Bovine Herpesvirus 1 UL49.5 Interacts with gM and VP22 To Ensure Virus Cell-to-Cell Spread and Virion Incorporation: Novel Role for VP22 in gM-Independent UL49.5 Virion Incorporation.

Alphaherpesvirus envelope glycoprotein N (gN) and gM form a covalently linked complex. Bovine herpesvirus type 1 (BHV-1) UL49.5 (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 UL49.5/gM complex and to characterize the functional significance of the UL49.5/gM complex, we constructed and analyzed C42S and C78S substitution mutants in either a BHV-1 wild type (wt) or BHV-1 UL49.5 cytoplasmic tail-null (CT-null) virus background. The results demonstrated that BHV-1 UL49.5 residue C42 but not C78 was essential for the formation of the covalently linked functional UL49.5/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 UL49.5 virion incorporation. However, when both of the mutations were introduced simultaneously, the UL49.5 C42S/CT-null protein virion incorporation was severely reduced. Incidentally, the anti-VP22 antibody coimmunoprecipitated the UL49.5 C42S/CT-null mutant protein at a noticeably reduced level compared to that of the individual UL49.5 C42S and CT-null mutant proteins. As expected, in a dual UL49.5 C42S/VP22Δ virus with deletion of VP22 (VP22Δ), the UL49.5 C42S virion incorporation was also severely reduced while in a gMΔ virus, UL49.5 virion incorporation was affected only slightly. Together, these results suggested that UL49.5 virion incorporation is mediated redundantly, by both UL49.5/gM functional complex and VP22, through a putative gM-independent novel UL49.5 and VP22 interaction.IMPORTANCE Bovine herpesvirus 1 (BHV-1) envelope protein UL49.5 is an important virulence determinant because it downregulates major histocompatibility complex class I (MHC-I). UL49.5 also forms a covalently linked complex with gM. The results of this study demonstrate that UL49.5 regulates gM maturation and virus cell-to-cell spread since gM maturation in the Golgi compartment depends on covalently linked UL49.5/gM complex. The results also show that the UL49.5 residue cysteine 42 (C42) mediates the formation of the covalently linked UL49.5-gM interaction. Furthermore, a C42S mutant virus in which UL49.5 cannot interact with gM has defective cell-to-cell spread. Interestingly, UL49.5 also interacts with the tegument protein VP22 via its cytoplasmic tail (CT). The putative UL49.5 CT-VP22 interaction is essential for a gM-independent UL49.5 virion incorporation and is revealed when UL49.5 and gM are not linked. Therefore, UL49.5 virion incorporation is mediated by UL49.5-gM complex interaction and through a gM-independent interaction between UL49.5 and VP22.

Us3 and Us9 proteins contribute to the stromal invasion of bovine herpesvirus 1 in the respiratory mucosa.

Bovine herpesvirus 1 (BHV-1) infection may lead to conjunctivitis, upper respiratory tract problems, pneumonia, genital disorders and abortion. BHV-1 is able to spread quickly in a plaque-wise manner and invade by breaching the basement membrane (BM) barrier in the respiratory mucosa. BHV-1 Us3, a serine/threonine kinase, induces a dramatic cytoskeletal reorganization and BHV-1 Us9, a tail-anchored membrane protein, is required for axonal transport of viruses in neurons. In this study, we investigated the role of Us3 and Us9 during BHV-1 infection in the respiratory mucosa. First, we constructed and characterized BHV-1 Us3 null, Us9 null and revertant viruses. Then, we analysed the viral replication and plaque size (latitude) in Madin-Darby bovine kidney (MDBK) cells and the respiratory mucosa as well as viral penetration depth underneath the BM of the respiratory mucosa when inoculated with these recombinant viruses. Knockout of Us3 resulted in a 1 log10 reduction in viral titre and plaque size (latitude) in MDBK cells and the trachea mucosa. There were no defects in the cell-to-cell spread observed for BHV-1 Us9 null virus. Both BHV-1 Us3 null and Us9 null viruses showed a significant reduction of plaque penetration underneath the BM; however, penetration was not completely inhibited. In conclusion, the current findings demonstrated that Us3 and Us9 play an important role in the invasion of BHV-1 through the BM of the respiratory mucosa, which shows the way forward for research-based attenuation of viruses in order to make safer and better-performing vaccines.

Protection induced by a glycoprotein E-deleted bovine herpesvirus type 1 marker strain used either as an inactivated or live attenuated vaccine in cattle.

