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Background: Bovine herpesvirus 1 (BoHV-1) is a major pathogen that affects the global bovine population, primarily inducing respiratory and reproductive disorders. Its ability to establish latent infections in neuronal cells and to reactivate under certain conditions poses a continual threat to uninfected hosts. In this study, we aimed to analyze the replication characteristics of BoHV-1 in neuronal cells, as well as the effects of viral replication on host cell immunity and physiology., Methods: Using the Neuro-2a neuronal-origin cell line as a model, we explored the dynamics of BoHV-1 replication and analyzed differential gene expression profiles post-BoHV-1 infection using high-throughput RNA sequencing., Results: BoHV-1 demonstrated restricted replication in Neuro-2a cells. BoHV-1 induced apoptotic pathways and enhanced the transcription of interferon-stimulated genes and interferon regulatory factors while suppressing the complement cascade in Neuro-2a cells., Conclusions: Different from BoHV-1 infection in other non-highly differentiated somatic cells result in viral dominance, BoHV-1 regulated the innate immune response in neuronal cells formed a "virus-nerve cell" relative equilibrium state, which may account for the restricted replication of BoHV-1 in neuronal cells, leading to a latent infection. These findings provide a foundation for further research into the mechanism underlying BoHV-1-induced latent infection in nerve cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bovine herpesvirus type 1 (BoHV-1) causes severe diseases in bovine species and great economic burden to the cattle industry worldwide. Due to its complex life cycle, many host factors that affect BoHV-1 replication remain to be explored. To understand the possible roles that the Oct1 cellular protein could play in this process, we first created Oct1-deficient MDBK cells using CRISPR/Cas9-mediated genome editing. Upon infection, the absence of Oct1 in MDBK cells significantly impacted BoHV-1 replication, a phenotype rescued by over-expressing the wild-type Oct1 protein in the deficient cells. We further found that the expression of all three classes of temporal genes, including essential and non-essential viral genes, were significantly reduced in Oct1 knockout MDBK cells, following both high and low multiplicity of infection. In summary, our findings confirm that the bovine Oct1 protein acts as a pro-viral factor for BoHV-1 replication by promoting its viral gene transcription in MDBK cells.

Bovine herpesvirus type 1 (BoHV-1) establishes lifelong latency in trigeminal ganglionic (TG) neurons following intranasal and ocular infection in cattle. Periodically, the latent virus reactivates in the TG due to stress and is transported anterogradely to nerve endings in the nasal epithelium, where the virus replicates and sheds. Consequently, BoHV-1 is transmitted to susceptible animals and maintained in the cattle population. Modified live BoHV-1 vaccine strains (BoHV-1 MLV) also have a similar latency reactivation. Therefore, they circulate and are maintained in cattle herds. Additionally, they can regain virulence and cause vaccine outbreaks because they mutate and recombine with other circulating field wild-type (wt) strains. Recently, we constructed a BoHV-1 quadruple mutant virus (BoHV-1qmv) that lacks immune evasive properties due to UL49.5 and glycoprotein G (gG) deletions. In addition, it also lacks the gE cytoplasmic tail (gE CT) and Us9 gene sequences designed to make it safe, increase its vaccine efficacy against BoHV-1, and restrict its anterograde neuronal transport noted above. Further, we engineered the BoHV-1qmv-vector to serve as a subunit vaccine against the Rift Valley fever virus (BoHV-1qmv Sub-RVFV) (doi: 10.3390/v15112183). In this study, we determined the latency reactivation and nasal virus shedding properties of BoHV-1qmv (vector) and BoHV-1qmv-vectored subunit RVFV (BoHV-1qmv sub-RVFV) vaccine virus in calves in comparison to the BoHV-1 wild-type (wt) following intranasal inoculation. The real-time PCR results showed that BoHV-1 wt- but not the BoHV-1qmv vector- and BoHV-1qmv Sub-RVFV-inoculated calves shed virus in the nose following dexamethasone-induced latency reactivation; however, like the BoHV-1 wt, both the BoHV-1qmv vector and BoHV-1qmv Sub-RVFV viruses established latency, were reactivated, and replicated in the TG neurons. These results are consistent with the anterograde neurotransport function of the gE CT and Us9 sequences, which are deleted in the BoHV-1qmv and BoHV-1qmv Sub-RVFV.

Background: Widely regarded as one of the chief causes of diseases in cattle population, bovine herpesvirus-1 (BoHV-1) has the potential to infect sheep and goat, making them potential reservoirs or hosts for this virus. Thus, preventive measures against BoHV-1 in cattle should not overlook the ability of this virus to infect other animals., Aims: Therefore, the focal point of this study was to ascertain the seroprevalence of BoHV-1 in 300 healthy goats, the relationship between host and the environmental determinants of infection, and the contributing role of goats in the epidemiology of the BoHV-1., Materials & Methods: In order to pinpoint the existing antibodies to BoHV-1, the obtained sera were analyzed by Virus Neutralization test., Results: According to this test, the seroprevalence of BoHV-1 appeared to be 64.33% in southwestern Iran. What logistic regression disclosed was that the odds ratio between age and infection with BoHV-1 was 0.83 (p = 0.01), representing a decrease of 17% as goats grew one year older. In addition, females manifested a higher relative frequency of infection compared to males, with the odds of infection in female goats being registered at 1.88, compared to those in males (p = 0.2). Moreover, contrasted with goats lacking any history of abortion, those with a history of abortion featured 1.1 as the odds ratio (p = 0.87). The seroprevalence in Hendijan, Ahvaz, Shushtar and Dasht e Azadegan was detected to stand at 73.24, 71.30, 55.56 and 47.06 percent, respectively, with 6% of fluctuation in the infection rates being attributed to various geographical locations under the scrutiny of this study (p = 0.003)., Discussion and Conclusion: Having attested the marked seroprevalence of BoHV-1, the definitive role of goats in the epidemiology of this virus as a secondary host or reservoir was confirmed by the present study, necessitating the strict monitoring of BoHV-1 in goats by animal health authorities in areas where BoHV-1 abounds in cattle., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Bovine alphaherpesvirus 1 (BoHV-1) infection causes respiratory tract disorders and immune suppression and may induce bacterial pneumonia. BoHV-1 establishes lifelong latency in sensory neurons after acute infection. Reactivation from latency consistently occurs following stress or intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators necessary for productive infection and reactivation from latency. The IEtu1 promoter contains two glucocorticoid receptor (GR) responsive elements (GREs) that are transactivated by activated GR. GC-rich motifs, including consensus binding sites for specificity protein 1 (Sp1), are in the IEtu1 promoter sequences. E2F family members bind a consensus sequence (TTTCCCGC) and certain specificity protein 1 (Sp1) sites. Consequently, we hypothesized that certain E2F family members activate IEtu1 promoter activity. DEX treatment of latently infected calves increased the number of E2F2 + TG neurons. GR and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivate a 436-bp cis -regulatory module in the IEtu1 promoter that contains both GREs. A luciferase reporter construct containing a 222-bp fragment downstream of the GREs was transactivated by E2F2 unless two adjacent Sp1 binding sites were mutated. Chromatin immunoprecipitation studies revealed that E2F2 occupied IEtu1 promoter sequences when the BoHV-1 genome was transfected into mouse neuroblastoma (Neuro-2A) or monkey kidney (CV-1) cells. In summary, these findings revealed that GR and E2F2 cooperatively transactivate IEtu1 promoter activity, which is predicted to influence the early stages of BoHV-1 reactivation from latency., Importance: Bovine alpha-herpesvirus 1 (BoHV-1) acute infection in cattle leads to establishment of latency in sensory neurons in the trigeminal ganglia (TG). A synthetic corticosteroid dexamethasone consistently initiates BoHV-1 reactivation in latently infected calves. The BoHV-1 immediate early transcription unit 1 (IEtu1) promoter regulates expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators. Hence, the IEtu1 promoter must be activated for the reactivation to occur. The number of TG neurons expressing E2F2, a transcription factor and cell cycle regulator, increased during early stages of reactivation from latency. The glucocorticoid receptor (GR) and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivated a 436-bp cis -regulatory module (CRM) in the IEtu1 promoter that contains two GR responsive elements. Chromatin immunoprecipitation studies revealed that E2F2 occupies IEtu1 promoter sequences in cultured cells. GR and E2F2 mediate cooperative transactivation of IEtu1 promoter activity, which is predicted to stimulate viral replication following stressful stimuli., Competing Interests: The authors declare no conflict of interest.

