Search Results (20)

Orthoherpesviridae is a large family of enveloped DNA virus. Among the most significant animal-infecting viruses are bovine alphaherpesvirus 1 (BoAHV1), caprine alphaherpesvirus 1 (CpAHV1) and equid alphaherpesvirus 1 (EqAHV1). Research into new methods to combat herpesvirus infections is ongoing. The aim of this study was to evaluate the antiviral activity of three extracts of the Helleborus bocconei roots against BoAHV1, CpAHV1 and EqAHV1. The roots were air-dried, extracted with methanol (MeOH) and then partitioned between n-butanol (n-BuOH) and water. All three extracts were tested for cytotoxicity on MDBK and RK-13 cells, and for antiviral activity. Two non-cytotoxic concentrations were assessed for their anti-BoAHV1, anti-CpAHV1 and anti-EqAHV1effects. Cells were incubated with the extracts for 72 h under three experimental conditions: pretreatment before viral infection, treatment post virus infection and simultaneous viral infection and treatment with extracts. The n-BuOH extract (BE) at 0.62 µg/mL inhibited the cytopathic effects of all three viruses in the simultaneous assay. Additionally, no cytopathic effect was observed in MDBK cells infected with CpAHV1and treated with 0.31 µg/mL BE post virus infection. Therefore, the BE contains molecules or groups of molecules potentially useful for developing an alternative therapy against herpesvirus (HV) infection. Full article

Bovine alphaherpesvirus 1 (BoHV-1) acute infection leads to latently infected sensory neurons in trigeminal ganglia. During lytic infection, the immediate early expression of infected cell protein 0 (bICP0) and bICP4 is regulated by an immediate early transcription unit 1 (IEtu1) promoter. A separate bICP0 early (E) promoter drives bICP0 as an early viral gene, presumably to sustain high levels during productive infection. Notably, bICP0 protein expression is detected before bICP4 during reactivation from latency, suggesting the bICP0 E promoter drives bICP0 protein expression during the early phases of reactivation from latency. The glucocorticoid receptor (GR) and Krüppel-like factor 4 (KLF4) cooperatively transactivate the bICP0 E promoter despite this promoter lacks a consensus GR response element (GRE). KLF and specificity protein (Sp) family members comprise a “super-family” of transcription factors. Consequently, we hypothesized Sp1 and Sp3 transactivated the bICP0 E promoter. These studies revealed GR and Sp3 or Sp1 cooperatively transactivated bICP0 E promoter activity. KLF4 and Sp3, but not Sp1, had an additive effect on bICP0 E promoter activity. Mutating the consensus Sp1 and CACCC binding sites proximal to the TATA box impaired promoter activity more than the Sp1 sites further upstream from the TATA box. Full article

Bovine alpha-herpesvirus 1 (BoHV-1) is a significant problem for the cattle industry, in part because the virus establishes latency, and stressful stimuli increase the incidence of reactivation from latency. Sensory neurons in trigeminal ganglia and unknown cells in pharyngeal tonsils are important
sites for latency. Reactivation from latency can lead to reproductive problems in pregnant cows, virus transmission to young calves, suppression of immune responses, and bacterial pneumonia. BoHV-1 is also a significant cofactor in bovine respiratory disease (BRD). Stress, as mimicked by the synthetic corticosteroid dexamethasone, reproducibly initiates reactivation from latency. Stress-mediated activation of the glucocorticoid receptor (GR) stimulates viral replication and transactivation of viral promoters that drive the expression of infected cell protein 0 (bICP0) and bICP4. Notably, GR and Krüppel-like factor 15 (KLF15) form a feed-forward transcription loop that cooperatively transactivates immediate early transcription unit 1 (IEtu1 promoter). Two  pioneer transcription factors, GR and KLF4, cooperatively transactivate the bICP0 early promoter. Pioneer transcription factors bind silent viral  heterochromatin, remodel chromatin, and activate gene expression. Thus, we
predict that these novel transcription factors mediate early stages of BoHV-1 reactivation from latency. Full article

Depopulation is frequently employed during outbreaks of high-impact animal diseases. Security breaches in sites managing mortality may jeopardize pathogen control efforts as infected carcasses can serve as an infection source. This study evaluated the viability and nucleic acid detection of veterinary-relevant viruses or their surrogates in decomposing tissues. The used viruses were: Senecavirus A1 (SVA), feline calicivirus (FCV), bovine viral diarrhea virus (BVDV), porcine epidemic diarrhea virus (PEDV), bovine alphaherpesvirus 1 (BoHV-1), and swinepox virus (SwPV). Viruses were spiked in three decomposing tissues (swine bone marrow and spleen, and bovine bone marrow) and maintained for 90 days. Samples were kept under two temperature conditions resembling the average soil temperature in central Oklahoma, US, during the winter and summer (5.5 °C and 29.4 °C). At 5.5 °C, SVA and FCV remained viable over the 90 days of the study, followed by BVDV (75 days), BoHV-1 and SwPV (60 days), and PEDV (10 days). At 29.4 °C, SVA remained viable for 45 days, followed by BVDV and BoHV-1 (14 days). SwPV was viable for 10 days, whereas FCV and PEDV were viable for 5 days. Overall, viral nucleic acid detection was not significantly altered during the study. These findings support decision-making and risk management in sites overseeing animal mortality. Full article