BACKGROUND: 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 aim of the present study was the development and evaluation of safety and efficacy of a glycoprotein E-deleted (gE-) BoHV-1 marker vaccine strain (BoHV-1ΔgEßgal) generated by homologous recombination, replacing the viral gE gene with the ß-galactosidase (ßgal) gene. RESULTS: In vitro growth kinetics of the BoHV-1ΔgEßgal virus was similar to BoHV-1 LA. The immune response triggered by the new recombinant strain in cattle was characterized both as live attenuated vaccine (LAV) and as an inactivated vaccine. BoHV-1ΔgEßgal was highly immunogenic in both formulations, inducing specific humoral and cellular immune responses. Antibody titers found in animals vaccinated with the inactivated vaccine based on BoHV-1ΔgEßgal was similar to the titers found for the control vaccine (BoHV-1 LA). In the same way, titers of inactivated vaccine groups were significantly higher than any of the LAV immunized groups, independently of the inoculation route (p < 0.001). Levels of IFN-γ were significantly higher (p < 0.001) in those animals that received the LAV compared to those that received the inactivated vaccine. BoHV-1ΔgEßgal exhibited an evident attenuation when administered as a LAV; no virus was detected in nasal secretions of vaccinated or sentinel animals during the post-vaccination period. BoHV-1ΔgEßgal, when used in either formulation, elicited an efficient immune response that protected animals against challenge with virulent wild-type BoHV-1. Also, the deletion of the gE gene served as an immunological marker to differentiate vaccinated animals from infected animals. All animals vaccinated with the BoHV-1ΔgE ßgal strain were protected against disease after challenge and shed significantly less virus than control calves, regardless of the route and formulation they were inoculated. CONCLUSIONS: Based on its attenuation, immunogenicity and protective effect after challenge, BoHV-1ΔgEßgal virus is an efficient and safe vaccine candidate when used either as inactivated or as live attenuated forms.

Live Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Undergoes an Abortive Replication Cycle in the TG Neurons following Latency Reactivation.

Like other alpha herpesviruses, pseudorabies virus (PRV) establishes lifelong latency in trigeminal ganglionic (TG) neurons. Upon stress, the latent viruses in the TG neurons reactivate and are transported anterograde from the neuron cell bodies to the nerve endings in the nasal mucosa, where they replicate and are discharged in the nasal and oral secretions. Consequently, the virus is transmitted to other naïve animals. This cycle of latency and reactivation continues until the animal dies or is slaughtered. We have constructed a PRV triple mutant virus (PRVtmv) and used it as a live subunit vaccine vector against porcine circovirus 2b (PCV2b) and classical swine fever virus (CSFV) (PRVtmv+). We compared the latency reactivation properties of PRVtmv+ with its parent wild-type (wt) Becker strain following intranasal infection. The results showed that PRV wt and PRVtmv+ established latency in the TG neurons. Based on nasal virus shedding, immediate early (infected cell protein 0; ICP0) and late genes, MCP (major capsid protein) and gC (glycoprotein C) transcriptions, and viral DNA copy numbers in the TGs of latently infected and dexamethasone (Dex)-treated pigs, both PRV wt and PRVtmv+ reactivated from latency. We noticed that PRV wt virus replicated productively in the terminally differentiated, postmitotic TG neurons, but PRVtmv+ failed to replicate and, therefore, there was no virus production in the TG. In addition, we found that only the PRV wt virus was shed in the nasal secretions following the Dex-induced reactivation. Our results demonstrated that the PRVtmv+ is safe as a live viral subunit vaccine vector without the possibility of productive replication in the TG upon reactivation from latency and without subsequent nasal virus shedding. This property of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protect Pigs against a Virulent CSFV Challenge.

Classical swine fever (CSF) remains one of the most economically significant viral diseases affecting domestic pigs and wild boars worldwide. To develop a safe and effective vaccine against CSF, we have constructed a triple gene-deleted pseudorabies virus (PRVtmv)-vectored bivalent subunit vaccine against porcine circovirus type 2b (PCV2b) and CSFV (PRVtmv+). In this study, we determined the protective efficacy of the PRVtmv+ against virulent CSFV challenge in pigs. The results revealed that the sham-vaccinated control group pigs developed severe CSFV-specific clinical signs characterized by pyrexia and diarrhea, and became moribund on or before the seventh day post challenge (dpc). However, the PRVtmv+-vaccinated pigs survived until the day of euthanasia at 21 dpc. A few vaccinated pigs showed transient diarrhea but recovered within a day or two. One pig had a low-grade fever for a day but recovered. The sham-vaccinated control group pigs had a high level of viremia, severe lymphocytopenia, and thrombocytopenia. In contrast, the vaccinated pigs had a low-moderate degree of lymphocytopenia and thrombocytopenia on four dpc, but recovered by seven dpc. Based on the gross pathology, none of the vaccinated pigs had any CSFV-specific lesions. Therefore, our results demonstrated that the PRVtmv+ vaccinated pigs are protected against virulent CSFV challenge.