Bovine viral diarrhea virus (BVDV) is considered to be the most common agent of severe diarrhea in cattle worldwide, causing fever, diarrhea, ulcers, and abortion. Bovine herpesvirus 1 (BoHV-1) is also a major bovine respiratory disease agent that spreads worldwide and causes extensive damage to the livestock industry. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method with the advantages of high efficiency, rapidity and sensitivity, which has been widely used in the diagnosis of infectious diseases. A dual RPA assay was developed for the simultaneous detection of BVDV and BoHV-1. The assay was completed at a constant temperature of 37 °C for 30 min. It was highly sensitive and had no cross-reactivity with other common bovine viruses. The detection rate of BVDV RPA in clinical samples (36.67%) was higher than that of PCR (33.33%), the detection rate of BoHV-1 RPA and PCR were equal. Therefore, the established dual RPA assay for BVDV and BoHV-1 could be a potential candidate for use as an immediate diagnostic., (© 2024. The Author(s).)

Bovine herpesvirus 1 (BoHV-1) is a highly contagious pathogen which causes infectious bovine rhinotracheitis in cattle worldwide. Although it has the ability to evade the host's antiviral innate immune response and establish persistent latent infections, the mechanisms are not fully understood, especially the function of the tegument protein to escape innate immunity and participate in viral replication. In this study, we showed that overexpression of tegument protein UL3 facilitates BoHV-1 replication and suppresses the expression of type-I interferon (IFN-I) and IFN-stimulated genes. Then, STING was identified as the target by which UL3 inhibits the IFN-I signaling pathway, and STING was degraded through the UL3-induced autophagy pathway. Furthermore, overexpression of UL3 promotes the expression of the autophagy-related protein ATG101, thereby inducing autophagy. Further study showed that UL3 enhances the interaction between ATG101 and STING, and then the degradation of STING was reversed following ATG101 silencing in UL3-overexpressing cells during BoHV-1 infection. Our research results demonstrate a novel function of UL3 in regulating host's antiviral response and provide a potential mechanism for BoHV-1 immune evasion., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Compare immune responses induced by 2 commercial intranasal (IN) modified-live viral (MLV) vaccines given individually or coadministered and evaluate prevention of infection and lung pathology following bovine herpesvirus-1 (BHV-1) challenge., Animals: 36 male Holstein calves (ages, 5 to 12 days)., Methods: In a randomized complete block design, each calf received an IN injection of either vaccine diluent (Placebo), an MLV vaccine containing bovine herpesvirus-1 (BHV-1; N3), bovine coronavirus vaccine (BC), or both N3 and BC (BC + N3) with a booster 4 weeks later. Nasal secretions and blood were collected weekly. Three weeks after the booster, the calves were challenged with BHV-1, sampled for virus shedding, and euthanized 10 days later to quantify lung pathology. The study period was September 7, 2020, to April 6, 2021., Results: Calves were seropositive for BHV-1 and BC before vaccination. No significant difference in BC-specific serum immunoglobin G and nasal immunoglobin A antibody responses in the BC versus BC + N3 group or BHV-1-specific serum immunoglobin G and nasal immunoglobin A antibody responses in the N3 versus BC + N3 group. Cytokine responses to BHV-1 and BC did not differ among groups. BHV-1 shedding after challenge was significantly reduced in N3 groups versus Placebo and BC. There was a significant reduction in lung pathology in the N3 + BC group versus Placebo., Clinical Relevance: This study provides evidence an MLV vaccine containing BHV-1 and an MLV BC vaccine can be coadministered to neonatal calves without significantly altering immune responses to the 2 viruses or compromising the prevention of BHV-1 respiratory disease. Calves receiving the BC + N3 vaccine had a significant reduction in lung pathology after BHV-1 aerosol challenge.

Objective: To determine the efficacy of primary or booster intranasal vaccination of beef steers on clinical protection and pathogen detection following simultaneous challenge with bovine respiratory syncytial virus and bovine herpes virus 1., Methods: 30 beef steers were randomly allocated to 3 different treatment groups starting at 2 months of age. Group A (n = 10) was administered a single dose of a parenteral modified-live vaccine and was moved to a separate pasture. Groups B (n = 10) and C (10) remained unvaccinated. At 6 months of age, all steers were weaned and transported. Subsequently, groups A and B received a single dose of an intranasal modified-live vaccine vaccine while group C remained unvaccinated. Group C was housed separately until challenge. Two days following vaccination, all steers were challenged with bovine respiratory syncytial virus and bovine herpes virus 1 and housed in a single pen. Clinical and antibody response outcomes and the presence of nasal pathogens were evaluated., Results: The odds of clinical disease were lower in group A compared with group C on day 7 postchallenge; however, antibody responses and pathogen detection were not significantly different between groups before and following viral challenge. All calves remained negative for Histophilus somni and Mycoplasma bovis; however, significantly greater loads of Mannheimia haemolytica and Pasteurella multocida were detected on day 7 postchallenge compared with day -2 prechallenge., Clinical Relevance: Intranasal booster vaccination of beef steers at 6 months of age reduced clinical disease early after viral challenge. Weaning, transport, and viral infection promoted increased detection rates of M haemolytica and P multocida regardless of vaccination status.

