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Glycoprotein gIV, a structural component of bovine herpesvirus type 1, stimulates high titers of virus-neutralizing antibody. The protein contains three potential sites for the addition of N-linked carbohydrates. Three mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Thr/Ser to Ser-X-Thr/Ser. A fourth mutant was altered at two sites. The altered forms of the gIV gene were cloned into a vaccinia virus transfer vector to generate recombinant vaccinia viruses expressing mutant proteins. Analysis of these mutants revealed that only two (residues 41 and 102) of the three (residues 41, 102, and 411) potential sites for the addition of N-linked glycans are actually utilized. Absence of glycans at residue 41 (gN1) showed no significant effect on the conformation of the protein or induction of a serum neutralizing antibody response. However, mutant proteins lacking glycans at residue 102 (gN2) or residues 41 and 102 (gN1N2) showed altered reactivity with conformation-dependent gIV-specific monoclonal antibodies. These mutants also induced significantly lower serum neutralizing antibody responses than wild-type gIV. Nonetheless, each of the mutant proteins were modified by the addition of O-glycans and transported to the cell surface. Our results demonstrate that absence of N-linked glycans at one (residue 102) or both (residues 41 and 102) utilized N-linked glycosylation sites alters the conformation but does not prevent processing and transport of gIV to the cell surface.

High levels of recombinant bovine herpesvirus-1 (BHV-1) glycoprotein IV were produced in baculovirus, adenovirus, vaccinia virus and Escherichia coli expression systems. The different recombinant forms as well as authentic gIV were injected intramuscularly into seronegative calves. With the exception of E. coli-produced gIV, all forms of gIV induced high levels of neutralizing antibodies both in the serum and in the nasal superficial mucosa. Animals immunized with gIV produced in insect or mammalian cells were completely protected from infection with BHV-1, as demonstrated by the absence of temperature responses, clinical signs or detectable virus in the nasal secretions after challenge exposure. The E. coli-derived gIV induced partial protection from clinical disease, even though it was not glycosylated and did not induce appreciable levels of neutralizing antibodies. This study demonstrated that all forms of glycosylated gIV, whether authentic or recombinant, confer protection from BHV-1 infection and thus may be useful as an effective subunit vaccine.

The haemagglutinin-esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.

The major glycoprotein complex gl of bovine herpesvirus-1 was expressed at high levels (36 micrograms per 1 x 10(6) cells) in insect cells using a recombinant baculovirus. The recombinant gl had an apparent molecular weight of 116 kDa and was partially cleaved to yield 63-kDa (glb) and 52-kDa (glc) subunits. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells, even though the cleavage sites of authentic and recombinant gl were shown to be identical. The oligosaccharide linkages were mostly endoglycosidase-H-sensitive, in contrast to those of authentic gl, which has mostly endoglycosidase-H-resistant linkages and an apparent molecular weight of 130/74/55 kDa. Despite the reduced cleavage and altered glycosylation, the recombinant glycoprotein was transported and expressed on the surface of infected insect cells. These surface molecules were biologically active as demonstrated by their ability to induce cell-cell fusion. Fusion was inhibited by three monoclonal antibodies specific for antigenic domains I and IV on gl. Domain I maps to the extracellular region of the carboxy terminal fragment glc and domain IV to the very amino terminus of the glb fragment, indicating that domains mapping in two distinct regions of gl function in cell fusion. Monoclonal antibodies specific for eight different epitopes recognized recombinant gl, indicating that the antigenic characteristics of the recombinant and authentic glycoproteins are similar. In addition, the recombinant gl was as immunogenic as the authentic gl, resulting in the induction of gl-specific antibodies in cattle.

Two conformation-dependent neutralizing epitopes, A and B, have been mapped to the S1 subunit of the S spike glycoprotein of bovine coronavirus (BCV). In order to characterize the structure of these antigenic sites, we constructed a series of cDNA clones encoding deleted or truncated S1 derivatives and expressed the modified genes in insect cells using recombinant baculoviruses. Monoclonal antibodies directed against epitopes A and B recognized only the mutant S1 polypeptides containing amino acids 324-720, as demonstrated by immunoprecipitation and Western blot analysis in the absence of beta-mercaptoethanol. In addition, two domains within this region were identified and only mutants containing both domains were immunoreactive, indicating that both were critical in the formation of the antigenic determinants. One domain was localized between residues 324 and 403 and the other at residues 517-720. Deletion of either domain inhibited extracellular secretion of the mutant proteins whereas mutants containing both or none of the domains were secreted efficiently. This observation suggests a vital function of the native conformation of the S1 protein in both antigenic structure and intracellular transport. Antigenic determinants A and B were not distinguished, but these determinants appeared to require both domains for epitope formation. Our results suggest that the antigenic determinants formed by two domains are likely associated with the probable polymorphic region of the BCV S1 subunit.

