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Bovine herpesvirus envelope glycoprotein E (gE) and, in particular, the gE cytoplasmic tail (CT) is a virulence determinant in cattle. Also, the gE CT contributes to virus cell-to-cell spread and anterograde neuronal transport. In this study, our goal was to map the gE CT sub-domains that contribute to virus cell-to-cell spread property. A panel of gE-CT specific mutant viruses was constructed and characterized, in vitro, with respect to their plaque phenotypes, gE recycling and gE basolateral membrane targeting. The results revealed that disruption of the tyrosine-based motifs, 467 YTSL 470 and 563 YTVV 566 , individually produced smaller plaque phenotypes than the wild type. However, they were slightly larger than the gE CT-null virus plaques. The Y467A mutation affected the gE endocytosis, gE trans-Golgi network (TGN) recycling, and gE virion incorporation properties. However, the Y563A mutation affected only the gE basolateral cell-surface redistribution function. Notably, the simultaneous Y467A/Y563A mutations produced gE CT-null virus-like plaque phenotypes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Pannhorst et al.)

We constructed a recombinant bovine herpesvirus type 1 triple mutant virus (BoHV-1 tmv) that lacks UL49.5 residues 30-32 and 80-96, gE cytoplasmic tail (gE CT) residues 452-575 and the entire 435 bp long Us9 ORF. To develop a gE CT-specific blocking ELISA test that is necessary to distinguish the BoHV-1 tmv vaccinated calves from the wild-type (wt) virus-infected calves, a mouse monoclonal antibody (mAb) 2H8F3 was generated by using the Escherichia coli expressed gE CT residues 452-575. Further, by performing a PEPSCAN analysis of 12 mer overlapping peptides spanning the entire gE CT, the epitope sequence recognized by the mAb2H8F3 was mapped within the gE CT residues 499SDDDGPASN507. A blocking ELISA test was then developed for detecting antibodies in wild-type BoHV-1 infected calves against the gE CT epitope specified by 499SDDDGPASN507. The assay is based on the use of HRP conjugated mAb2H8F3 and the E. coli expressed gE CT protein as an indicator antibody and a coating antigen, respectively. In this assay, serum from entire gE-deleted and BoHV-1 tmv-infected calves scored negative, whereas serum from calves infected with BoHV-1 wt scored positive. Therefore, the gE CT-ELISA, based on the mAb2H8F3 and E. coli expressed gE CT protein, is suitable for differentiating the wt virus-infected and BoHV-1 tmv-vaccinated cattle., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bovine herpesvirus 1 (BoHV-1) causes respiratory infections and abortions in cattle, and is an important component of bovine respiratory disease complex, which causes a considerable economic loss worldwide. Several efforts have been made to produce safer and more effective vaccines. One of these vaccines is a glycoprotein E (gE)-deleted marker vaccine which is currently mandated for use in EU countries. In the present study, we have constructed a three-gene-mutated BoHV-1 vaccine virus (UL49.5 luminal domain residues 30-32 and cytoplasmic tail residues 80-96 deleted, gE cytoplasmic tail- and entire Us9-deleted) and compared its protective vaccine efficacy in calves after intranasal vaccination with that of a gE-deleted virus. Following vaccination, both the triple mutant and gE-deleted vaccine virus replicated well in the nasal epithelium of the calves. The vaccinated calves did not show any clinical signs. Four weeks post-vaccination, the animals were challenged intranasally with a virulent BoHV-1 wild-type virus. Based on clinical signs, both the gE-deleted and triple mutant group were protected equally against the virulent BoHV-1 challenge. However, based on the quantity and duration of nasal viral shedding, virus neutralizing antibody and cellular immune responses, the triple mutant virus vaccine induced a significantly better protective immune response than the gE-deleted virus vaccine. Notably, after the virulent BoHV-1 challenge, the triple mutant virus vaccinated group cleared the challenge virus three days earlier than the BoHV-1 gE-deleted virus vaccinated group., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Bovine herpesvirus type 1 (BHV-1) is an important component of the bovine respiratory disease complex (BRDC) in cattle. Following primary intranasal and ocular infection of cattle, BHV-1 establishes lifelong latent infection in trigeminal ganglia (TG). Upon reactivation from latency, the virus is transported from neuronal cell bodies in the TG to projected nerve endings in nose and cornea of latently infected cattle where the virus shedding occurs. This property of BHV-1 plays a significant role in the pathogenesis of BRDC and maintenance of BHV-1 in the cattle population. Recently, we have reported that a glycoprotein E (gE) cytoplasmic tail-truncated BHV-1 (BHV-1 gEAm453) did not reactivate from latency and was not shed in the nasal and ocular secretions of calves and rabbits. Here we describe the methods to establish rabbit primary dorsal root ganglia (DRG) neuron cultures in a microfluidic chamber system and to characterize in vitro anterograde and retrograde axonal transport properties of BHV-1 gE-deleted and BHV-1 cytoplasmic tail-truncated gEAm453 mutant viruses relative to BHV-1 gEAm453-rescued/wild-type viruses. The results clearly demonstrated that whereas the BHV-1 gE-deleted, BHV-1 gEAm453, and BHV-1 gEAm453-rescued/wild-type viruses were transported equally efficiently in the retrograde direction, only the BHV-1 gEAm453-rescued/wild-type virus was transported anterogradely. Therefore, we have concluded that sequences within the BHV-1 gE cytoplasmic tail are essential for anterograde axonal transport and that primary rabbit DRG neuronal cultures in the microfluidic chambers are suitable for BHV-1 neuronal transport studies.