There is sufficient evidence that both bovine herpesvirus (BoHV-1) and bubaline herpesvirus (BuHV-1) can overcome the species barrier represented by their respective hosts, cattle and buffalo. Although several studies have focused on the impact of BoHV-1 on buffalo, little is known about the impact of BuHV-1 on cattle. In this work, we evaluated the seroprevalence of BuHV-1 in the cattle population in an area where intensive buffalo farming is highly developed (Campania region, Italy). BuHV-1 seroprevalence of cattle sampled in this study was estimated to be 21.4% using a specific commercial ELISA for the detection of antibodies against glycoprotein E of the virus. Risk factor assessment by univariate analysis revealed a correlation between housing type and higher prevalence. Similarly, cattle housed with buffalo and adult animals had a higher likelihood of being seropositive. BoHV-1 vaccination did not prove to be a protective factor against BuHV-1 exposure. The role of age, grazing, and co-living with buffalo in influencing BuHV-1 exposure was also confirmed by multivariate analysis. All BuHV-1 positive animals were also tested with cross-serum neutralization aimed at evaluating the specific antibody titers against BoHV-1 and BuHV-1. We, therefore, assessed the potential cross-reaction between BoHV-1 and BuHV-1, the co-infection rate, and the agreement of the assays used. This study described the presence of BuHV-1 in the cattle population of the Campania region (Italy) and indicated the requirement to take BuHV-1 into consideration for any measures and control and/or eradication plans to be applied against BoHV-1., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine herpesvirus 1(BoHV-1) is an important bovine pathogen that causes great economic loss to cattle farms worldwide. The virus-productive infection in bovine kidney (MDBK) cells results in ATP depletion. The mechanisms are not well understood. Mitochondrial fatty acid β-oxidation (FAO) is an important energy source in many tissues with high energy demand. Since carnitine palmitoyl-transferase 1 A (CPT1A) is the rate-limiting enzyme of FAO, we investigated the interactions between virus-productive infection and CPT1A signaling. Here, we found that virus-productive infection at the later stage significantly decreased CPT1A protein levels in all the detected cells, including MDBK, A549, and Neuro-2A cells, differentially altered the accumulation of CPT1A proteins in the nucleus and cytosol, and re-localized the protein in the nucleus. Etomoxir (ETO), an irreversible inhibitor of CPT1A, inhibited viral replication and partially interfered with the ability of BoHV-1 to alter CPT1A accumulation in the nucleus but not in the cytosol. Furthermore, ETO consistently reduced RNA levels of two viral regulatory proteins (bICP0 and bICP22) and protein expression of virion-associated proteins during productive infection, further supporting the important roles of CPT1A signaling in BoHV-1 productive infection. These data, for the first time, suggest that CPT1A is potentially involved in BoHV-1 productive infection., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle with a worldwide distribution. It occurs as a subclinical, mild or severe disease. The clinical signs may vary widely with respiratory, genital, ocular and encephalomyelitis form. This cross-sectional study was carried out between May 2019 and March 2020 with the aim to estimate the seroprevalence of bovine herpesvirus 1 (BHV-1) and to identify related potential risk factors in dairy cattle in central and southern Ethiopia. A total of 954 serum samples were obtained from randomly selected dairy cattle in 98 herds. The samples were collected from animals over 6 months old and tested using a BHV-1 antibody blocking enzyme linked immunosorbent assay (b-ELISA). The study showed that the animal- and herd-level seroprevalence of BHV-1 was 30.0 % (95 % CI: 21.7, 39.9) and 75.5 % (95 % CI: 65.9, 83.1), respectively. Multiple logistic regression model demonstrated that adult animals (> 2.5 years) (OR = 2.4, 95 % CI: 1.1, 5.5) had higher seroprevalence of BHV-1 compared to their counterparts (p < 0.05). Cattle in farms using artificial insemination (AI), and both AI and bulls had a 3.9 (95 % CI: 1.2, 13.3) and 5.1 (95 % CI: 1.8, 14.8) odds of being seropositive, respectively, compared to farms using bulls only. Arrangement of animals in a tail-to-tail fashion appeared to be protective against BHV-1 infection (p < 0.05). However, source of the animal was not associated with BHV-1 serostatus (p > 0.05). The animal- and herd-level prevalence recorded in our study confirms that BHV-1 infection is widespread and remains endemic in dairy cattle of central and southern Ethiopia., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Bovine alphaherpesvirus 1 (BoHV-1) is a serious disease with severe negative economic effects on the global cattle sector, especially in Iran., Objective: This cross-sectional study was carried out to examine the seroprevalence and associated risk factors of BoHV-1 infection with progesterone levels and embryo death in 30-day pregnant dairy cattle at Zagros Industrial Dairy Farm in Shahrekord, Iran., Methods: Between December 2017 to February 2018, blood samples were obtained from 60 dairy cow herds. To determine whether BoHV-1 was present, serum samples were examined using the ELISA for serum antibodies. To find progesterone (P4) in blood, the progesterone ELISA test was used., Results: 96.7 % of sera tested positive for BoHV-1 antibodies, according to the findings. Additionally, 60.34 % of blood samples that tested positive had an experience of abortion and significantly more inseminations that resulted in pregnancy, consistent with findings from other studies conducted in Iran and other nations., Conclusions: Since this study is the first to document the risk factor for BoHV-1 infection in Shahrekord, Iran, we could infer that the virus is extensively dispersed in this area., (© 2023 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

BoHV-5 is a worldwide distributed pathogen usually associated with a lethal neurological disease in dairy and beef cattle resulting in important economic losses due to the cattle industry. Using recombinant gD5, we evaluated the long-duration humoral immunity of the recombinant vaccines in a cattle model. Here we report that two doses of intramuscular immunization, particularly with the rgD5ISA vaccine, induce long-lasting antibody responses. Recombinant gD5 antigen elicited tightly mRNA transcription of the Bcl6 and the chemokine receptor CXCR5 which mediate memory B cells and long-lived plasma cells in germinal centers. In addition, using an in-house indirect ELISA we observed higher and earlier responses of rgD5-specific IgG antibody and the upregulation of mRNA transcription of IL2, IL4, IL10, IL15, and IFN-γ in rgD5 vaccinated cattle, indicating a mixed immune response. We further show that rgD5 immunization protects against both BoHV -1 and -5. Our findings indicate that the rgD5-based vaccine represents an effective vaccine strategy to induce an efficient control of herpesviruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bubaline alphaherpesvirus 1 (BuHV-1) is a pathogen of water buffaloes responsible for economic loss worldwide. MicroRNAs (miRNAs) regulate gene expression produced by alphaherpesviruses and hosts. This study aimed at (a) unravelling the ability of BuHV-1 to produce miRNAs, including hv1-miR-B6, hv1-miR-B8, hv1-miR-B9; (b) measuring the host immune-related miRNAs associated to herpesvirus infection, including miR-210-3p, miR-490-3p, miR-17-5p, miR-148a-3p, miR-338-3p, miR-370-3p, by RT-qPCR; (c) identifying candidate markers of infection by receiver-operating characteristic (ROC) curves; (d) exploiting the biological functions by pathway enrichment analyses. Five water buffaloes BuHV-1 and Bovine alphaherpesvirus 1 (BoHV-1) free were immunized against Infectious Bovine Rhinotracheitis (IBR). Five additional water buffaloes served as negative controls. All animals were challenged with a virulent wild-type (wt) BuHV-1 via the intranasal route 120 days after the first vaccination. Nasal swabs were obtained at days (d) 0, 2, 4, 7, 10, 15, 30, and 63 post-challenge (pc). The animals of both groups shed wt BuHV-1 up to d7 pc. Results demonstrated that (a) miRNAs produced by the host and BuHV-1 could be efficiently quantified in the nasal secretion up to d63 and d15 pc, respectively; b) the levels of host and BuHV-1 miRNAs are different between vaccinated and control buffaloes; c) miR-370-3p discriminated vaccinated and control animals; d) host immune-related miRNAs may modulate genes involved in the cell adhesion pathway of the neuronal and immune system. Overall, the present study provides evidence that miRNAs can be detected in nasal secretions of water buffaloes and that their expression is modulated by BuHV-1., (© 2023. The Author(s).)