The hemagglutinin/esterase (HE), spike precursor (S) and the S1 and S2 subunits of the spike precursor protein of bovine coronavirus were expressed in Spodoptera frugiperda (Sf9) cells, and the cell-fusing activity of each recombinant glycoprotein was examined. Extensive syncytia formation was observed in cells infected with the S2 recombinant but not with the HE or S1 recombinant baculoviruses. Fusion of Sf9 cells expressing the intact S protein precursor was evident after trypsin treatment. These results demonstrate that proteolytic cleavage of the S spike precursor is required for fusion induction and that the fusion is mediated by the S2 subunit. These observations may reflect the biological role of the S2 subunit in fusion-penetration during bovine coronavirus infection.

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.

A cDNA fragment representing the hemagglutinin-esterase (HE) gene of bovine coronavirus (BCV) was inserted into the genome of Autographa californica nuclear polyhedrosis virus. Infection of insect cells with the recombinant virus resulted in the production of a 120-kilodalton disulfide-linked dimeric form of the BCV HE polypeptide. Deletion of the carboxy-terminal hydrophobic domain from the HE polypeptide resulted in secretion of a dimeric form of the truncated HE polypeptide. The acetylesterase activity of the BCV HE was detectable in insect cells expressing the BCV hemagglutinin and was inhibited by two monoclonal antibodies which also inhibit hemagglutination.

Partial N-terminal amino acid analyses of five radiolabelled non-structural (ns) proteins specified by Kunjin (KUN) virus provided positive identification of NS3, NS5 and three previously hypothetical ns proteins of flaviviruses, ns2a, ns2b and ns4b. Their correct gene order was obtained from their deduced amino acid sequences. Thus the gene order for KUN virus relative to that proposed for yellow fever (YF) virus was as follows: KUN 5'...GP44.P19.P10.P71.(?).P21.P98-3', YF 5'...NS1.ns2a.ns2b.NS3.ns4a.ns4b.NS5 -3'. The identity of GP44 as NS1 was assumed from the known nucleotide and deduced amino acid sequences; ns4a was not identified. The cleavage sites in the polyprotein for KUN NS2B, NS3 and NS5 were identical, Lys-Arg decreased Gly, similar in form to the sequence Arg-Arg decreased Ser defined at the cleavage sites of YF NS3 and NS5. A new consensus cleavage site for NS1, NS2A and NS4B in the form Val-X-Ala decreased, where X is any one of several uncharged amino acids, was found at corresponding sites homologous to those of KUN virus in all published flavivirus sequences (a total of 18 sites). NS1 and NS4B, but not NS2A, were preceded by a putative signal sequence.

Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.

A Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5'-C.GP20.E.GP44.P19.P10.P71.(?).P21.P98-3' YF 5'-C.prM.E.NS1.ns2a.ns2b.NS3.ns4a.ns4b. NS5-3'. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site Val-X-Ala decreases where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg decreases Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85% homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN greater than Murray Valley greater than Japanese encephalitis greater than St Louis encephalitis viruses within this closely related serological complex. A non-coding 5' sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3' sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87% homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.

Autographa californica nuclear polyhedrosis viral recombinants containing coding information for the Rift Valley fever virus (RVFV) envelope glycoproteins (G1 and G2) and varying amounts of preglycoprotein coding sequences were prepared by using transfer vectors pAc373 or pAcYM1. Expression products were processed to yield proteins indistinguishable from authentic G1 and G2 by gel electrophoresis. The immunogenic properties of the expressed proteins were assessed by immunizing mice and challenging with RVFV. A single inoculation with lysates of cells infected with recombinants expressing both G1 and G2 induced neutralizing antibody responses in mice and protected them from an otherwise lethal challenge with RVFV. Lysates of cells infected with a recombinant expressing only G2 also induced a protective response after two immunizations. Survivors displayed elevated antibody titers to G1 and G2 and also developed antibodies to the RVFV nucleocapsid protein, the latter allowing discrimination from vaccinated mice and indicating that animals had survived infection. Nonimmune mice were protected from lethal RVFV infection by passive transfer of sera from animals immunized with recombinant antigens, indicating that a humoral immune response is sufficient to protect against RVFV.