Infectious bovine rhinotracheitis (IBR) and bovine meningoencephalitis are caused by Bovine alphaherpesvirus (BoHV) types 1 and 5, which seriously threaten the global cattle industry. Vaccination to improve immunity is the most direct and effective means to prevent these conditions. Glycoprotein B (gB) is essential for the attachment of both viruses to permissive cells, and is a major target of the host immune system, inducing a strong humoral response. The aim of this study was to evaluate, in a murine model, the immune response of a candidate vaccine formulation composed of a chimeric BoHV-1 and BoHV-5 gB (DgB), expressed in Komagataella phaffii. The chimeric DgB vaccine adjuvanted with Montanide 50 ISA V2 or aluminum hydroxide was administered intramuscularly or subcutaneously. A control group and a group that received a commercial vaccine were inoculated subcutaneously. Higher titers of neutralizing antibodies against BoHV-1, BoHV-5, and a natural BoHV-1/5 recombinant strain were obtained with the oil-based candidate vaccine formulation administered intramuscularly. The results demonstrated that the chimeric DgB conserved important epitopes that were able to stimulate a humoral immune response capable of neutralizing BoHV-1, BoHV-5, and the recombinant strain, suggesting that the vaccine antigen is a promising candidate to be further evaluated in cattle. Full article

European regulations on the control of infectious diseases provide measures to control Bovine alphaherpesvirus 1 (BoHV-1) infection in both cattle and buffalo. Owing to the reported serological cross-reactivity between BoHV-1 and Bubaline alphaherpesvirus 1 (BuHV-1), we hypothesized a new immunization protocol using BoHV-1 gE-deleted marker vaccines could protect water buffalo against BuHV-1. Five water buffaloes devoid of BoHV-1/BuHV-1-neutralizing antibodies were immunized with two commercial BoHV-1 gE-deleted marker vaccines at 0, 30, 210, and 240 post-vaccination days (PVDs). Five additional water buffaloes were used as controls. At 270 PVD (0 post-challenge days (PCDs), all animals were challenged intranasally with wild-type (wt) BuHV-1. The vaccinated animals produced humoral immunity (HI) as early as PVD 30 whereas, in control animals, antibodies were detected on PCD 10. After challenge infection, HI significantly increased in vaccinated animals compared to that in controls. Real-time PCR for gB revealed viral shedding in vaccinated animals from PCDs 2 to 10. In contrast, positive results were observed from PCDs 2 to 15 in the unvaccinated control group. Although the findings indicated the possible protection capabilities of the tested protocol, these findings did not support its protective roles in water buffaloes against wt-BuHV-1. Full article

Bovine Alphaherpesvirus 1 (BoHV-1) is one of the major respiratory pathogens in cattle worldwide. Infection often leads to a compromised host immune response that contributes to the development of the polymicrobial disease known as “bovine respiratory disease”. After an initial transient phase of immunosuppression, cattle recover from the disease. This is due to the development of both innate and adaptive immune responses. With respect to adaptive immunity, both humoral and cell-mediated immunity are required to control infection. Thus, several BoHV-1 vaccines are designed to trigger both branches of the adaptive immune system. In this review, we summarize the current knowledge on cell-mediated immune responses directed against BoHV-1 infection and vaccination. Full article

Bovine alphaherpesvirus 1 (BoHV-1) is a persistent and recurring disease that affects cattle worldwide. It is a major contributor to bovine respiratory disease and reproductive failure in the US. A major complication of BoHV-1 arises from the lifelong latent infection established in the sensory ganglia of the peripheral nervous system following acute infection. Lifelong latency is marked by periodic reactivation from latency that leads to virus transmission and transient immunosuppression. Physiological and environmental stress, along with hormone fluctuations, can drive virus reactivation from latency, allowing the virus to spread rapidly. This review discusses the mechanisms of the latency/reactivation cycle, with particular emphasis on how different hormones directly regulate BoHV-1 gene expression and productive infection. Glucocorticoids, including the synthetic corticosteroid dexamethasone, are major effectors of the stress response. Stress directly regulates BoHV-1 gene expression through multiple pathways, including β-catenin dependent Wnt signaling, and the glucocorticoid receptor. Related type 1 nuclear hormone receptors, the androgen and progesterone receptors, also drive BoHV-1 gene expression and productive infection. These receptors form feed-forward transcription loops with the stress-induced Krüppel-like transcription factors KLF4 and KLF15. Understanding these molecular pathways is critical for developing novel therapeutics designed to block reactivation and reduce virus spread and disease. Full article