Bovine gammaherpesvirus 4 (BoHV-4) is a common virus detected in bovine with respiratory disease worldwide. In this study, we identified and characterized a novel BoHV-4 strain, referred as HB-ZJK, in vaginal swabs collected from cattle in China, 2022. The long unique region (LUR) of HB-ZJK is 10,9811 bp in length. It shares 99.17% to 99.38% nucleotide identity to five BoHV-4 strains available in GenBank and the highest similarity was seen with BoHV-4V. test (JN133502.1) strain (99.38%). Mutations, insertions or deletions were observed mainly in HB-ZJK gB (ORF8), TK (ORF21), gH (ORF22), MCP (ORF25), PK (ORF36), gM (ORF39), and gL (ORF47) genes compared to its genomic coordinates. Phylogenetic analyses of gB and TK genes showed that HB-ZJK clustered with China 512 (2019), B6010 (2009), and J4034 (2009) strains, demonstrating that the isolated HB-ZJK belongs to genotype 1. This is the first report that has revealed a comprehensive genome profile of BoHV-4 strain in China. This study will provide foundation for epidemiological investigations of BoHV-4 and contribute to the molecular and pathogenic studies of BoHV-4., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Water buffaloes (Bubalus bubalis) have been introduced in many regions of the world as a source of animal protein. In many instances, bubaline cattle are reared close to or mixed with bovine or zebuine cattle. However, little is known about infectious diseases of bubaline and the interactions that may arise involving the microbiota of those species. Alphaherpesviruses of ruminants (bovine alphaherpesviruses types 1 and 5, BoHV-1, BoHV-5; bubaline alphaherpesvirus 1, BuHV-1) are highly cross-reactive in serological assays performed with bovine or zebuine sera. However, the profile of reactivity of bubaline cattle sera to alphaherpesviruses remains unknown. As such, it is not known which virus strain (or strains) would be most appropriate to be used as the challenge virus in the laboratory in search for alphaherpesvirus-neutralizing antibodies. In this study, the profile of neutralizing antibodies to alphaherpesviruses in bubaline sera was determined against different types/subtypes of bovine and bubaline alphaherpesviruses. Sera (n=339) were screened in a 24-h serum neutralization test (SN) against 100 TCID 50 of each of the challenge viruses. From those, 159 (46.9 %) neutralized at least one of the viruses assayed; 131 (38.6%) sera neutralized the three viral strains used for screening. The viral strain that was neutralized by the largest number of sera was BoHV-5b A663 (149/159; 93.7%). A few sera neutralized only one of the challenge viruses: four sera neutralized BoHV-1 LA only; another neutralized BoHV-5 A663 only and four others neutralized BuHV-1 b6 only. SN testing with two additional strains gave rise to similar results, where maximum sensitivity (defined here as the largest number of sera that neutralized the challenge viruses) was obtained by adding positive results attained with three of the challenge strains. Differences in neutralizing antibody titers were not significant to allow inferences on which would be the most likely virus that induced the antibody responses detected here., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Bovine herpesvirus (BoAHV) types 1 and 5 are closely-related neurotropic alpha-herpesviruses. BoAHV-1 generally causes respiratory and genital disease but can occasionally cause encephalitis. BoAHV-5 is the causative agent of non suppurative meningoencephalitis in calves. During neuroinvasion, both viruses reach the central and peripheral nervous system. While brain alterations are well-described, the changes that occur in the medulla have not been fully detailed. In this work, we integrated and analyzed the virological findings, the microscopic lesions and the changes that occur in the expression of genes related to the innate immunity, cell cycle and apoptosis in the cervical medulla of calves experimentally-infected with BoAHV-1 and BoAHV-5. This will contribute to the understanding of the differential neuropathogenesis of these alpha-herpesviruses of cattle., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest on the research, authorship and publication of the manuscript., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Infections caused by bovine herpesvirus 1 (BoHV-1) remain a serious global issue to the health and welfare of the bovine industry. Monitoring of neutralizing antibodies is essential not only for epidemic diagnosis, but also to assess vaccination efficacy. In this study, we generated a neutralizing monoclonal antibody, termed as 3F8, targeting glycoprotein D (gD) of BoHV-1. This monoclonal antibody could neutralize BoHV-1 with a 50% inhibitory concentration (IC 50 ) of 37.82 ng/mL. Furthermore, 3F8 could inhibit BoHV-1 infection and cell-to-cell spread at the prebinding stage. A blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies against BoHV-1 was then developed based on 3F8 and protein gD generated using a baculovirus expression system. The sensitivity and specificity of the test were estimated to be 94.59% and 93.42%, respectively. A significant correlation (R 2  = 0.9583, p < 0.01) was observed between the results obtained with the blocking ELISA and a virus neutralization test, which suggested that the blocking ELISA could detect neutralizing antibodies against BoHV-1. A serological survey was carried out in the dairy farms in Beijing district using 3F8-based blocking ELISA to monitor the annual neutralization antibody against BoHV-1 during 2012-2020. It revealed that the dairy farms in Beijing were at high risk of BoHV-1 infection during 2012-2017 but were protected since 2018 upon implementation of an immunization program. Our results demonstrated that this assay is suitable for BoHV-1 surveillance and vaccination efficacy in cattle as a replacement for the virus neutralization test. KEY POINTS: • Prevention of BoHV-1 infection requires the monitoring of neutralizing antibodies. • A blocking ELISA for the neutralizing antibody was developed based on mAb 3F8 against BoHV-1 gD. • It can replace the labor-intensive and time-consuming viral neutralizing tests., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bovine herpesvirus 1 (BoHV-1) is a significant risk factor for the bovine respiratory disease complex (BRDC), a severe disease causing great economic losses to the cattle industry worldwide. Previous studies have reported that both phospholipase C-γ1 (PLC-γ1) and β-catenin are activated during BoHV-1 infection for efficient replication. However, the interplay between PLC-γ1 and β-catenin as a consequence of virus infection remains to be elucidated. Here, we reported that PLC-γ1 interacted with β-catenin, which was enhanced following virus infection. PLC-γ1-specific inhibitor, U73122, significantly reduced the mRNA levels of β-catenin in BoHV-1-infected cells; however, the steady-state protein levels were not affected due to the virus infection. Interestingly, the treatment of virus-infected cells with U73122 reduced the accumulation of activated β-catenin [p-β-catenin(S552)] in fractions of the cytoplasmic membrane as that observed with the treatment of methyl-β-cyclodextrin (MβCD), which can disrupt cytoplasmic membrane structure via sequestering cholesterol. Nucleus accumulation of p-β-catenin(S552) was increased following U73122 treatment in virus-infected cells. In addition, the association of p-β-catenin(S552) with cytoplasmic membrane induced by the virus infection was significantly disrupted by the treatment of U73122 and MβCD. These data indicated that the PLC-γ1 signaling is potentially involved in the regulation of β-catenin signaling stimulated by BoHV-1 infection partially via affecting the subcellular localization of p-β-catenin(S552)., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine herpesvirus type 1 (BoHV 1) is a major bovine pathogen spreading worldwide and causing extensive damage to the livestock industry. BoHV causes respiratory, genital, and neurological disorders. A cross-sectional study was performed for the first