Bovine Herpesvirus 1 Infection Research Articles

Schisandra chinensis inhibits the entry of BoHV-1 by blocking PI3K-Akt pathway and enhances the m6A methylation of gD to inhibit the entry of progeny virus.

Schisandra chinensis, a traditional Chinese medicine known for its antitussive and sedative effects, has shown promise in preventing various viral infections. Bovine herpesvirus-1 (BoHV-1) is an enveloped DNA virus that causes respiratory disease in cattle, leading to significant economic losses in the industry. Because the lack of previous reports on Schisandra chinensis resisting BoHV-1 infection, this study aimed to investigate the specific mechanisms involved. Results from TCID50, qPCR, IFA, and western blot analyses demonstrated that Schisandra chinensis could inhibit BoHV-1 entry into MDBK cells, primarily through its extract Methylgomisin O (Meth O). The specific mechanism involved Meth O blocking BoHV-1 entry into cells via clathrin- and caveolin-mediated endocytosis by suppressing the activation of PI3K-Akt signaling pathway. Additionally, findings from TCID50, qPCR, co-immunoprecipitation and western blot assays revealed that Schisandra chinensis blocked BoHV-1 gD transcription through enhancing m6A methylation of gD after virus entry, thereby hindering gD protein expression and preventing progeny virus entry into cells and ultimately inhibiting BoHV-1 replication. Overall, these results suggest that Schisandra chinensis can resist BoHV-1 infection by targeting the PI3K-Akt signaling pathway and inhibiting gD transcription.

Tegument protein UL3 of bovine herpesvirus 1 suppresses antiviral IFN-I signaling by targeting STING for autophagic degradation

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.

Impact of bovine herpesvirus-1 infection on fertility in dairy cattle

Bovine herpesvirus-1 (BoHV-1) is endemic in the UK dairy herd and can have an important negative impact on fertility. As well as being the cause of infectious pustular vulvovaginitis and infectious balanoposthitis, BoHV-1 can reduce conception rate following introduction of the virus in contaminated semen at the time of artificial insemination. Its ability to cause abortions, particularly in the last trimester, is well-documented and the incidence of abortions can be high following the introduction of infection to naïve herds. The impact of BoHV-1 on herd fertility will depend on the degree of herd immunity, route of infection and strain of BoHV-1. Vaccination has been shown to reduce spread of BoHV-1 within and between herds, and to significantly reduce the risk of abortion. In light of the cost of a single abortion, and the impact of BoHV-1 on milk yield, routine vaccination against BoHV-1 offers a cost-effective control strategy suitable for most UK dairy herds.

Seroprevalence and risk factors associated with bovine herpesvirus 1 (BHV-1) infection in dairy cattle in southern and central Ethiopia.

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.

Development of Matrix-Embedded Bovine Tracheal Organoids to Study the Innate Immune Response against Bovine Respiratory Disease

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle. Bovine herpesvirus-1 (BHV-1) is one of the main culprits of BRD; however, research on BHV-1 is hampered by the lack of suitable models for infection and drug testing. In this study, we established a novel bovine tracheal organoid culture grown in a basement membrane extract type 2 (BME2) matrix and compared it with the air–liquid interface (ALI) culture system. After differentiation, the matrix-embedded organoids developed beating cilia and demonstrated a transcriptomic profile similar to the ALI culture system. The matrix-embedded organoids were also highly susceptible to BHV-1 infection and immune stimulation by Pam2Cys, an immunomodulator, which resulted in robust cytokine production and tracheal antimicrobial peptide mRNA upregulation. However, treatment of bovine tracheal organoid cultures with Pam2Cys was not sufficient to inhibit viral infection or replication, suggesting a role of the non-epithelial cellular microenvironment in vivo.

Characterization of BoHV-4 ORF45.

