Bovine Herpesvirus-1 Glycoprotein Research Articles

Bovine Herpesvirus-1 Glycoprotein M Mediates the Translocation to the Golgi Apparatus and Packaging of VP8.

VP8, the most abundant tegument protein of bovine herpesvirus-1 (BoHV-1), plays an important role in viral replication. According to our previous studies, VP8 localizes to the Golgi apparatus of BoHV-1-infected cells where it can be packaged into the virus; however, Golgi localization of VP8 does not occur outside of the context of infection. The goal of this study was to identify the viral factor(s) involved in the tropism of VP8 towards the Golgi. VP8 was found to interact with glycoprotein M (gM), and the VP8 and gM domains that are essential for this interaction were identified. VP8 and gM colocalized to the Golgi apparatus in BoHV-1-infected cells. In cells co-transfected with VP8- and gM-encoding plasmids, VP8 was also found to be localized to the Golgi, suggesting gM to be sufficient. The localization of VP8 to the Golgi was lost in cells infected with a gM deletion mutant, and the amount of VP8 incorporated into mature virus was significantly reduced. However, with the restoration of gM in a revertant virus, the localization to the Golgi and the amount of VP8 incorporated in the virions were restored. These results indicate that gM plays a critical role in VP8 subcellular localization to the Golgi and packaging into mature virions.

A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Serological study on the presence of some alpha-herpesviruses in goats of northern Anatolia, Turkey.

The aim of this study was to investigate the presence of caprine herpes virus-1 (CpHV-1) and bovine herpes virus-1 (BoHV-1) in 269 goat sera collected from small-scale family farms located in six provinces within the Black Sea region of northern Turkey. The overall seropositivity for alpha-herpesvirus in the native goats was found as 19.33% using BoHV-1 glycoprotein B (gB)-blocking enzyme-linked immunosorbent assay (ELISA). Additionally, the seroprevalence of BoHV-1 was determined in 5.20% of the goats using virus neutralization test. To distinguish between CpHV-1 and BoHV-1, the combinations of gB/gE-blocking ELISA tests were performed. Of tested samples, 15.24% were CpHV-1 seropositive; whereas, 4.09% were BoHV-1 seropositive. The results indicated that CpHV-1 is in circulation among local goats of northern Turkey. Considering the close relationship between BoHV-1 and CpHV-1, the transmission of BoHV-1 via goats may also be one of the predisposing factors involving in the spread of virus among the surrounding cattle.

An Improved DNA Vaccine Against Bovine Herpesvirus-1 Using CD40L and a Chemical Adjuvant Induces Specific Cytotoxicity in Mice.

Bovine herpesvirus-1 (BoHV-1) uses many mechanisms to elude the immune system; one of them is spreading intracellularly, even in the presence of specific antiviral antibodies. Cytotoxic T lymphocytes (CTLs) are necessary to eliminate the virus. The main preventive strategy is vaccination based on inactivated virus. These vaccines are poor inducers of cellular immune responses, and complicate serological diagnosis and determination of the real prevalence of infection. DNA vaccines are a good option because of the capacity of Differentiating Infected from Vaccinated Animals-(DIVA vaccine)-and may be the best way to induce cytotoxic responses. Although this type of vaccines leads to only weak "in vivo" expression and poor immune responses, incorporation of molecular and/or chemical adjuvants can improve the latter, both in magnitude and in direction. In this study, we have investigated the specific immune responses elicited in mice by DNA vaccines based on the BoHV-1 glycoprotein D (pCIgD) with and without two different adjuvants: a plasmid encoding for murine CD40L (pCD40L) or Montanide™ 1113101PR (101). Mice vaccinated with pCIgD+CD40L, pCIgD+101, and pCIgD+CD40L+101 developed significantly higher specific antibody titers against BoHV-1 than the pCIgD group (p < 0.01). The animals vaccinated with pCgD+pCD40L+101 raised significantly higher levels of IgG2a and IgG2b (p < 0.01 and p < 0.001, respectively) than mice vaccinated with pCIgD alone. On the contrary, when the activity of CTL against cells infected with BoHV-1 was measured, the vaccine pCgD+pCD40L+101 induced significantly higher levels of cytotoxicity activity (p < 0.001) than pCIgD alone. A significant increase in the CD4+ populations in the group receiving pCIgD+CD40L+101 in comparison with the pCIgD group was observed and, also, interferon gamma, interleukin (IL)-6, and IL-17A levels were higher. Considering the results obtained from this study for humoral and cellular responses in mice, the inclusion of pCD40L and 101 as adjuvants in a BoHV-1 DNA vaccine for cattle is highly recommendable.