time to estimate the seroreactivity to BoHV 1 and related risk factors among Iran's central desert dairy cattle. A total of 800 blood samples was randomly collected from 76 unvaccinated herds. Samples were tested with an indirect enzyme-linked immunosorbent assay (ELISA) commercial kit to detect BoHV 1 antibodies. The logistic regression model was used to analyze the data. BoHV 1 seroreactivity at animal and herd levels was 50% and 65%, respectively. Herd size was recognized as a risk factor (OR = 2.65, CI = 1.61-4.37) for seroreactivity to BoHV using GLM (p < 0.05). The high prevalence of BoHV 1 antibodies in the study area indicates the need to implement educational programs on the importance of the disease and design methods to control and prevent virus distribution., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Bovine alphaherpesviruses (BoHV) 1 and 5 share many genetic and structural characteristics; however, they differ in their ability to cause encephalitis. Previous research suggests that this difference might be caused by a differential modulation of apoptosis. In this study, we analyzed the mRNA expression of Bax, Bcl-2, Fas and caspases 3 and 8 in neural tissue sections of BoHV-1 and BoHV-5 experimentally-infected cattle. Overall, Fas and caspase 3 mRNA was up-regulated during BoHV-5 acute infection, latency, and reactivation. Conversely, caspase 3 mRNA levels increased only in the olfactory cortex during BoHV-1 acute infection, and it was down-regulated during reactivation, while Fas was only up-regulated during BoHV-1 acute infection and latency. Moreover, Bax/Bcl-2 ratio was lower than 1 during BoHV-1 acute infection except in the trigeminal ganglion, whereas many brain regions exhibit a ratio higher than 1 during BoHV-5 acute infection and reactivation. In summary, our findings suggest that during acute infection and reactivation, BoHV-5 induces a pro-apoptotic condition that could partially justify its increased ability to cause neurological damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bovine Herpesvirus Type 1 (BoHV-1) infection causes infectious bovine rhinotracheitis and genital disease in cattle, with significant economic and welfare impacts. However, the role of cellular host factors during viral replication remains poorly characterised. A previously performed genome-wide CRISPR knockout screen identified pro- and antiviral host factors acting during BoHV-1 replication. Herein we validate a pro-viral role for a candidate from this screen: the cellular protein tetracopeptide repeat protein 4 (TTC4). We show that TTC4 transcript production is upregulated during BoHV-1 infection. Depletion of TTC4 protein impairs BoHV-1 protein production but does not reduce production of infectious virions, whereas overexpression of exogenous TTC4 results in a significant increase in production of infectious BoHV-1 virions. TTC4 itself is poorly characterized (especially in the context of virus infection), but is a known co-chaperone of heat shock protein 90 (HSP90). HSP90 has a well-characterized pro-viral role during the replication of diverse herpesviruses, and we therefore hypothesized that HSP90 is also pro-viral for BoHV-1. Drug-mediated inhibition of HSP90 using geldanamycin at sub-cytotoxic concentrations inhibited both BoHV-1 protein production and viral genome replication, indicating a pro-viral role for HSP90 during BoHV-1 infection. Our data demonstrates pro-viral roles for both TTC4 and HSP90 during BoHV-1 replication; possibly, interactions between these two proteins are required for optimal BoHV-1 replication, or the two proteins may have independent pro-viral roles., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Bovine gammaherpesvirus 4 (BoHV-4) is ubiquitous in cattle worldwide, and it has been detected in animals exhibiting broad clinical presentations. The virus has been detected in the United States since the 1970s; however, its clinical relevance remains unknown. Here, we determined the complete genome sequences of two contemporary BoHV-4 isolates obtained from respiratory (SD16-38) or reproductive (SD16-49) tract specimens and assessed clinical, virological, and pathological outcomes upon intranasal (IN) inoculation of calves with the respiratory BoHV-4 isolate SD16-38. A slight and transient increase in body temperature was observed in BoHV-4-inoculated calves. Additionally, transient viremia and virus shedding in nasal secretions were observed in all inoculated calves. BoHV-4 DNA was detected by nested PCR in the tonsil and regional lymph nodes (LNs) of calves euthanized on day 5 post-inoculation (pi) and in the lungs of calves euthanized on day 10 pi. Calves euthanized on day 35 pi harbored BoHV-4 DNA in the respiratory tract (turbinates, trachea, lungs), regional lymphoid tissues, and trigeminal ganglia. Interestingly, in situ hybridization revealed the presence of BoHV-4 DNA in nerve bundles surrounding the trigeminal ganglia and retropharyngeal lymph nodes (day 35 pi). No histological changes were observed in the respiratory tract (turbinate, trachea, and lung), lymphoid tissues (tonsil, LNs, thymus, and spleen), or central nervous tissues (olfactory bulb and trigeminal ganglia) sampled throughout the animal studies (days 5, 10, and 35 pi). This study contributes to the understanding of the infection dynamics and tissue distribution of BoHV-4 following IN infection in calves. These results suggest that BoHV-4 SD16-38 used in our study has low pathogenicity in calves upon intranasal inoculation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Type III interferons (IFNs) are components of the innate immunity, with IFN lambda- (λ)3 having the most potent bioactivity in humans. IFN-λ has a predominant role in epithelial cells. However, antiviral function in certain infections of the central nervous system has also been demonstrated. IFN-λ3 expression in neural tissues of cattle has not been investigated. Thus, the aim of this study was to analyze whether an antiviral IFN-λ3 response is mounted after infection with bovine alphaherpesviruses (BoHV-1 and BoHV-5) in vitro, in neuronal-type cells, and in neural tissues from experimentally-infected calves. This study demonstrated that there is a strong IFN-λ3 response early after BoHV-1infection of undifferentiated neuroblastoma cells. During acute BoHV-1 and BoHV-5 infection of calves, low levels of IFN-λ3 expression were detected in the brain, which would favor virus spread within this tissue. Striking differences in the transcriptional levels of IFN-λ3 were observed in trigeminal ganglion, particularly in BoHV-1-acutely- and latently-infected calves. During reactivation, IFN-λ3 expression was down-regulated, which may be a requirement for virus replication and spread. Overall, different patterns of IFN-λ3 expression were detected during BoHV-1 and BoHV-5 infection, particularly during latency., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bovine alphaherpesvirus 1 (BoHV-1) is a pathogen causing respiratory and reproductive clinical signs in cattle. Infected animals may develop rhinotracheitis, vulvovaginitis, balanoposthitis, and abortion. Viral latency is generally established in neuronal ganglia simultaneously to a decrease in both genes or genome expression and viral replication. Under stressful conditions, infection is reactivated leading to viral replication and the manifestation of clinical signs. In this study, we evaluated both viral reactivation and apoptosis in trigeminal ganglia cells as BoHV-1 progressed from the latent to the acute phase of infection after dexamethasone administration in experimentally infected calves. To test ganglia cell death as a consequence of BoHV-1 infection, we stained the BoHV-1 samples with TUNEL after the viral shedding by the calves. RT-qPCR of apoptotic genes was also performed, showing the upregulation of the caspase 8 gene in the trigeminal ganglia from cattle experimentally infected with BoHV-1. These results showed the occurrence of apoptosis in ganglion cells of calves infected by BoHV-1., (© 2021. Sociedade Brasileira de Microbiologia.)

