Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections; causing a high economic loss in all continents. Use of marker vaccines in IBR eradication programs is widely accepted since it allows for protection of the animals against the disease while adding the possibility of differentiating vaccinated from infected animals.

The bovine respiratory disease complex (BRDC) remains a major problem for both beef and dairy cattle industries worldwide. BRDC frequently involves an initial viral respiratory infection resulting in immunosuppression, which creates a favorable condition for fatal secondary bacterial infection. Current polyvalent modified live vaccines against bovine herpesvirus type 1(BoHV-1) and bovine viral diarrhea virus (BVDV) have limitations concerning their safety and efficacy. To address these shortcomings and safety issues, we have constructed a quadruple gene mutated BoHV-1 vaccine vector (BoHV-1 QMV), which expresses BVDV type 2, chimeric E2 and Flag-tagged Erns-fused with bovine granulocyte monocyte colony-stimulating factor (GM-CSF) designated here as QMV-BVD2*. Here we compared the safety, immunogenicity, and protective efficacy of QMV-BVD2* vaccination in calves against BVDV-2 with Zoetis Bovi-shield Gold 3 trivalent (BoHV-1, BVDV types 1 and 2) vaccine. The QMV-BVD2* prototype subunit vaccine induced the BoHV-1 and BVDV-2 neutralizing antibody responses along with BVDV-1 and -2 cross-reactive cellular immune responses. Moreover, after a virulent BVDV-2 challenge, the QMV-BVD2* prototype subunit vaccine conferred a more rapid recall BVDV-2-specific neutralizing antibody response and considerably better recall BVDV types 1 and 2-cross protective cellular immune responses than that of the Zoetis Bovi-shield Gold 3.

Bovine herpesvirus type 1 (BHV-1) U_L49.5 inhibits transporter associated with antigen processing (TAP) and down-regulates cell-surface expression of major histocompatibility complex (MHC) class I molecules to promote immune evasion. We have constructed a BHV-1 U_L49.5 cytoplasmic tail (CT) null and several U_L49.5 luminal domain mutants in the backbone of wild-type BHV-1 or BHV-1 U_L49.5 CT- null viruses and determined their relative TAP mediated peptide transport inhibition and MHC-1 down-regulation properties compared with BHV-1 wt. Based on our results, the U_L49.5 luminal domain residues 30–32 and U_L49.5 CT residues, together, promote efficient TAP inhibition and MHC-I down-regulation functions. In vitro, BHV-1 U_L49.5 Δ30–32 CT-null virus growth property was similar to that of BHV-1 wt and like the wt U_L49.5, the mutant U_L49.5 was incorporated in the virion envelope and it formed a complex with gM in the infected cells.

Alphaherpesvirus envelope glycoprotein N (gN) and gM form a covalently linked complex. Bovine herpesvirus type 1 (BHV-1) U _L 49.5 (a gN homolog) contains two predicted cysteine residues, C42 and C78. The C42 is highly conserved among the alphaherpesvirus gN homologs (e.g., herpes simplex virus 1 and pseudorabies virus). To identify which cysteine residue is required for the formation of the U _L 49.5/gM complex and to characterize the functional significance of the U _L 49.5/gM complex, we constructed and analyzed C42S and C78S substitution mutants in either a BHV-1 wild type (wt) or BHV-1 U _L 49.5 cytoplasmic tail-null (CT-null) virus background. The results demonstrated that BHV-1 U _L 49.5 residue C42 but not C78 was essential for the formation of the covalently linked functional U _L 49.5/gM complex, gM maturation in the Golgi compartment, and efficient cell-to-cell spread of the virus. Interestingly, the C42S and CT-null mutations separately did not affect mutant U _L 49.5 virion incorporation. However, when both of the mutations were introduced simultaneously, the U _L 49.5 C42S/CT-null protein virion incorporation was severely reduced. Incidentally, the anti-VP22 antibody coimmunoprecipitated the U _L 49.5 C42S/CT-null mutant protein at a noticeably reduced level compared to that of the individual U _L 49.5 C42S and CT-null mutant proteins. As expected, in a dual U _L 49.5 C42S/VP22Δ virus with deletion of VP22 (VP22Δ), the U _L 49.5 C42S virion incorporation was also severely reduced while in a gMΔ virus, U _L 49.5 virion incorporation was affected only slightly. Together, these results suggested that U _L 49.5 virion incorporation is mediated redundantly, by both U _L 49.5/gM functional complex and VP22, through a putative gM-independent novel U _L 49.5 and VP22 interaction. IMPORTANCE Bovine herpesvirus 1 (BHV-1) envelope protein U _L 49.5 is an important virulence determinant because it downregulates major histocompatibility complex class I (MHC-I). U _L 49.5 also forms a covalently linked complex with gM. The results of this study demonstrate that U _L 49.5 regulates gM maturation and virus cell-to-cell spread since gM maturation in the Golgi compartment depends on covalently linked U _L 49.5/gM complex. The results also show that the U _L 49.5 residue cysteine 42 (C42) mediates the formation of the covalently linked U _L 49.5-gM interaction. Furthermore, a C42S mutant virus in which U _L 49.5 cannot interact with gM has defective cell-to-cell spread. Interestingly, U _L 49.5 also interacts with the tegument protein VP22 via its cytoplasmic tail (CT). The putative U _L 49.5 CT-VP22 interaction is essential for a gM-independent U _L 49.5 virion incorporation and is revealed when U _L 49.5 and gM are not linked. Therefore, U _L 49.5 virion incorporation is mediated by U _L 49.5-gM complex interaction and through a gM-independent interaction between U _L 49.5 and VP22.