A Novel Quadruple Gene-Deleted BoHV-1-Vectored RVFV Subunit Vaccine Induces Humoral and Cell-Mediated Immune Response against Rift Valley Fever in Calves.

Rift Valley fever virus (RVFV) is considered to be a high biodefense priority based on its threat to livestock and its ability to cause human hemorrhagic fever. RVFV-infected livestock are also a significant risk factor for human infection by direct contact with contaminated blood, tissues, and aborted fetal materials. Therefore, livestock vaccination in the affected regions has the direct dual benefit and one-health approach of protecting the lives of millions of animals and eliminating the risk of severe and sometimes lethal human Rift Valley fever (RVF) disease. Recently, we have developed a bovine herpesvirus type 1 (BoHV-1) quadruple gene mutant virus (BoHV-1qmv) vector that lacks virulence and immunosuppressive properties due to the deletion of envelope proteins UL49.5, glycoprotein G (gG), gE cytoplasmic tail, and US9 coding sequences. In the current study, we engineered the BoHV-1qmv further by incorporating a chimeric gene sequence to express a proteolytically cleavable polyprotein: RVFV envelope proteins Gn ectodomain sequence fused with bovine granulocyte-macrophage colony-stimulating factor (GMCSF) and Gc, resulting in a live BoHV-1qmv-vectored subunit vaccine against RVFV for livestock. In vitro, the resulting recombinant virus, BoHV-1qmv Sub-RVFV, was replicated in cell culture with high titers. The chimeric Gn-GMCSF and Gc proteins expressed by the vaccine virus formed the Gn-Gc complex. In calves, the BoHV-1qmv Sub-RVFV vaccination was safe and induced moderate levels of the RVFV vaccine strain, MP12-specific neutralizing antibody titers. Additionally, the peripheral blood mononuclear cells from the vaccinated calves had six-fold increased levels of interferon-gamma transcription compared with that of the BoHV-1qmv (vector)-vaccinated calves when stimulated with heat-inactivated MP12 antigen in vitro. Based on these findings, we believe that a single dose of BoHV-1qmv Sub-RVFV vaccine generated a protective RVFV-MP12-specific humoral and cellular immune response. Therefore, the BoHV-1qmv sub-RVFV can potentially be a protective subunit vaccine for cattle against RVFV.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protects Pigs against PCV2b Challenge and Induces Serum Neutralizing Antibody Response against CSFV.

Porcine circovirus type 2 (PCV2) is endemic worldwide. PCV2 causes immunosuppressive infection. Co-infection of pigs with other swine viruses, such as pseudorabies virus (PRV) and classical swine fever virus (CSFV), have fatal outcomes, causing the swine industry significant economic losses in many if not all pig-producing countries. Currently available inactivated/modified-live/vectored vaccines against PCV2/CSFV/PRV have safety and efficacy limitations. To address these shortcomings, we have constructed a triple gene (thymidine kinase, glycoprotein E [gE], and gG)-deleted (PRVtmv) vaccine vector expressing chimeric PCV2b-capsid, CSFV-E2, and chimeric Erns-fused with bovine granulocytic monocyte-colony stimulating factor (Erns-GM-CSF), designated as PRVtmv+, a trivalent vaccine. Here we compared this vaccine's immunogenicity and protective efficacy in pigs against wild-type PCV2b challenge with that of the inactivated Zoetis Fostera Gold PCV commercial vaccine. The live PRVtmv+ prototype trivalent subunit vaccine is safe and highly attenuated in pigs. Based on PCV2b-specific neutralizing antibody titers, viremia, viral load in lymphoid tissues, fecal-virus shedding, and leukocyte/lymphocyte count, the PRVtmv+ yielded better protection for vaccinated pigs than the commercial vaccine after the PCV2b challenge. Additionally, the PRVtmv+ vaccinated pigs generated low to moderate levels of CSFV-specific neutralizing antibodies.