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.

MANα1-2MAN decorated liposomes enhance the immunogenicity induced by a DNA vaccine against BoHV-1.

New technologies in the field of vaccinology arise as a necessity for the treatment and control of many diseases. Whole virus inactivated vaccines and modified live virus ones used against Bovine Herpesvirus-1 (BoHV-1) infection have several disadvantages. Previous works on DNA vaccines against BoHV-1 have demonstrated the capability to induce humoral and cellular immune responses. Nevertheless, 'naked' DNA induces low immunogenic response. Thus, loading of antigen encoding DNA sequences in liposomal formulations targeting dendritic cell receptors could be a promising strategy to better activate these antigen-presenting cells (APC). In this work, a DNA-based vaccine encoding the truncated version of BoHV-1 glycoprotein D (pCIgD) was evaluated alone and encapsulated in a liposomal formulation containing LPS and decorated with MANα1-2MAN-PEG-DOPE (pCIgD-Man-L). The vaccinations were performed in mice and bovines. The results showed that the use of pCIgD-Man-L enhanced the immune response in both animal models. For humoral immunity, significant differences were achieved when total antibody titres and isotypes were assayed in sera. Regarding cellular immunity, a significant increase in the proliferative response against BoHV-1 was detected in animals vaccinated with pCIgD-Man-L when compared to the response induced in animals vaccinated with pCIgD. In addition, upregulation of CD40 molecules on the surface of bovine dendritic cells (DCs) was observed when cells were stimulated and activated with the vaccine formulations. When viral challenge was performed, bovines vaccinated with MANα1-2MAN-PEG-DOPE elicited better protection which was evidenced by a lower viral excretion. These results demonstrate that the dendritic cell targeting using MANα1-2MAN decorated liposomes can boost the immunogenicity resulting in a long-lasting immunity. Liposomes decorated with MANα1-2MAN-PEG-DOPE were tested for the first time as a DNA vaccine nanovehicle in cattle as a preventive treatment against BoHV-1. These results open new perspectives for the design of vaccines for the control of bovine rhinotracheitis.

Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules.

Herpesvirus envelope glycoprotein B (gB) is one of the best-documented extracellular vesicle (EVs)-incorporated viral proteins. Regarding the sequence and structure conservation between gB homologs, we asked whether bovine herpesvirus-1 (BoHV-1) and pseudorabies virus (PRV)-encoded gB share the property of herpes simplex-1 (HSV-1) gB to be trafficked to EVs and affect major histocompatibility complex (MHC) class II. Our data highlight some conserved and differential features of the three gBs. We demonstrate that mature, fully processed BoHV-1 and PRV gBs localize to EVs isolated from constructed stable cell lines and EVs-enriched fractions from virus-infected cells. gB also shares the ability to co-localize with CD63 and MHC II in late endosomes. However, we report here a differential effect of the HSV-1, BoHV-1, and PRV glycoprotein on the surface MHC II levels, and MHC II loading to EVs in stable cell lines, which may result from their adverse ability to bind HLA-DR, with PRV gB being the most divergent. BoHV-1 and HSV-1 gB could retard HLA-DR exports to the plasma membrane. Our results confirm that the differential effect of gB on MHC II may require various mechanisms, either dependent on its complex formation or on inducing general alterations to the vesicular transport. EVs from virus-infected cells also contained other viral glycoproteins, like gD or gE, and they were enriched in MHC II. As shown for BoHV-1 gB- or BoHV-1-infected cell-derived vesicles, those EVs could bind anti-virus antibodies in ELISA, which supports the immunoregulatory potential of alphaherpesvirus gB.

Antiviral Immunotoxin Against Bovine herpesvirus-1: Targeted Inhibition of Viral Replication and Apoptosis of Infected Cell.

