDS-PAGE indicated that one protein (mol. wt. 31,000–34,000) exhibited marked variation. The NIA 4 and Bartha vaccine strains were identical and could be distinguished from other isolates.With 2 Figures     

Pseudorabies virus (PRV) is protected from complement attack by cellular factors and glycoprotein C (gC)

Swine kidney derived CPK cells were resistant to swine complement attack in vitro while rabbit kidney derived RK13 cells were destroyed by swine complement. To rabbit complement, RK13 cells were resistant but CPK cells were sensitive. This phenomenon was known as homologous restriction (Proc. Natl. Acad. Sci. USA 78 (1981) 5118). The gC deletion mutant of pseudorabies virus (PRVdlgC) grown in CPK cells was resistant to swine complement while the same virus grown in RK13 cells was neutralized by swine complement. PRVdlgC grown in RK13 cells was more resistant to rabbit complement than the virus grown in CPK cells. Hence, the sensitivity of PRVdlgC to swine or rabbit complement was similar to that of the cells in which the virus was grown. It would appear that cell derived factors were present on the virion and they were protective against homologous complement but not against heterologous complement. The expression of gC rendered PRV more resistant to swine or rabbit complement, but the protective effect of gC was much less than that of cell derived factors. The best protection against complement was obtained when gC and cell derived factors were coexistent on the virion.     

Differentiation of pseudorabies (Aujeszky's disease) virus strains by restriction endonuclease analysis

Summary Genomes of six virulent and three attenuated vaccine strains of pseudorabies (Aujeszky's disease) virus were compared by cleavage with five restriction endonucleases. Differences either in number or in size of DNA fragments could be detected among all nine strains.With 2 Figures     

A peptide-model for the heparin-binding property of pseudorabies virus glycoprotein III

The pseudorabies virus glycoprotein III (PrV-gIII) has been identified previously as the major viral component binding to a heparin-like receptor on the surface of target cells. The amino acid sequence of gIII contains three regions corresponding to consensus sequences for heparin binding. A synthetic peptide corresponding to amino acids 134 to 141 of PrV-gIII bound heparin in a dot blot assay. In contrast, a synthetic peptide derived from amino acids 290–299 of PrV-gIII did not bind heparin. We therefore conclude that the region containing amino acid 134–141 is involved in binding to the heparin-like cellular receptor.     

Influence of double infections on the induction of thymidine kinase by UV-irradiated herpes simplex virus types 1 and 2 and pseudorabies virus

Summary Simultaneous infection of primary rabbit kidney cells with HSV type 1 TK⁺ and a TK^– strain results in a mutual influence of both viruses on the induction of thymidine kinase (TK). TK⁺ virus has an enhancing and TK^– virus a depressing effect on TK induction by a superinfecting TK⁺ virus. The enzyme induction depends on the ratio of multiplicities of both viruses. The mutual influence on TK induction depends further on the time of addition of the superinfecting virus: the effect of the second virus can still be observed when given 6 hours after primary infection. Identical phenomena can be observed using combinations with HSV type 2 or Pseudorabies viruses.The ability of HSV to induce TK is progressively inactivated with increasing the time of UV-irradiation. The depressing effect of a TK^– strain and the stimulating effect of a TK⁺ strain on superinfecting TK⁺ strains is UV-sensitive: after 6 minutes of UV-irradiation neither inhibition nor stimulation of TK induction by a superinfecting TK⁺ strain can be observed. Infection by long-term (20 minutes) UV-irradiated TK⁺ strains results in a depression of TK induction by a superinfecting TK⁺ virus. Long-term irradiation of the TK^– virus does not show this effect.Cytosine-arabinoside has no effect on the mutual influence of TK induction by TK⁺ and TK^– strains; the phenomenon of mutual depression therefore has to be considered an early process.With 5 Figures     

Evaluation of a recombinant vaccinia virus containing pseudorabies (PR) virus glycoprotein genes gp50, gII,and gIII as a PR vaccine for pigs