Bovine herpesvirus 4 (BoHV-4) is a Gammaherpesvirus belonging to the Rhadinovirus genus. The bovine is BoHV-4's natural host, and the African buffalo is BoHV-4's natural reservoir. In any case, BoHV-4 infection is not associated with a specific disease. Genome structure and genes are well-conserved in Gammaherpesvirus, and the orf 45 gene and its product, ORF45, are one of those. BoHV-4 ORF45 has been suggested to be a tegument protein; however, its structure and function have not yet been experimentally characterized. The present study shows that BoHV-4 ORF45, despite its poor homology with other characterized Rhadinovirus ORF45s, is structurally related to Kaposi's sarcoma-associated herpesvirus (KSHV), is a phosphoprotein, and localizes in the host cell nuclei. Through the generation of an ORF45-null mutant BoHV-4 and its pararevertant, it was possible to demonstrate that ORF45 is essential for BoHV-4 lytic replication and is associated with the viral particles, as for the other characterized Rhadinovirus ORF45s. Finally, the impact of BoHV-4 ORF45 on cellular transcriptome was investigated, an aspect poorly explored or not at all for other Gammaherpesvirus. Many cellular transcriptional pathways were found to be altered, mainly those involving p90 ribosomal S6 kinase (RSK) and signal-regulated kinase (ERK) complex (RSK/ERK). It was concluded that BoHV-4 ORF45 has similar characteristics to those of KSHV ORF45, and its unique and incisive impact on the cell transcriptome paves the way for further investigations.

A Qualitative PCR Assay for the Discrimination of Bubaline Herpesvirus 1, Bovine Herpesvirus 1 and Bovine Herpesvirus 5.

Bubaline herpesvirus 1 (BuHV-1), Bovine herpesvirus 1 (BoHV-1) and Bovine herpesvirus 5 (BoHV-5) are classified in the genus Varicellovirus, subfamily Alphaherpesvirinae. BoHV-1 is the causative agent of infectious bovine rhinotracheitis, BoHV-5 induces moderate disease in adult cattle while BuHV-1 has instead been associated with a decline in livestock production of water buffaloes. The aim of this study was to develop a qualitative PCR assay that allows the discrimination of BuHV-1, BoHV-1 and BoHV-5. The alignment of homologous genes identified specific nucleotide sequences of BuHV- 1, BoHV-1 and BoHV-5. The design of the primers and the optimization of the PCR assay were focused on the target sequences located on the portions of gD, gE and gG genes. This assay involved the use of three different PCR end-points: the PCR of a portion of the gD gene identified only the presence of BoHV-1; the PCR of a portion of the gE gene confirmed the presence of both BoHV-5 and BuHV-1; the PCR of a portion of the gG gene discriminated between BoHV-5 and BuHV-1, as the amplification product was observed only for BoHV-5. This qualitative PCR assay allowed the differentiation of BoHV-1 and BoHV-5 infections both in cattle and water buffaloes and heterologous BuHV-1 infections in bovine.

Whole blood transcriptome analysis in dairy calves experimentally challenged with bovine herpesvirus 1 (BoHV-1) and comparison to a bovine respiratory syncytial virus (BRSV) challenge.

Bovine herpesvirus 1 (BoHV-1), is associated with several clinical syndromes in cattle, among which bovine respiratory disease (BRD) is of particular significance. Despite the importance of the disease, there is a lack of information on the molecular response to infection via experimental challenge with BoHV-1. The objective of this study was to investigate the whole-blood transcriptome of dairy calves experimentally challenged with BoHV-1. A secondary objective was to compare the gene expression results between two separate BRD pathogens using data from a similar challenge study with BRSV. Holstein-Friesian calves (mean age (SD) = 149.2 (23.8) days; mean weight (SD) = 174.6 (21.3) kg) were either administered BoHV-1 inoculate (1 × 107/mL × 8.5mL) (n = 12) or were mock challenged with sterile phosphate buffered saline (n = 6). Clinical signs were recorded daily from day (d) -1 to d 6 (post-challenge), and whole blood was collected in Tempus RNA tubes on d six post-challenge for RNA-sequencing. There were 488 differentially expressed (DE) genes (p < 0.05, False Discovery rate (FDR) < 0.10, fold change ≥2) between the two treatments. Enriched KEGG pathways (p < 0.05, FDR <0.05); included Influenza A, Cytokine-cytokine receptor interaction and NOD-like receptor signalling. Significant gene ontology terms (p < 0.05, FDR <0.05) included defence response to virus and inflammatory response. Genes that are highly DE in key pathways are potential therapeutic targets for the treatment of BoHV-1 infection. A comparison to data from a similar study with BRSV identified both similarities and differences in the immune response to differing BRD pathogens.