Side effects of oseltamivir in end-stage renal failure patients.

BACKGROUND: Patients with end-stage renal disease who developed H1N1 infections have an increased risk of morbidity and mortality. In light of the high incidence of H1N1 infections in renal replacement therapy patients in Brunei Darussalam, an Oseltamivir (Tamiflu) prophylactic dosing regimen of 75 mg every 5 days for renal replacement therapy patients was initiated by the Ministry of Health in August 2009. The regime was used to serve as a bridge towards an anticipated nationwide vaccination programme that was due in September 2009. This study aimed to evaluate the side effects, factors that might influence the side effects profile and compliance of the dialysis patients that had undergone the month-long chemoprophylactic regime. METHODS: A cross-sectional study was carried out on the dialysis patients that had undergone the oseltamivir prophylactic regime, which involved distribution of questionnaires to participants after the regime was completed. RESULTS: Three hundred and thirty-three patients participated in this study. 25.7% of sample participants reported at least one side effect (experienced during the regime). 97% of participants were found to have reported three side effect types or less. The most frequent side effects reported were nausea (9.4%), abdominal pain (9.1%) and dizziness (9.1%). Age, gender, dialysis types, serum haemoglobin, serum albumin and dialysis clearance measurements were found to have no significant associations with the frequency of participants that had reported side effects. 11.2% of sample participants made up the non-compliant group. The top two reasons for not completing the medication were participants' perceived side effects (24.3%) and forgetting to take their medications (56.8%). CONCLUSIONS: Side effects were found to be mild and tolerable by participants, with no life-threatening events. The study showed that high compliance of this regime can be achieved. These results, together with no incidence of H1N1 cases in the sample participants, showed that the dosing regimen of 75 mg every 5 days in both haemodialysis and continuous ambulatory peritoneal dialysis patients is both tolerable and effective and should be considered for future prophylactic regimes.

BoHV-1-Vectored BVDV-2 Subunit Vaccine Induces BVDV Cross-Reactive Cellular Immune Responses and Protects against BVDV-2 Challenge.

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.

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP.

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

Lipoprotein (a) level in pre-eclampsia patients.

This study was carried in BSMMU from July 2001 to June 2003. During the study period, 60 pregnant women were studied. Thirty patients were preeclamptic and thirty were normal healthy pregnant women served as control. Serum lipoprotein(a) was found significantly higher in preeclamptic women 56.63 +/- 22.6 mg/dl and found within limit in normal healthy pregnant women, 12.89 +/- 4.59 mg/dl. Result is statistically highly significant (P < 0.001). Mean systolic blood pressure was 163.33 +/- 29.63 mmHg and 117.00 +/- 11.19 mmHg in case and control and Diastolic Blood Pressure was 108.53 +/- 14.54 mmHg and 76.00 +/- 6.87 mmHg respectively in case and control group. Result was highly significant as P < 0.001. The mean (+/- SD) serum lipoprotein(a) concentration in normal pregnancies and preeclampsia were found to be 12.91 +/- 4.94 and 56.65 +/- 22.62. Moderate proteinuria was found in 77.5% and severe proteinuria in 22.2% cases of preeclampsia respectively. Regardless of mechanism and pathophysiology of preeclampsia, we found high serum level of lipoprotein (a) in preeclampsia patients. These high levels of lipoprotein (a) significantly correlated with blood pressure and proteinuria.

Bovine herpesvirus 1 (BHV-1) envelope protein gE subcellular trafficking is contributed by two separate YXXL/Φ motifs within the cytoplasmic tail which together promote efficient virus cell-to-cell spread.

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, 467YTSL470 and 563YTVV566, 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.

Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport.

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs 467YTSL470 and 563YTVV566, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport.

The zinc RING finger of bovine herpesvirus 1-encoded bICP0 protein is crucial for viral replication and virulence.