Bovine alphaherpesvirus 1 (subtypes 1.1, 1.2a, and 1.2b), type 5 (subtypes 5a, 5b, and 5c), and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity (κ = 0.93; PPV = 95.0%; NPV = 97.3%). The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses. Full article

Three commercially available infectious bovine rhinotracheitis (IBR) live marker vaccines were evaluated for their ability to provide clinical protection to vaccinated calves against wild-type (wt) Bovine alphaherpesvirus-1 (BoHV-1) challenge and their possible effect on wt BoHV-1 latency reactivation following the challenge. On 35 post-vaccination days (PVDs), all animals were challenged with wt BoHV-1. Only the calves in the control group developed severe forms of IBR. The reactivation of latent BoHV-1 was induced by dexamethasone (DMS) treatment on 28 post-challenge days (PCDs). All animals showed IBR clinical signs on three post-DMS treatment days (PDTDs). On PVD 14, all vaccinated animals developed neutralizing antibodies (NAs), whereas in control animals, the NAs appeared post-challenge. The positivity for glycoprotein-B (gB) was detected using real-time polymerase chain reactions in all animals from PCDs 1 to 7. In contrast, the gB-positivity was observed in the immunized calves from PDTDs 3 to 10. Positive expression of gD and gE was observed in nasal swabs of all calves on PDTD 7. These findings suggested that the IBR marker vaccines evaluated in this study protected against wt BoHV-1-induced disease but not against wt BoHV-1-induced latency reactivation, indicating the necessity of developing new products to protect animals from wt BoHV-1-induced latency. Full article

In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet’s agreement coefficient of 0.99 (95% confidence interval (CI): 0.96–1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3–100%) and 97.5% (95% CI: 91.3–99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen’s κ: 0.96, 95% CI: 0.78–1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples. Full article

In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the circ gene is expressed with immediate–early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene (bicp4) for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the bicp22 gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism. Full article

Infectious bovine rhinotracheitis (IBR), caused by bovine alphaherpesvirus 1 (BoHV-1), is an important disease affecting cattle worldwide resulting in great economic losses. Marker vaccines are effective in controlling infectious diseases including IBR, because they allow the discrimination between the natural infection and the vaccination. Therefore, a triple gene deleted strain BoHV-1 gG-/tk-/gE- was developed and evaluated in vivo and in vitro as a marker vaccine. In cell culture, this triple mutant virus showed significantly slower growth kinetics and smaller plaques when compared to wild-type (wt) BoHV-1 and double mutant BoHV-1 gG-/tk- (p < 0.01). On proteomic level, it revealed downregulation of some virulence related proteins including thymidine kinase, glycoproteins G, E, I, and K when compared to the wt. In vitro, the triple mutant virus showed a significantly lower and shorter viral shedding period (p < 0.001) in calves compared to double mutant. Moreover, the immunized calves with triple mutant virus showed protection rates of 64.2% and 68.6% against wt BoHV-1 and wt BoHV-5 challenge, respectively, without reactivation of latency after dexamethasone injection. In conclusion, BoHV-1 gG-/tk-/gE- is a safer marker vaccine against IBR although its immunogenicity in calves was decreased when compared to double mutant virus. Full article

The Madin–Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 10⁷ cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log₁₀TCID₅₀/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine. Full article

Bovine alphaherpesvirus 1 is ubiquitous in cattle populations and is associated with several clinical syndromes, including respiratory disease, genital disease, infertility and abortions. Control of the virus in many parts of the world is achieved primarily through vaccination with either inactivated or live modified viral vaccines. The objective of this study was to evaluate the performance of four commercially available BoHV-1 vaccines commonly used in Central and South America. Animals were divided into eight groups and vaccinated on days 0 and 30. Groups 1 to 4 received two doses of four different BoHV-1 commercial vaccines (named A to D). Groups 5 and 6 received vaccine D plus a vaccine for either Clostridial or Food-and-Mouth-Disease (FMD), respectively. Group 7 received one dose of two different brands of reproductive vaccines. Serum samples were collected from all animals on days 0, 30 and 60 to evaluate neutralizing and isotype-specific (IgG1 and IgG2) antibodies. Of the four commercial vaccines evaluated, only vaccine A induced neutralizing antibodies to titers ≥ 1:8 in 13/15 (86%) of the animals 60 days post-vaccination. Levels of IgG2 antibody increased in all groups, except for group 2 after the first dose of vaccine B. These results show that only vaccine A induced significant and detectable levels of BoHV-1-neutralizing antibodies. The combination of vaccine D with Clostridial or FMD vaccines did not affect neutralizing antibody responses to BoHV-1. The antibody responses of three of the four commercial vaccines analyzed here were lower than admissible by vaccine A. These results may be from vaccination failure, but means to identify the immune signatures predictive of clinical protection against BoHV-1 in cattle should also be considered. Full article