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A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.

Pseudorabies virus (PRV) is an alphaherpesvirus which causes an economically important disease of swine. One of the PRV glycoproteins, gp50, was previously identified as the sequence homolog of herpes simplex virus glycoprotein gD (E.A. Petrovskis, J.G. Timmins, M.A. Armentrout, C.C. Marchioli, R.J. Yancey, Jr., and L.E. Post, J. Virol. 59:216-223, 1986). gp50 was evaluated as a PRV subunit vaccine candidate. gp50 protected mice from PRV-induced mortality either when delivered via infection with a recombinant vaccinia virus or when administered as a subunit vaccine produced in a eucaryotic cell line, Chinese hamster ovary (CHO) cells. In addition, gp50 synthesized in CHO cells protected pigs from lethal infection with PRV. This result demonstrates that a single viral glycoprotein could induce a protective immune response in the natural host of a herpesvirus infection.

A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality. Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV. Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli. Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus. Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs. The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs. The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)

The DNA sequence was determined for a region of the pseudorabies virus (PRV) genome to which a mutation defining resistance to a monoclonal antibody has been mapped (M. W. Wathen and L. M. K. Wathen, J. Virol., 51:57-62, 1984). This sequence was found to contain an open reading frame that did not include an amino acid sequence directing N-linked glycosylation. This open reading frame was expressed in uninfected Chinese hamster ovary cells to produce the PRV glycoprotein gp50. When PRV-infected Vero cells were incubated in the presence of tunicamycin, the gp50 that was produced had an identical molecular weight to that produced in the absence of drug. When infected cells were incubated in the presence of monensin, the molecular weight of gp50 was reduced from 60,000 to 45,000, but was not sensitive to endo-beta-N-acetylglucosaminidase H. These observations led to the conclusion that gp50 does not contain N-linked carbohydrate, as predicted from the DNA sequence. A region of the amino acid sequence and the positions of the cysteine residues of PRV gp50 are homologous to glycoprotein D of herpes simplex virus.

Pseudorabies virus (PRV) produces a glycoprotein, gX, that accumulates in the medium of infected cells. The gX gene was expressed in Chinese hamster ovary cells (CHOgX cells) using the cytomegalovirus Towne major immediate early promoter. Like PRV-infected cells, CHOgX cells produced gX and exported it into the medium. Tunicamycin reduced the molecular weight of the gX in the medium to 89 kDa, compared with 99 kDa for gX made in the absence of drug. In the presence of tunicamycin gX produced by both PRV-infected cells and CHOgX cells was still glycosylated, as indicated by incorporation of [14C]glucosamine. The most likely form of this glycosylation is O-linked. In a pulse-chase experiment, gX first appeared in a 90-kDa form, then a 115-kDa form. This 115-kDa form is probably cleaved to give the 99-kDa form of gX that is released into the medium. The 115-kDa form was much more persistent in the PRV-infected Vero cells than in the CHOgX cells. In both cell types, gX was labeled by [35S]sulfate in the presence and absence of tunicamycin.

The pseudorabies virus vaccine strains Norden and Bartha each have been reported to have deletions in the small unique component of the genome (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). The deletion in Norden was shown to delete the entire coding region for gI but not any of the coding sequences for gp63. However, gp63 in Norden-infected cells was only 36 kilodaltons, and a 44-kilodalton form of gp63 was released into the medium. In Bartha, the deletion removed the coding region for all but 89 amino acids of gp63, and no gp63 was detected in either Bartha-infected cells or medium.

An open reading frame coding for an 11-kDa protein was located downstream from the gI gene of pseudorabies virus (PRV). This open reading frame is homologous to an open reading frame (US9) in an analogous position in herpes simplex virus and to an open reading frame (US1) in a different position in varicella zoster virus. The open reading frame encoding the 11-kDa protein is in a region known to be deleted in live attenuated vaccine strains of PRV.

RNA from pseudorabies virus (PRV)-infected cells was translated in a reticulocyte lysate with and without the addition of dog pancreas microsomes. Upon addition of the microsomes to the translation reaction, an additional prominent protein product was observed that was not present when microsomes were omitted. The gene coding for this processed protein and its lower-molecular-weight precursor was mapped within the small unique region of the genome by hybridization of mRNA to cloned fragments of PRV DNA and translation of the selected mRNAs. A fragment of the coding region of this gene was inserted into an open reading frame cloning vector to express part of this gene as a hybrid protein in Escherichia coli. This hybrid protein was injected into mice to raise an antiserum which was found to precipitate the glycoprotein which accumulates in the medium of PRV-infected cells. This allows us to conclude that the gene for the "excreted" glycoprotein (gX) maps to the small unique region of the genome, and that the precursor of this glycoprotein is readily processed by dog pancreas microsomes. The region of the PRV genome which codes for this glycoprotein was sequenced and found to include an open reading frame coding for 498 amino acids, flanked by sequences which contain features common to eucaryotic promoters and polyadenylation signals. The predicted protein sequence includes a hydrophobic sequence at the N-terminus which could be a signal sequence, and a hydrophobic sequence followed by a hydrophilic sequence at the C-terminus.

Pseudorabies virus (PRV) glycoprotein gX accumulates in the medium of infected cells. In an attempt to study the function of gX, two viruses were constructed that lacked a functional gX gene. One virus, PRV delta GX1, was derived by insertion of the herpes simplex virus thymidine kinase gene into the gX-coding region. The other virus, PRV delta GXTK-, was derived by subsequent deletion of the inserted herpes simplex virus thymidine kinase gene. Both viruses replicated in cell cultures but produced no gX. Furthermore, PRV delta GX1 was capable of killing mice with a 50% lethal dose of less than 100 PFU.