Summary Pigs vaccinated twice intramuscularly with a highly attenuated strain of vaccinia virus (NYVAC) containing gene inserts for pseudorabies virus (PRV) glycoproteins gp50, gII, and gIII produced neutralizing antibodies for PRV and were less clinically affected than were nonvaccinated pigs following oronasal exposure to virulent PRV. Also, following oronasal exposure to virulent PRV the duration of virulent virus shedding by pigs that had been vaccinated intramuscularly with the recombinant virus was statistically less (p<0.05) than that of nonvaccinated pigs and like that of pigs vaccinated twice intramuscularly with inactivated PR vaccine. Intramuscular vaccination with the recombinant virus was compatible with the most commonly used differential diagnostic tests, namely those based on PRV glycoproteins gX and gI. Serum antibodies for these glycoproteins were absent from the sera of all pigs before and after vaccination with recombinant virus; whereas, they were present in the sera of all of the same pigs after they were exposed to virulent PRV. In contrast to the effectiveness of the recombinant virus administered intramuscularly, neither serum antibody nor clinical protection against PRV was detected when aliquots of the same recombinant virus preparation were administered either orally or intranasally. The latter finding suggests that recombinant virus replicates poorly, if at all, at these sites. If so, the dissemination of recombinant virus from vaccinated pigs to nonvaccinated pigs or other animals in contact seems unlikely.     

A comparative study of 63 strains of ECHO virus type 30

Summary Thirty-three European strains and 30 American strains of ECHO virus type 30 were examined. The strains were representative isolates from 8 distinct outbreaks of infection in various geographical areas between 1959 and 1966. Primary human amnion and WI 38 cells supported the growth of these viruses much more satisfactorily than primary monkey kidney cells. Thirty-one of the total of 63 strains showed haemagglutinating activity with human group 0 erythrocytes. In both neutralization tests in tissue cultures and in haemagglutination inhibition tests the strains showed marked antigenic differences within the serotype. They fell into two broad groups one of which contained the prototype strain. In general, strains isolated in the same outbreak resembled each other, but a few individual strains were antigenically quite unlike other strains from the same outbreak. Antiserum to the Frater strain appears to be capable of neutralizing rather more strains of the virus than antiserum to the prototype. The difference between the prototype strain and the Frater strain is not great. The antigenic variation within the serotype is such that it is unlikely that any single strain is antigenically broad enough to constitute an ideal strain for antiserum production.     

Construction of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB,gC, gD,and gE

Summary We have improved the method for constructing recombinants of bovine herpesvirus type-1 (BHV-1). Using this method, we constructed three recombinants in which the pseudorabies virus (PRV) thymidine kinase (tk) gene was inserted at three different sites in the unique short region of BHV-1. These three sites are located in the open reading frame of gE, gG and gI genes. Previously, two sites (tk and gC) had been used to insert foreign DNA fragments to BHV-1 genome. Therefore we now have 5 sites in BHV-1 where DNA can be inserted. The gB, gC, gD, gE and gI genes of PRV were successfully inserted at the tk or the gC gene of BHV-1 genome and Western blot analyses confirmed that the recombinants express PRV gB, gC, gD and gE. Anti-PRV gB and gC antibodies as well as anti-PRV polyclonal serum neutralized BHV-1 recombinants which express PRV gB and gC. The latter was neutralized more strongly. However, anti-gD monoclonal antibody and anti-PRV polyclonal serum failed to neutralize gD-expressing recombinants. This suggests that PRV gC and some gB are integrated into the viral envelope of the recombinants, but very little gD is present in the viral envelope.     

Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA

Pseudorabies virus glycoprotein E (PRV gE) has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE epitopes was expressed in Pichia pastoris expression system. SDS-PAGE and Western blotting revealed that the expression product was two recombinant proteins, approximately 38 and 32 kDa, in the culture supernatant of P. pastoris integrant 72 h after induction. Protein concentration assay showed the expression product amounted to 106.7 mg/l, accounting for 66.67% of total culture supernatant proteins. An indirect PRV gE-ELISA was then established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. Reproducibility experiment displayed good consistency. Comparison of detection results of 348 field serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed there was no significant difference between these two methods (P > 0.05).     