Bovine herpesvirus 1 (BoHV-1) is a highly contagious viral pathogen which causes infectious bovine rhinotracheitis in cattle worldwide. Currently, there is no antiviral prophylactic treatment available capable of mitigating the disease impact and facilitating recovery from latent infection. In this study, we have engineered a novel recombinant anti-BoHV-1 immunotoxin construct termed “BoScFv-PE38” that consists of a single-chain monoclonal antibody fragment (scFv) fused with an active domain of Pseudomonas exotoxin A as a toxic effector (PE38). The recombinant BoScFv-PE38 immunotoxin expressed in a prokaryotic expression system has specific binding affinity for BoHV-1 glycoprotein D (gD) with a dissociation constant (Kd) of 12.81 nM and for BoHV-1 virus particles with a Kd value of 97.63 nM. We demonstrate that the recombinant BoScFv-PE38 is internalized into MDBK cell compartments that inhibit BoHV-1 replication with a half-maximal inhibitory concentration (IC50) of 4.95 ± 0.33 nM and a selective index (SI) of 456 ± 31. Furthermore, the BoScFv-PE38 exerted a cytotoxic effect through the induction of ATP and ammonia, leading to apoptosis of BoHV-1-infected cells and the inhibition of BoHV-1 replication in MDBK cells. Collectively, we show that BoScFv-PE38 can potentially be employed as a therapeutic agent for the treatment of BoHV-1 infection.

Long-term study (2005-2010) on the vaccination with BoHV-1 glycoprotein E-deleted marker vaccine in selected two dairy herds in Turkey.

A follow-up study from 2005 to 2010 was carried out in two herds where eradication programme for the bovine herpes virus-1 (BoHV-1) infection depends on the vaccination with inactivated glycoprotein E-deleted vaccine that was started in 2001 following the vaccination with inactivated conventional vaccine between 1999 and 2001. For serological screening, a total of 12,976 sera sampled over several sampling times approximately 6months of interval during 5years (2005-2010) were tested for glycoprotein E (gE)- and glycoprotein B-specific antibodies using ELISA. According to the serological evidence, the long-term persistence of BoHV-1 antibodies, success of marker vaccine, first vaccination time of the calves in herds regularly vaccinated, etc. were discussed in this paper. In conclusion, the vaccination programme using gE (-) marker vaccines, with making efforts to prevent the other factors about transmission of infection, was suggested for the eradication of BoHV-1 infection in Turkey as many EU countries. This is the first report on the BoHV-1 eradication programme in some dairy cattle in Turkey.

A Universal Surface for Label-Free Electrical and Optical Sensing of Disease Markers

Quantitative point-of-care (POC) diagnostic tools are crucial for next-generation human (and animal) health monitoring and disease diagnosis to rapidly infer effective treatment decisions. ELISA based disease diagnostics is often performed off-site in centralized labs, requiring long turnaround times (>72hrs), reducing the effective period for disease intervention. In this study, a gold sensing surface is shown to provide ELISA-equivalent results across multiple label-free and rapid biosensing platforms including potentiometric field-effect transistor (FET) biosensors, electrochemical impedance spectroscopy (EIS) biosensors and surface plasmon resonance (SPR) devices. Using the model pathogen Bovine Herpes Virus 1 (BHV-1) (a major source of economic loss in the agri-food industry globally 1), BHV-1 glycoprotein E (gE) was attached to sensor surfaces to detect the immunological interaction against anti-BHV-1 antibodies. The use of a gold sensing surface offers access to the well-established thiol surface functionalization and protein immobilization via amine coupling. This functionalized gold sensing surface is used to compare the response of ELISA in four different sensing platforms: (1) as the gate electrode for graphene ion-sensitive field-effect transistor (ISFET), (2) as the gate electrode for extended-gate field-effect transistor (EGFET) sensors (3) as the working electrode (WE) for EIS sensors, and (4) as the sensing surface for SPR. The results show that the sensitivity of the EGFET biosensors is equivalent to the ISFETs.2 Moreover, EGFET biosensors provide many advantages including improved reliability by separating the sensor and transducer components, low-cost and easy fabrication, disposable chip design and high compatibility with state-of-art semiconductor technologies.3-5 While the mechanism for EIS is quite different than potentiometric FET-based sensing, the dynamic response is found to be equivalent. Similarly, the same Au surface can be used in optical SPR biosensors using the same surface chemistry. The universality of a Au sensing surface allows detection using multi-platforms but with a single chip and, therefore, simplifies the sample preparation and reduces the required sample volumes for diagnostics. All three electrical sensors demonstrate performance equivalent to SPR and ELISA, with dynamic range approaching the limit of detection for sera dilutions < 10-3of anti-BHV-1 antiserum. However, the potentiometric and EIS sensors provide results much faster (~10 min.) than SPR (~2 hr.) and ELISA (> 20 hr.) Additionally, the electronic biosensors are compact in size and are promising for POC devices. In conclusion, a gold surface is shown to provide universal and equivalent sensing across multiple platforms using well-established surface chemistry. Furthermore, the use of one surface to facilitate integration of multiple sensing mechanisms can potentially improve specificity. Among all of the available biosensor technologies, electrical biosensors provide rapid, label-free and cost-efficient sensing capability in a compact size, which is promising for quantitative point-of-care devices. 1G. D. Snowder et al. Journal of Animal Science 85, 1885 (2007). 2A. Tarasov et al. 2D Materials 2, 044008 (2015). 3L.-L. Chi et al. Materials Chemistry and Physics 63 , 19 (2000). 4P. Dak et al. Device Research Conference (DRC), 2013 71st Annual, 105 (2013). 5W. Guan et al. Biosensors and Bioelectronics 51, 225 (2014).