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., (© 2020 Blackwell Verlag GmbH.)

A stochastic quantitative risk assessment model was developed to estimate the annual probability of introduction of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) on 127 dairy farms through indirect contacts. Vehicles transporting calves, cattle to slaughterhouse, dead animals, and mixture of feed, as well as visits by veterinarians and hoof trimmers, farm workers and contacts with neighbors were considered in the model. Data from biosecurity questionnaires of each farm, scientific literature and expert opinion from field veterinarians, animal vehicle drivers, hoof trimmers and personnel from rendering transport companies were used to estimate values for input parameters. Results showed that the annual probability of introducing BVDV or BoHV-1 through indirect contacts was very heterogeneous. The overall distribution of median values for each farm ranged from 0.5 to 14.6% and from 1.0 to 24.9% for BVDV and BoHV-1, respectively. The model identified that providing protective clothing and boots to visits, not allowing the animal vehicle driver to come into contact with animals present on the farm and ensuring that calf vehicles arrived empty, were the measures with the highest impact on the probability of infection for most farms. This model could be a useful tool to show the impact of the measures to farmers and veterinarians, thus increasing their awareness on biosecurity. In addition, it could support decision making on which measures should be prioritized in dairy cattle herds to reduce the probability of introduction of diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples., (Copyright © 2020. Published by Elsevier Ltd.)

Bovine herpesvirus type 1 (BoHV-1) is one of the most critical pathogens in cattle and is prevalent in China. BoHV-1 is divided into two gene types, BoHV-1.1 and 1.2, which are further differentiated into two subtypes, BoHV-1.2a and 1.2b. However, the phylogenetic analysis of BoHV-1 isolates has not been reported in China. To perform a molecular epidemiological survey based on isolates from cattle in China, 102 lung tissue samples of calves under ten months of age with respiratory disease (BRD) that died from 2016 to 2019 in China were used to isolate BoHV-1 with Madin-Darby bovine kidney (MDBK) cells. Part of the BoHV-1 isolates were applied to the phylogenetic analysis based on the region of the glycoprotein C (gC) gene of BoHV-1. Thirty BoHV-1 isolates were obtained, and the gC gene of 13 isolates was amplified by polymerase chain reaction (PCR) methods and sequenced. The result of the phylogenetic analysis according to the 451-nucleotide portion of the gC gene found that all of 13 isolates belonged to the BoHV-1.2b gene subtype, but these isolates had located two different phylogenetic tree branches. The gC gene sequence homology of isolates in group1 was higher with a reference strain of BoHV-1.2b EVI14 up to 98.0-100%, while in group 2, this was higher with reference strain BoHV-1.2b B589 up to 97.8-99.8%. The deduced amino acid sequence of gC from isolates in group 2 had two amino acid mutations with interference strain BoHV-1.2b K22 or BoHV-1.1 COOPER. The cytopathic effects (CPEs) of BoHV-1 isolates in group 2 were ulcered on the centration like a volcano on MDBK cell, and different from traditional CPEs of BoHV-1. Overall, BoHV-1.2b seems to be the primary strain of BoHV-1 in cattle in China and is also a critical cause of BRD. These BoHV-1.2b isolates had significant genetic variations., (Copyright © 2020. Published by Elsevier B.V.)

Background: The dairy sector is one of the leading in agricultural production sectors in the world and the bovine herpesvirus 1 (BoHV-1) is an important pathogen that causes great losses in most production systems. Moreover, BoLA DRB3 immunological gene presents different alleles related to protection against many pathogens., Methods: Serological diagnosis was carried out to determine the BoHV-1 infection and through PCR-RFLP 506 Holstein cows from several municipalities of Antioquia were genotyped for BoLA DRB3.2 gene polymorphisms., Results: Alleles 8, 16, 22, and 24 were the most common out of the 42 alleles found. By indirect ELISA technique, a 58.7% prevalence of BoHV-1 infection in this population was diagnosed and Odd ratios for found alleles were calculated by logistic regression; the only significant association was held for allele 37, which showed that it effects confers susceptibility to infection. On the other hand, by using generalized linear models, a significant association between BoLA DRB3.2 gene and milk and fat yield in primiparous and services per conception in multiparous was found, with the most favorable alleles being 11 and 28 in primiparous and 22 and 28 in multiparous; allele 37 was unfavorable only in primiparous., Conclusion: BoLA DRB3.2 gene polymorphisms have shown high variability and significant effects on Holstein cattle and their performance in production systems in Antioquia are at both sanitary or health and productive levels., Competing Interests: The authors declare that there is no conflict of interest.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes great economic losses in the cattle industry. Herpesvirus infection generally induces endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) in infected cells. However, it is not clear whether ER stress and UPR can be induced by BoHV-1 infection. Here, we found that ER stress induced by BoHV-1 infection could activate all three UPR sensors (the activating transcription factor 6 (ATF6), the inositol-requiring enzyme 1 (IRE1), and the protein kinase RNA-like ER kinase (PERK)) in MDBK cells. During BoHV-1 infection, the ATF6 pathway of UPR did not affect viral replication. However, both knockdown and specific chemical inhibition of PERK attenuated the BoHV-1 proliferation, and chemical inhibition of PERK significantly reduced the viral replication at the post-entry step of the BoHV-1 life cycle. Furthermore, knockdown of IRE1 inhibits BoHV-1 replication, indicating that the IRE1 pathway may promote viral replication. Further study revealed that BoHV-1 replication was enhanced by IRE1 RNase activity inhibition at the stage of virus post-entry in MDBK cells. Furthermore, IRE1 kinase activity inhibition and RNase activity enhancement decrease BoHV1 replication via affecting the virus post-entry step. Our study revealed that BoHV-1 infection activated all three UPR signaling pathways in MDBK cells, and BoHV-1-induced PERK and IRE1 pathways may promote viral replication. This study provides a new perspective for the interactions of BoHV-1 and UPR, which is helpful to further elucidate the mechanism of BoHV-1 pathogenesis.

