One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3to 20-fold) than does wild-type virus. The interactions of gIImutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of glll. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gUI, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII.. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIIIor wild-type virus-infected rabbit kidney cells, release of a gIII-/gIdouble mutant from the cells occurred less readily than did release of a glmutant. The glH-/gIand gIIImutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIll on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIll gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIll affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.

Recombinant strains of herpesvirus of turkeys (HVT) were constructed that contain either the fusion protein gene or the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) inserted into a nonessential gene of HVT. Expression of the NDV antigens was regulated from a strong promoter element derived from the Rous sarcoma virus long terminal repeat. Recombinant HVT strains were stable and fully infectious in cell culture and in chickens. Chickens receiving a single intra-abdominal inoculation at 1 day of age with recombinant HVT expressing the NDV fusion protein had an immunological response and were protected (> 90%) against lethal intramuscular challenge at 28 days of age with the neurotropic velogenic NDV strain Texas GB. Recombinant HVT expressing the NDV hemagglutinin-neuraminidase provided partial protection (47%) against the same challenge. Chickens vaccinated with recombinant HVT vaccines had low levels of protection against NDV replication in the trachea when challenged ocularly. Recombinant HVT vaccines and the parent HVT strain provided similar levels of protection to chickens challenged with the very virulent RB1B strain of Marek's disease virus, indicating that insertion of foreign sequences into the HVT genome did not compromise the ability of HVT to protect against Marek's disease.

The Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome which includes the genes that code for glycoproteins gl and gp63 (E.

To ascertain the biological functions of different glycoproteins that are nonessential for pseudorabies virus growth in vitro, we have constructed mutants defective in one (or a combination) of these glycoproteins and have examined various aspects of their role in the infective process. We made the following two observations. (i) Glycoproteins gI and gp63 are noncovalently complexed to each other. They are coprecipitated by antisera against either one of these glycoproteins but do not share antigenic determinants: monoclonal antibodies against gp63 do not immunoprecipitate gI from extracts of gp63mutant-infected cells, and monoclonal antibodies against gI do not immunoprecipitate gp63 from extracts of glmutant-infected cells. (ii) Mutants unable to synthesize either gI or gp63 have some common biological characteristics; they have a growth advantage in primary chicken embryo fibroblasts. Furthermore, we have shown previously that in conjunction with glycoprotein gIII, gI and gp63 are necessary for the expression of virulence (T. C. Mettenleiter, C.

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