Development and validation of an indirect ELISA as a confirmatory test for surveillance of infectious bovine rhinotracheitis in vaccinated herds.

BackgroundBovine herpesvirus 1 (BoHV1) is a member of the viral subfamily of Alphaherpesvirinae that infects various species, including cattle, sheep, and goats. The virus causes infectious bovine rhinotracheitis (IBR), which is included in a European list of diseases that may require control and eradication programs. The lack of confirmatory tests affects the validity of diagnostic tools, especially those used for vaccinated herds. In this study, we report the development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) based on BoHV1 glycoprotein E, which was expressed as a secreted recombinant antigen in a mammalian cell system. The performance of the new rec-gE ELISA was compared with that of commercially available indirect and/or blocking ELISAs.ResultsThe sample set included blood sera from animals from IBR-positive farms, IBR-free farms, and marker-vaccinated farms. The indirect ELISA proposed in this study is based on antibody reactivity against BoHV1 gE, and showed high sensitivity and specificity (98.41 and 99.76 %, respectively).ConclusionsThe ELISA performed well, in terms of both its diagnostic sensitivity and specificity, and as a confirmatory methodology, and therefore should improve the diagnostic protocols used for IBR surveillance.

Development of a SYBR Green I based duplex real-time PCR for detection of bovine herpesvirus-1 in semen

Bovine herpesvirus-1 (BoHV-1) is a viral pathogen found in infected bull semen, which is transmitted to inseminated cows by artificial insemination. BoHV-1 infection can cause reproductive disorders leading to significant economic loss to cattle industry. To detect BoHV-1 in semen, in this study, a SYBR Green I based duplex real-time PCR was developed. The assay included primers from BoHV-1 glycoprotein C (gC) and bovine growth hormone (bGH) genes for simultaneous detection in single tube. The result was interpreted by analysing melting temperature (Tm) peaks obtained after melt curve analysis of the amplified products at the end of reaction. The Tm peaks for BoHV-1-gC indicated presence of BoHV-1 while the bGH peak indicated reaction without inhibition. The sensitivity of the assay was to detect ten BoHV-1 genome copies per reaction. The analytical sensitivity was to detect 0.21 TCID50 infectious BoHV-1 in spiked semen. The assay was validated with 80 semen samples collected from breeding bulls. The diagnostic sensitivity and specificity of the assay was 100% with OIE recommended TaqMan probe based real-time PCR.

A triple gene mutant of BoHV-1 administered intranasally is significantly more efficacious than a BoHV-1 glycoprotein E-deleted virus against a virulent BoHV-1 challenge

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.

Baculovirus Mediated Expression of Bovine Herpesvirus-1 Glycoprotein-C in Insect Cells

Bovine herpesvirus-1 (BHV-1) glycoprotein C (gC) is an immunodominant protein that is believed to play a role in initial viral attachment. In the present study a recombinant baculovirus was constructed by incorporating BHV-1 gC coding gene to characterize the expression of the glycoprotein in infected Spodoptera frugiperda cells. The BHV-1 complete gC gene was cloned into pENTR/SD/D Directional TOPO vector (entry clone). The purified entry clone plasmid was subjected to LR recombination with the linear baculovirus DNA which was then transfected into S. frugiperda cells. The recombinant baculovirus carrying the gC coding gene was selected against ganciclovirus in the S. frugiperda cells (first passage), serially passaged for two more generations and the third passage viral stock was used to infect fresh S. frugiperda cells for gene expression study. The reactivity of polyclonal antibody with the S. frugiperda cell expressed gC was detected by immunoperoxidase test. The recombinant gC was purified by Ni-NTA column chromatography and immunoprecipitation. Protein bands of molecular weight 54 and 28 kDa were detected consistently with both monoclonal and polyclonal antibodies, when the recombinant protein was subjected to SDS-PAGE and Western blot analysis. Expression of gC gene in the infected S. frugiperda cells was further confirmed by dot-ELISA indicating its potential use as a coating antigen in an indirect ELISA for diagnosis of BHV-1, the causative agent of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis.