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.)

Background: Fenbendazole, a dewormer drug, is used widely in the clinical treatment of parasite infections in animals. Recent studies have shown that fenbendazole has substantial effects on tumor growth, immune responses, and inflammatory responses, suggesting that fenbendazole is a pluripotent drug. Nevertheless, the antiviral effects have not been reported. Fenbendazole can disrupt microtubules, which are essential for multiple viruses infections, suggesting that fenbendazole might have antiviral effects., Objectives: This study examined whether fenbendazole could inhibit bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures., Methods: The effects of fenbendazole on viral production, transcription of the immediate early (IE) genes, viron-associated protein expression, and the cellular signaling PLC-γ1/Akt pathway were assessed using distinct methods., Results: Fenbendazole could inhibit BoHV-1 productive infections significantly in MDBK cells in a dose-dependent manner. A time-of-addition assay indicated that fenbendazole affected both the early and late stages in the virus replication cycles. The transcription of IE genes, including BoHV-1 infected cell protein 0 ( bICP0 ), bCP4 , and bICP22 , as well as the synthesis of viron-associated proteins, were disrupted differentially by the fenbendazole treatment. The treatment did not affect the cellular signaling pathway of PLC-γ1/Akt, a known cascade playing important roles in virus infection., Conclusions: Overall, fenbendazole has antiviral effects on BoHV-1 replication., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Korean Society of Veterinary Science.)

A quantitative risk assessment model was developed to estimate the annual probability of introducing bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) at the farm level through animal movements. Data from 2017 official animal movements, biosecurity questionnaires, scientific literature, and expert opinion from field veterinarians were taken into consideration for model input parameters. Purchasing or introducing cattle, rearing replacement heifers offsite, showing cattle at competitions, sharing transport vehicles with other herds, and transporting cattle in vehicles that have not been cleaned and disinfected were considered in the model. The annual probability of introducing BVDV or BoHV-1 through infected animals was very heterogeneous between farms. The median likelihoods of BVDV and BoHV-1introduction were 12 and 9%, respectively. Farms that purchased cattle from within their region (i.e., local movements) and shared transport with other farms had a higher probability for BVDV and BoHV-1 introduction. This model can be a useful tool to support decision-making on biosecurity measures that should be prioritized to reduce the probability of introduction of these 2 diseases in dairy herds., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

A duplex real‑time PCR was developed and validated for the simultaneous detection of Brucella and bovine alphaherpesvirus‑1 (BoHV‑1) from bovine clinical specimens. The bcsp31 gene of Brucella and gB gene of BoHV‑1 were used as targets in the assay. The limit of detection for BoHV‑1 was 0.03 TCID50 of virus and 10 plasmid copies containing the target gene while for Brucella it was 4.1 × 101 CFUs. Intra‑assay and inter‑assay values showed high repeatability and reproducibility of the assay. The diagnostic sensitivity (dsn) and diagnostic specificity (dsp) of the duplex assay were determined by screening 443 clinical specimens and comparing the results with the respective individual assays. The dsn and dsp for detection of Brucella were found to be 95.24% and 95.65%, respectively whereas for BoHV‑1, the dsn (100%) and dsp (99.47%) were slightly higher. The duplex assay had a very good degree of agreement with the respective individual real‑time PCR test {kappa value 0.97 for Brucella and 0.95 for BoHV‑1}. The results of the current study suggest that the duplex assay would be a cost‑effective and time‑saving alternative for the individual real‑time PCR assay for the detection of Brucella and BoHV‑1.

The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are two closely related alphaherpesviruses. BoHV-1 causes several syndromes in cattle, including respiratory disease and sporadic cases of encephalitis, whereas BoHV-5 is responsible for meningoencephalitis in calves. Although both viruses are neurotropic, they differ in their neuropathogenic potential. This review summarizes the findings on the specific mechanisms and pathways known to modulate the pathogenesis of BoHV-1 and BoHV-5, particularly in relation to respiratory and neurological syndromes, which characterize BoHV-1 and BoHV-5 infections, respectively.

Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, can cause abortions in pregnant cows. Progesterone maintains pregnancy and promotes spermiogenesis and testosterone biosynthesis in males: furthermore, progesterone is a neuro-steroid. Recent published studies demonstrated progesterone stimulated the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, and two glucocorticoid receptor response elements within the promoter were required for progesterone mediated transactivation. In this study, we tested whether progesterone induces reactivation from latency in rabbits. As expected, the synthetic corticosteroid dexamethasone consistently induced reactivation from latency in males and females. While progesterone induced reactivation from latency in approximately one-half of male rabbits, virus shedding was sporadic compared to dexamethasone and less efficient in female rabbits. Progesterone significantly increased productive infection in rabbit skin cells, which correlated with stimulating reactivation. These studies suggest progesterone promotes BoHV-1 spread in cattle, in part, by increasing the frequency of reactivation from latency., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE Hind III site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

Bovine alphaherpesvirus 1 (BoHV-1) as a cusative agent for some diseases in cattle infects sheep and goat; and it is believed that these animals may be reservoir host for this virus. Thus, BoHV-1 infection in sheep and goat should be considerd when there is a program for control and eradication of this virus in cattle. Therefore, the aim of this study was to determine the seroprevalence of BoHV-1 in sheep, relationship between host and environmental factors with infection, and the role of sheep in the epidemiology of the BoHV-1. Blood samples were randomly collected from 310 healthy sheep in 6 cities of Khuzestan province (Southwest of Iran) including Ahvaz, Hendijan, Shushtar, Dezful, Masjed Soleyman and Behbahan. Sera were analyzed by virus neutralisation (VN) test for detection antibodies to BoHV-1. According to VN test, apparent and true seroprevalence seroprevalence of BoHV-1 infection was 28.4 % (95%CI: 23.4-33.4%) and 28.4 % (95%CI: 23.3-33.4%), respectively. Logistic regression revealed that the odds of infection between the age was 1.06 (95%CI: 0.9-1.25) (P > 0.05), implying that the odds of infection increased 6 % with rising one year of age. Besides, the relative frequency of infection in males was more than females', and the odds of infection in male sheep was identified to be 1.13 (95%CI: 0.47-2.71) (P > 0.05), compared to that in females. Moreover, in comparison to sheep with no history of abortion, the odds of infection in sheep with a history of abortion was 1.28 (95%CI: 0.57-2.87) (P > 0.05). The seroprevalence in Shushtar, Masjed Soleyman, Dezful, Ahvaz, Hendijan, and Behbahan were found to be 48.3, 46.7, 31.7, 20, 16.7, and 12 percent, respectively and 13.1 of fluctuation in infection can be justified by different geographical locations investigated in this study (P < 0.001). Considering the significant seroprevalence of BoHV-1, present study confirmed the role of sheep in the epidemiology of this virus and control of BoHV-1 in sheep should be considered by animal health authorities in areas where BoHV-1 is prevalent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine alphaherpesvirus 2 (BoHV-2) is the etiologic agent of bovine mammillitis (BM) and pseudo-lumpy skin disease. BM is also important because its clinical presentation can be confused with foot-and-mouth disease (FMD), making it necessary to establish differential diagnoses and perform additional laboratory tests. The objective of this work was to use a validated real-time PCR assay to test for the presence of BoHV-2 in samples from cattle and buffalo with suspected vesicular disease in Brazil. The method could detect the virus at a concentration of 0.5 fg/μL and had 99.4% amplification efficiency, a repeatability error of only 4.1%, and good reproducibility with other reagents. No evidence of BoHV-2 causing vesicular disease in cattle and buffalo was found in this work. This study was able to validate a new methodology for detection of BoHV-2 and evaluate its usefulness for investigating outbreaks of vesicular disease Brazil. The importance of BoHV-2 in cases involving other clinical signs should still be studied using the qPCR developed in this work.