Bovine herpesvirus 1 (BHV-1) infected cell protein 0 (bICP0) stimulates productive infection, in part by activating viral gene expression. The C(3)HC(4) zinc RING finger of bICP0 is crucial for activating viral transcription and productive infection. In this study, we used a bacterial artificial chromosome containing a wild-type (wt) virulent BHV-1 strain to generate a single amino acid mutation in the C(3)HC(4) zinc RING finger of bICP0. This virus (the 51g mutant) contains a cysteine-to-glycine mutation (51st amino acid) in the C(3)HC(4) zinc RING finger of bICP0. A plasmid expressing the 51g mutant protein did not transactivate viral promoter activity as efficiently as wt bICP0. The 51g mutant virus expressed higher levels of the bICP0 protein than did the 51g rescued virus (51gR) but yielded reduced virus titers following infection of permissive bovine cells. The 51g mutant virus, but not the 51gR virus, grew poorly in bovine cells pretreated with imiquimod to stimulate interferon production. During acute infection of calves, levels of infectious virus were 2 to 3 logs lower in ocular or nasal swabs with 51g than with 51gR. Calves latently infected with the 51g mutant did not reactivate from latency because virus shedding did not occur in ocular or nasal cavities. As expected, calves latently infected with 51gR reactivated from latency following dexamethasone treatment. These studies demonstrate that mutation of a single well-conserved cysteine residue in the C(3)HC(4) zinc RING finger of bICP0 has dramatic effects on the growth properties of BHV-1.

Bovine herpesvirus type 1 (BoHV-1) anterograde neuronal transport from trigeminal ganglia to nose and eye requires glycoprotein E.

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.

A review of the biology of bovine herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines.

Infection of cattle by bovine herpesvirus type 1 (BHV-1) can lead to upper respiratory tract disorders, conjunctivitis, genital disorders and immune suppression. BHV-1-induced immune suppression initiates bovine respiratory disease complex (BRDC), which costs the US cattle industry approximately 3 billion dollars annually. BHV-1 encodes at least three proteins that can inhibit specific arms of the immune system: (i) bICP0 inhibits interferon-dependent transcription, (ii) the UL41.5 protein inhibits CD8+ T-cell recognition of infected cells by preventing trafficking of viral peptides to the surface of the cells and (iii) glycoprotein G is a chemokine-binding protein that prevents homing of lymphocytes to sights of infection. Following acute infection of calves, BHV-1 can also infect and induce high levels of apoptosis of CD4+ T-cells. Consequently, the ability of BHV-1 to impair the immune response can lead to BRDC. Following acute infection, BHV-1 establishes latency in sensory neurons of trigeminal ganglia (TG) and germinal centers of pharyngeal tonsil. Periodically BHV-1 reactivates from latency, virus is shed, and consequently virus transmission occurs. Two viral genes, the latency related gene and ORF-E are abundantly expressed during latency, suggesting that they regulate the latency-reactivation cycle. The ability of BHV-1 to enter permissive cells, infect sensory neurons and promote virus spread from sensory neurons to mucosal surfaces following reactivation from latency is also regulated by several viral glycoproteins. The focus of this review is to summarize the biology of BHV-1 and how this relates to BRDC.

Immunization of bighorn sheep against Mannheimia haemolytica with a bovine herpesvirus 1-vectored vaccine.

Mannheimia haemolytica is an important pathogen of pneumonia in bighorn sheep (BHS), consistently causing 100% mortality under experimental conditions. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, a vaccine containing leukotoxin and surface antigens of M. haemolytica induced 100% protection in BHS, but required multiple booster doses. Vaccination of wildlife is difficult. BHS, however, can be vaccinated at the time of transplantation, but administration of booster doses is impossible. A vaccine that does not require booster doses, therefore, is ideal for vaccination of BHS. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation which obviates the need for booster administration. The objective of this study was to evaluate the potential of bovine herpesvirus 1 (BHV-1) as a vector encoding M. haemolytica immunogens. As the first step towards this goal, the permissiveness of BHS for BHV-1 infection was determined. BHS inoculated with wild-type BHV-1 shed the virus following infection. The lytic phase of infection was superseded by latency, and treatment of latently-infected BHS with dexamethasone reactivated the virus. A recombinant BHV-1-vectored vaccine encoding a leukotoxin-neutralizing epitope and an immuno-dominant epitope of the outer membrane protein PlpE was developed by replacing the viral glycoprotein C gene with a leukotoxin-plpE chimeric gene. Four of six BHS vaccinated with the recombinant virus developed significant leukotoxin-neutralizing antibodies at day 21 post-vaccination, while two of six BHS developed significant surface antigen antibodies at day 17 post-vaccination. These antibodies, however, were inadequate for protection of BHS against M. haemolytica challenge. These data indicate that BHV-1 is a suitable vector for immunization of BHS, but additional experimentation with the chimeric insert is necessary for development of a more efficacious vaccine.

Identification of an epitope within the Bovine herpesvirus 1 glycoprotein E cytoplasmic tail and use of a monoclonal antibody directed against the epitope for the differentiation between vaccinated and infected animals.

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.