Glycoprotein C gene based molecular subtyping of a bovine herpesvirus -1 isolate from uttar pradesh, India.

Bovine herpesvirus -1 (BHV-1) is the etiological agent of many clinical syndromes in cattle which causes huge economic losses to the animal husbandry sector annually. Since the first report of its presence in India in 1976, the disease is considered to be endemic in the country. In the present study, a case of keratoconjunctivitis in a cow was investigated to find out the underlying cause of the condition. The clinical material (ocular swab) was tested by BHV-1 glycoprotein D gene specific PCR using in house designed primers and found to be positive by the presence of a 212bp DNA product in agarose gel electrophoresis. The virus was isolated in MDBK cell line in the third passage and the serum from the animal, was positive for antibodies against BHV-1 by ELISA. A 575bp segment of the glycoprotein C gene of the isolate was amplified by PCR, cloned and sequenced. On phylogenetic analysis, it was seen that the sequence matched with published BHV-1.1 sequences from USA and Uruguay whereas it was divergent from Brazilian BHV-1.1 isolates. This study highlights the isolation, rapid and sensitive detection of BHV-1 virus from clinical cases and its subtyping by nucleotide sequencing and subsequent phylogenetic analysis which gives invaluable information about the molecular epidemiology of BHV-1 subtypes prevalent in the country.

Detection of bovine herpesvirus 1sequences in yaks (Bos grunniens) with keratoconjunctivitis, using a highly sensitive nested polymerase chain reaction

Thirty-seven yaks (Bos grunniens) with keratoconjunctivitis and 22 healthy yaks were used to investigate the role of bovine herpesvirus 1 (BoHV-1) in keratoconjunctivitis in yaks. Nucleic acid sequences of BoHV-1 glycoproteins B and E were detected in conjunctival swabs from all yaks with keratoconjunctivitis using a nested polymerase chain reaction (PCR). In 21 yaks, BoHV-1 sequences were detected along with Moraxella bovis (M. bovis) and Neisseria spp. The amplified BoHV-1 sequences were identical, and no nucleotide variation was observed when compared with a BoHV-1 reference strain using single-strand conformation polymorphism analysis of the amplified DNA sequences. Interestingly, BoHV-1 sequences could not be detected in samples from healthy yaks. However, conjunctival swabs from two healthy yaks (9.09%) yielded M. bovis and Neisseria spp. Samples from 35 yaks with keratoconjunctivitis showed positive reactions in an avidin biotin enzyme-linked immunosorbent assay for BoHV-1 antibodies; all the healthy yaks were seronegative. This is the first report of a possible association of BoHV-1 with keratoconjunctivitis in yaks.

Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1

Bovine herpesvirus-1 (BHV-1) is a major cause of respiratory tract diseases in cattle. Vaccination of cattle against BHV-1 is a high priority. A major concern of currently modified live BHV-1 vaccines is their ability to cause latent infection and subsequent reactivation resulting in many outbreaks. Thus, there is a need for alternative strategies. We generated two recombinant Newcastle disease viruses (NDVs) expressing the glycoprotein D (gD) of BHV-1 from an added gene. One recombinant, rLaSota/gDFL, expressed gD without any modification. The other recombinant, rLaSota/gDF, expressed a chimeric gD in which the ectodomain of gD was fused with the transmembrane domain and cytoplasmic tail of the NDV fusion F glycoprotein. Remarkably, the native gD expressed by rLaSota/gDFL virus was incorporated into the NDV virion 2.5-fold more efficiently than the native NDV proteins, whereas the chimeric gD was not detectably incorporated even though it was abundantly expressed on the infected cell surface. The expression of gD did not increase the virulence of the rNDV vectors in chickens. A single intranasal and intratracheal inoculation of calves with either recombinant NDV elicited mucosal and systemic antibodies specific to BHV-1, with the responses to rLaSota/gDFL being higher than those to rLaSota/gDF. Following challenge with BHV-1, calves immunized with the recombinant NDVs had lower titers and earlier clearance of challenge virus compared to the empty vector control, and reduced disease was observed with rLaSota/gDFL. Following challenge, the titers of serum antibodies specific to BHV-1 were higher in the animals immunized with the rNDV vaccines compared to the rNDV parent virus, indicating that the vaccines primed for secondary responses. Our data suggest that NDV can be used as a vaccine vector in bovines and that BHV-1 gD may be useful in mucosal vaccine against BHV-1 infection, but might require augmentation by a second dose or the inclusion of additional BHV-1 antigens.