Bovine alphaherpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses. BoHV-5 causes non-suppurative meningoencephalitis in calves. BoHV-1 is associated with several syndromes and, occasionally, can cause encephalitis. Although both viruses are neurotropic and they share similar biological properties, it is unknown why these alphaherpesviruses differ in their ability to cause neurological disease. Neural tissue samples were collected from BoHV-1- and BoHV-5-intranasally inoculated calves during acute infection, latency and reactivation and the levels of cyclins mRNA expression were analyzed by qRT-PCR. Striking differences in the levels of cyclins mRNA were particularly detected in trigeminal ganglion (TG). The expression levels of cyclins in TG during BoHV-5 latency suggest that these viruses utilize different strategies to persist in the host. It is apparent that a relationship between virus loads and cyclin mRNA levels can be established only during acute infection and other factors might be involved in the regulation of cell cycle components during BoHV latency and reactivation. Bovine alphaherpesviruses neuropathogenicity might be influenced by the differential control of cell cycle components by these herpesviruses. This is the first report on BoHV-5 modulation of cyclins expression in neural tissues from its natural host., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Cooper and Los Angeles (LA) strains were the two original respiratory strains of bovine herpesvirus type 1.1 (BoHV-1.1) isolated in the 1950s from cattle with infectious bovine rhinotracheitis. We report the complete genome sequence for the BoHV-1.1 LA strain and compare it to the prototype Cooper strain and six wild-type BoHV-1.1 isolates. A nucleotide sequence divergence of 0.74% was noted across the two complete genomes, caused by 19 single-nucleotide polymorphisms (SNPs) involving 12 genes and insertions/deletions that primarily affected the number of repeats within reiterated repeat regions of the genome. Phylogenetic analysis revealed that Cooper and LA strains are genetically the most ancient strains from which all of the more-recently isolated field strains of BoHV-1.1 evolved.

Bovine herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5) are two alphaherpesviruses that affect cattle with two different syndromes. While BoHV-1 mainly produces respiratory symptoms, BoHV-5 is highly neuropathogenic and responsible for meningoencephalitis in young cattle. The latency-related (LR) gene, which is not conserved between these two herpesviruses, is the only viral gene abundantly expressed in latently infected neurons. The antiapoptotic action of this gene has been demonstrated during acute infection and reactivation from latency and seems to be mainly mediated by a LR protein (ORF-2) which is truncated in amino acid 51 in the case of BoHV-5. In this work, we show that the BoHV-5 LR gene is less efficient at cell survival and apoptosis inhibition in transient as well as in established neuronal cell lines compared to its BoHV-1 homolog. We hypothesize that the BoHV-5 LR gene may have novel functions that are lacking in the BoHV-1 LR gene and that these differences may contribute to its enhanced neuropathogenesis.

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNβ, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNβ, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNβ in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Infection of cattle by bovine herpesvirus 1 (BoHV-1) can culminate in upper respiratory tract disorders, conjunctivitis, or genital disorders. Infection also consistently leads to transient immune-suppression. BoHV-1 is the number one infectious agent in cattle that is associated with abortions in cattle. BoHV-1, as other α-herpesvirinae subfamily members, establishes latency in sensory neurons. Stressful stimuli, mimicked by the synthetic corticosteroid dexamethasone, consistently induce reactivation from latency in latently infected calves and rabbits. Increased corticosteroid levels due to stress have a two-pronged effect on reactivation from latency by: (1) directly stimulating viral gene expression and replication, and (2) impairing antiviral immune responses, thus enhancing virus spread and transmission. BoHV-1 encodes several proteins, bICP0, bICP27, gG, UL49.5, and VP8, which interfere with key antiviral innate immune responses in the absence of other viral genes. Furthermore, the ability of BoHV-1 to infect lymphocytes and induce apoptosis, in particular CD4+ T cells, has negative impacts on immune responses during acute infection. BoHV-1 induced immune-suppression can initiate the poly-microbial disorder known as bovine respiratory disease complex, which costs the US cattle industry more than one billion dollars annually. Furthermore, interfering with antiviral responses may promote viral spread to ovaries and the developing fetus, thus enhancing reproductive issues associated with BoHV-1 infection of cows or pregnant cows. The focus of this review is to describe the known mechanisms, direct and indirect, by which BoHV-1 interferes with antiviral immune responses during the course of infection.

Bovine herpesvirus 1 (BoHV-1) is a causative agent of respiratory diseases in cattle, and infection with BoHV-1 can cause reproductive failure. There are few studies regarding infections in natural conditions in the reproductive organs of bovine animals. In this context, this study investigated the presence of BoHV-1 in the uterus, oviducts, and ovarian tissues of naturally infected cows. The three genital structures were evaluated for the presence or absence of BoHV-1 by immunofluorescence assay using confocal scanning laser microscopy. Blood and genital organ samples of 75 cows unvaccinated against BoHV-1 were used. Fragments of uterus, oviduct, and ovarian tissue were processed and analyzed by confocal scanning laser microscopy. Neutralization by antibodies was observed in 54.7% (41/75) of the serum samples tested. BoHV-1 were detected in the uterus of all the seropositive cows. The oviducts contained BoHV-1 in 73.2% of the samples and the ovaries contained BoHV-1 in 58.5% of the samples from seropositive animals. The presence of the virus was not observed in any of the genital organs of seronegative animals. There was no correlation between the antibody titer and the detection of BoHV-1 in positive tissue in the different genital organs or with the number of infected structures per animal. The detection of BoHV-1 in 100% of the uterus samples from seropositive cows suggests that this organ may be a source of infection for the fetus, resulting in abortion. Further studies on the mechanism by which BoHV-1 infects the fetus via the uterine route should be performed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.