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEDelta), thymidine kinase (BoHV-5TKDelta) and both proteins (BoHV-5gEDeltaTKDelta). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEDelta developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKDelta (N = 8) or BoHV-5gEDeltaTKDelta (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKDelta and BoHV-5gEDeltaTKDelta are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

The requirement of bovine herpesvirus type 1 (BoHV-1) envelope protein gE (Us8 homolog) for establishment of latency and reactivation in trigeminal ganglia (TG) was examined. Although BHV-1 gE-rescued and gE-deleted viruses were isolated from nasal or ocular swabs during primary infection, only the gE-rescued virus was isolated following dexamethasone-induced reactivation. Furthermore, gC protein expression, which requires viral DNA replication for its expression, was detected in TG of calves infected with either virus following reactivation. These studies suggest that gE is required for anterograde transport of BoHV-1 from neuronal cell bodies in the TG to their nerve processes.

The alphaherpesvirus envelope protein Us9 is a type II viral membrane protein that is required for anterograde spread of bovine herpesvirus 5 (BHV-5) infection from the olfactory receptor neurons to the brain. In a rabbit seizure model, Us9-deleted BHV-5 failed to invade the central nervous system (CNS) following intranasal infection. However, when injected directly into the olfactory bulb, retrograde-spread infection from the olfactory bulb (OB) to the piriform cortex and other areas connected to the OB was not affected. In contrast to BHV-5, wild-type BHV-1 failed to invade the CNS following intranasal infection. In this study, we show that mature BHV-1 Us9 is a 30- to 32-kDa protein, whereas mature BHV-5 Us9 is an 18- to 20-kDa protein. In vitro, BHV-1 Us9 is expressed at 3 h postinfection (hpi), whereas BHV-5 Us9 is expressed at 6 hpi. Despite these differences, BHV-1 Us9 not only complemented for BHV-5 Us9 and rescued the anterograde-spread defect of the BHV-5 Us9-deleted virus but conferred increased neurovirulence and neuroinvasiveness in our rabbit seizure model. Rabbits infected with BHV-5 expressing BHV-1 Us9 showed severe neurological signs at 5 days postinfection, which was 1 to 2 days earlier than BHV-5 wild-type or Us9-reverted BHV-5 virus. The data underscore the importance of both Us9 genes for virion anterograde transport and neuroinvasiveness. However, Us9 is not the determinant of the differential neuropathogenesis of BHV-1 and BHV-5.

Bovine herpesvirus glycoprotein C (gC) functions as a major virus attachment protein. Here, two BHV-1 gC-specific epitopes that are specified by complement-dependent neutralizing MAbs are mapped. The BHV-1 gC-specific peptides and MAbs were used to specifically localize continuous epitopes by direct binding to the MAbs and by blocking the Mab reactivity (competitive ELISA) to authentic viral antigen. The results of competitive ELISA indicated that the complement-dependent neutralizing epitopes recognized by MAbs F2 and 24 were located between BHV-1 gC amino acids (aa) 47-69 and (aa) 109-119, respectively.

Herpesvirus glycoprotein C (gC) functions as a major virus attachment protein. The gC sequence of the neurovirulent bovine herpesvirus type 5 (BHV-5) virus was determined and compared with the gC sequence of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gC ORFs showed that the amino-terminal third of the protein differed between the two viruses. Whole or subgenomic fragments of gC coding regions from both viruses were expressed as trpE-gC fusion proteins in Escherichia coli to map linear epitopes defined by type-specific murine monoclonal antibodies (MAbs). Based on the reactivity of BHV-1-specific MAbs with the recombinant proteins, two epitopes were mapped between BHV-1 gC residues 22 and 172. Undirectional deletion of these residues at the carboxy end mapped one within residues 22-69 and the other within residues 103-122. Two BHV-5-specific MAbs identified an epitope coding region within BHV-5 gC residues 31-78. Bovine antisera against BHV-1 and BHV-5 showed specificity to BHV-1 gC residues 22-69 and to BHV-5 gC residues 31-78, respectively, in a type-specific manner.