Should the domestic buffalo (Bubalus bubalis) be considered in the epidemiology of Bovine Herpesvirus 1 infection?

Only limited information is available on the epidemiology and pathogenesis of Bovine Herpesvirus 1 (BoHV-1) in domestic buffalos. In this study, a virulent BoHV-1 field strain isolated from cattle was inoculated into buffaloes to evaluate their susceptibility to the virus and to investigate the establishment of viral latency through clinical, virological and serological investigations. Latency was also studied by attempting viral reactivation using pharmacological induction. Six of seven male, 5 months old buffaloes were intranasally inoculated with BoHV-1; the other animal was kept as negative control. The animals were clinically monitored during the post-infection (P.I.) and the post-pharmacological induction (P.P.) periods. During these periods, nasal and rectal swabs, and blood samples, with and without anticoagulant, were collected at 2–3 day intervals. On culling the animals, 206 days P.I., their trigeminal ganglia and tonsils were collected. No clinical signs referable to BoHV-1 were observed throughout the experimental period. However, seropositivity was detected in all infected animals within day 20 P.I., using BoHV-1 glycoprotein E and glycoprotein B competitive ELISAs (IDEXX) and virus neutralisation test. In real-time PCR (RT-PCR), five of these animals were positive, at least once, for nasal or rectal swabs, during the P.I. period. The sixth infected animal was found positive only in the trigeminal ganglia after culling. Ganglia were also positive for two other animals. Virus isolation in permissive cell-lines was successful for a part of the RT-PCR positive samples. The detected viruses were confirmed by genetic analysis as identical to the inoculated strain. No evidence of infection was observed in the negative control. This study represents the first experimental transmission of BoHV-1 in buffaloes, confirming their susceptibility to infection and their possible role as host/reservoirs of BoHV-1.

A chimeric baculovirus displaying bovine herpesvirus-1 (BHV-1) glycoprotein D on its surface and their immunological properties

The ability of a recombinant baculovirus containing the ectodomain of the mature sequence of glycoprotein D (gD) fused to the amino-terminus of baculoviral glycoprotein gp64 to display gD on its surface and to serve as an improved immunogen against bovine herpesvirus-1 was tested. The gD-gp64 fusion protein was correctly expressed on the virus particles as revealed by immunomicroscopy assays. Mice immunized with 5 x 10(8) plaque forming units developed antibodies that specifically reacted in an enzyme-linked immunosorbent assay with recombinant gD and whole bovine herpesvirus-1. These antibodies were able to neutralize bovine herpesvirus-1 in vitro, whereas those elicited by a version of gD expressed in Escherichia coli did not. Our data demonstrated that the display on the virion surface of recombinant baculovirus can provide a tool for the development of recombinant vaccines against bovine herpesvirus-1.

The cell-mediated immune response induced by plasmid encoding bovine herpesvirus 1 glycoprotein B is enhanced by plasmid encoding IL-12 when delivered intramuscularly or by gene gun, but not after intradermal injection

Bovine herpesvirus 1 (BHV-1) causes respiratory and genital infections in cattle. Previously we demonstrated that a DNA vaccine encoding a truncated, secreted form of BHV-1 glycoprotein B (tgB) induces cytotoxic T lymphocyte (CTL) responses in C3H mice. In this study we investigated the potential of interleukin 12 (IL-12) to further enhance the CTL response. C3H mice were immunized with a plasmid encoding tgB or with plasmids encoding tgB and murine IL-12. When the plasmid encoding tgB was delivered intramuscularly or epidermally by a gene gun, co-administration with IL-12 plasmid stimulated the synthesis of more IgG2a, the production of higher levels of IFN-γ, and more effective killing by CTLs. In contrast, after intradermal delivery no effect of co-administration of IL-12 encoding plasmid was observed. Further investigation suggested that antigen and IL-12 need to be expressed in the draining lymph nodes, where IL-12 can have a direct effect on T cells.