Bovid herpesvirus type-1 (Infectious bovine rhinotracheitis virus) induced thymidine kinase

This communication reports the presence of a new thymidine kinase (dTK) in bovine kidney cells infected with Bovid herpesvirus 1(BHV-1). The virus-induced enzyme activity differs from the host cell enzyme with respect to substrate specificity, thermostability, and the ability to use phosphate donors other than ATP. Thus, the BHV-1-induced dTK could be selectively assayed when CTP was used in place of ATP as the phosphate donor in the enzyme reaction. The virus-induced activity can phosphorylate thymidine, bromodeoxyuridine, bromovinyldeoxyuridine, and methylmethoxydeoxyuridine but not deoxycytidine, bromodeoxycytidine, deoxyguanosine, or acycloguanosine.     

Comparative pathogenesis of three strains of pseudorabies virus in pigs

Three strains of pseudorabies virus were intranasally inoculated into 10-week-old pigs and the pathogenesis of the infection was compared. Virulent NIA-3 virus caused widespread necrotic lesions in nasal mucosa, rapidly invading the stroma and infecting axons of olfactory nerves within 24 h of inoculation. Intermediate virulent virus 2.4N3A, a mutant strain derived from NIA-3, caused less necrosis of the mucosa and did not reach axons of olfactory nerves until 72 h after inoculation. Bartha virus strain K, a non-virulent virus strain, caused a mild infection in the superficial layers of nasal epithelium. Viral antigens were not detected in stromal fibroblasts or nerve cells. The inflammatory response of the pigs varied with the virus strains used: after infection with NIA-3 virus mainly neutrophils infiltrated the nasal mucosa, whereas after infection with 2.4N3A virus and Bartha virus, mainly macrophages and lymphocytes infiltrated the nasal mucosa.     

Expression of porcine pseudorabies virus genes by a bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) vector

Summary Recombinant DNA techniques were used to insert foreign genes into bovine herpesvirus-1 [infectious bovine rhinotracheitis virus (IBRV)] vectors which were attenuated by deletion and/or insertion mutations in the IBRV thymidine kinase (tk) gene. In one recombinant, the regulatory and coding sequences of the late pseudorabies virus (PRV) glycoprotein gIII gene, were inserted into the early IBRV tk gene. This recombinant efficiently expressed the PRV gIII gene indicating that immediate early IBRV proteins were competent to transactivate the late PRV gIII gene. IBRV vector viruses were also prepared in which the coding sequences of the early PRV tk gene, the late PRV gIII gene, and theE. coli -galactosidase gene were ligated to the late IBRV gIII promoter. Genotypes and phenotypes of the recombinant viruses were verified by restriction endonuclease and molecular hybridization experiments, thymidine plaque autoradiography, -gal plaque assays, and by immunoprecipitation experiments on extracts from³H-mannose-labelled cells. The recombinant IBRV expressing -gal from the IBRV gIII promoter has been useful as an intermediate in the construction of IBRV vectors harboring foreign DNA sequences. The infectivity of the IBRV recombinant that expressed PRV gIII from the IBRV gIII promoter, was neutralized by polyclonal PRV antisera and by monoclonal antibodies to PRV gIII. The PRV gIII glycoprotein synthesized by the preceding recombinant has been used to coat microtiter test plate wells in a PRV gIII differential diagnostic test kit.     

A rapid method for identifying the thymidine kinase genes of avipoxviruses

The thymidine kinase (TK) genes of poxviruses can be rapidly located without using TK⁻ mutants or having to restriction map and clone the viral genomes. Identification of the TK gene is based on in situ gel hybridization with an end-labelled degenerate oligonucleotide probe, representing a consensus sequence near the 3' end of the gene. Restriction fragments of the viral DNAs are electrophoresed in agarose gels and annealed with the probe. Using this method, the TK genes of fowl pox (FPV) and quail pox (QPV) viruses were initially localized to HindIII fragments of approximately 3.8 and 6.7 kb, respectively. After inserting these fragments into pUC 19, recombinant plasmids containing the TK genes were screened by a modified in situ gel annealing procedure. Restriction mapping of the two cloned fragments and subsequent hybridization analysis more precisely placed at least the 3' portion of the FPV and QPV TK genes within a 1.4 kb ClaI-XbaI and 1.7 kb ClaI-PstI fragment, respectively. The site of the FPV TK gene was verified by comparison to the mapped position of the similar gene in an Australian FPV. The location of the QPV TK gene was confirmed by hybridization with the FPV TK gene, despite the apparent divergency of these two genes.