Cell entry of Bovine herpesvirus-1 through clathrin- and caveolin-mediated endocytosis requires activation of PI3K-Akt-NF-κB and Ras-p38 MAPK pathways as well as the interaction of BoHV-1 gD with cellular receptor nectin-1

Bovine herpesvirus-1 (BoHV-1) can infect all breeds of cattle and cause severe respiratory organs and genital tract diseases. However, the mechanism of BoHV-1 entering the cells remains unclear. In this study, we explored the mechanism of BoHV-1 entering MDBK cells. We found that the entry of BoHV-1 was blocked by NH4Cl and bafilomycin A1, indicating that BoHV-1 entry is dependent on the acidic environment of endosome. Specific inhibitor dynasore and small interfering RNA (siRNA) knockdown of dynamin-2 inhibited BoHV-1 entry, showing that dynamin is required in BoHV-1 entry. The results of specific inhibitor, siRNA knockdown and co-localization indicating clathrin- and caveolin- mediated endocytosis play a role in BoHV-1 entry. BoHV-1 infection was not affected by EIPA which is a specific inhibitor of macropinocytosis. In addition, we found that BoHV-1 triggered PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways to induce clathrin-mediated and caveolin-mediated endocytosis at the early stage of BoHV-1 infection. BoHV-1 binding was sufficient to activate the endocytic signaling pathways and promote viral entry. These two signaling pathways were activated by transfection of viral gD protein, and were inhibited by deletion of viral gD protein and the siRNA knockdown of cellular receptor nectin-1. The results of co-localization indicating the entered BoHV-1 is traced to late endosomes via early endosomes. Our results suggested the interaction of viral gD protein and cellular receptor nectin-1 triggered the PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways and induced clathrin-mediated and caveolin-mediated endocytosis to promote BoHV-1 entry into MDBK cells at the early stage of BoHV-1 infection.

Phospholipase C-γ1 potentially facilitates subcellular localization of activated β-catenin, p-β-catenin(S552), during bovine herpesvirus 1 productive infection in MDBK cells

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).

A neutralizing monoclonal antibody-based blocking ELISA to detect bovine herpesvirus 1 and vaccination efficacy.

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 (IC50) of 37.82ng/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 (R2 = 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 developedbased on mAb 3F8 against BoHV-1 gD. • It can replace the labor-intensive and time-consuming viral neutralizing tests.

Molecular Investigation of Bovine Viral Diarrhea Virus, Bovine Herpes Virus-1 and Bovine Herpes Virus-4 Infections in Abortion Cases of Cattle in Van District, Turkey

Abortions, fetal mummification, calf anomalies, and infertility problems constitute most of the reproductive problems in cattle. Viruses play a significant role in the cause of these cases. In cattle, these agents are known as primary abortion agents and the most common of these agents are Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus Type 1 (BoHV-1), and Bovine Herpes Virus Type 4 (BoHV-4). The objective of this research is to determine the potential role of BVDV, BoHV-1, and BoHV-4 as viral abortion agents in cattle housed in the Van district. For this, a total of 115 animal specimens (blood, serum, vaginal swab, vaginal fluid discharge, nasal swab, and abortion material) from 100 abortion, early embryonic deaths, and infertility cases in cattle over the age of 2-5 years old were collected. All samples for detection of BVDV, BoHV-1 and BoHV-4 genomes were tested by the Polymerase Chain Reaction (PCR) technique using specific primers encoding Panpesti 5'-UTR, Glycoprotein C (gC) and Glycoprotein B (gB) genes, respectively. Result out of the samples tested, 41.73% were positive for BVDV and all samples were negative for BoHV-1 and BoHV-4. In conclusion, the presence of BVDV in cattle in the Van region and its role in the occurrence of abortion cases was emphasized for the first time. It is necessary to the consideration of viral abortions and determine the etiology of abortion cases and genital system problems. According to this, we need to focus on the detection of persistently infected (PI) animals for prevention and control of infection and the most effective way of vaccinating susceptible populations.

Stress Triggers Expression of Bovine Herpesvirus 1 Infected Cell Protein 4 (bICP4) RNA during Early Stages of Reactivation from Latency in Pharyngeal Tonsil.

Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, establishes lifelong latency in sensory neurons within trigeminal ganglia (TG) after acute infection. The BoHV-1 latency-reactivation cycle, like other alphaherpesvirinae subfamily members, is essential for viral persistence and transmission. Notably, cells within pharyngeal tonsil (PT) also support a quiescent or latent BoHV-1 infection. The synthetic corticosteroid dexamethasone, which mimics the effects of stress, consistently induces BoHV-1 reactivation from latency allowing early stages of viral reactivation to be examined in the natural host. Based on previous studies, we hypothesized that stress-induced cellular factors trigger expression of key viral transcriptional regulatory genes. To explore this hypothesis, RNA-sequencing studies compared viral gene expression in PT during early stages of dexamethasone-induced reactivation from latency. Strikingly, RNA encoding infected cell protein 4 (bICP4), which is translated into an essential viral transcriptional regulatory protein, was detected 30 min after dexamethasone treatment. Ninety minutes after dexamethasone treatment bICP4 and, to a lesser extent, bICP0 RNA were detected in PT. All lytic cycle viral transcripts were detected within 3 h after dexamethasone treatment. Surprisingly, the latency related (LR) gene, the only viral gene abundantly expressed in latently infected TG neurons, was not detected in PT during latency. In TG neurons, bICP0 and the viral tegument protein VP16 are expressed before bICP4 during reactivation, suggesting distinct viral regulatory genes mediate reactivation from latency in PT versus TG neurons. Finally, these studies confirm PT is a biologically relevant site for BoHV-1 latency, reactivation from latency, and virus transmission. IMPORTANCE BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. RNA-sequencing studies revealed the viral infected cell protein 4 (bICP4) RNA was expressed in PT of latently infected calves within 30 min after dexamethasone was used to initiate reactivation. As expected, bICP4 RNA was not detected during latency. All lytic cycle viral genes were expressed within 3 h after dexamethasone treatment. Conversely, bICP0 and the viral tegument protein VP16 are expressed prior to bICP4 in trigeminal ganglionic neurons during reactivation. The viral latency related gene, which is abundantly expressed in latently infected neurons, was not abundantly expressed in PT during latency. These studies provide new evidence PT is a biologically relevant site for BoHV-1 latency and reactivation. Finally, we predict other alphaherpesvirinae subfamily members utilize PT as a site for latency and reactivation.

Mannheimia haemolytica Negatively Affects Bovine Herpesvirus Type 1.1 Replication Capacity In Vitro.

Bovine Respiratory Disease (BRD) is a multifactorial condition affecting cattle worldwide resulting in high rates of morbidity and mortality. The disease can be triggered by Bovine Herpesvirus-1 (BoHV-1) infection, stress, and the subsequent proliferation and lung colonization by commensal bacteria such as Mannheimia haemolytica, ultimately inducing severe pneumonic inflammation. Due to its polymicrobial nature, the study of BRD microbes requires co-infection models. While several past studies have mostly focused on the effects of co-infection on host gene expression, we focused on the relationship between BRD pathogens during co-infection, specifically on M. haemolytica's effect on BoHV-1 replication. This study shows that M. haemolytica negatively impacts BoHV-1 replication in a dose-dependent manner in different in vitro models. The negative effect was observed at very low bacterial doses while increasing the viral dose counteracted this effect. Viral suppression was also dependent on the time at which each microbe was introduced to the cell culture. While acidification of the culture medium did not grossly affect cell viability, it significantly inhibited viral replication. We conclude that M. haemolytica and BoHV-1 interaction is dose and time-sensitive, wherein M. haemolytica proliferation induces significant viral suppression when the viral replication program is not fully established.

Sero-survey of bovine herpes virus-1 in dromedary camels and associated risk factors

Infectious bovine rhinotracheitis (IBR) is a major animal health hazard in many countries throughout the world, caused by bovine herpesvirus-1 (BoHV-1). The study’s goal was to evaluate the prevalence of BoHV-1 seropositivity among dromedary camels in three governorates in northern Egypt, as well as to identify risk variables related with BoHV-1 seropositivity. A total of 321 blood samples were collected randomly from dromedary camels living in the selected governorates and examined for presence of BoHV-1 antibody using ELISA test. The overall seroprevalence of BoHV-1 among examined camels was 5.92% (95%CI: 3.82–9.06). Univariable analysis confirmed that the significant association (P < 0.05) between sex, history of abortion, contact with small ruminants and herd size and BoHV-1 seropositivity. Using multiple logistic regression analysis, the following risk factors were identified to be related with the presence of BoHV-1 infection: sex (OR = 2.54, 95%CI: 0.63–10.22), history of abortion (OR = 4.16, 95%CI: 1.30–13.27), contact with small ruminants (OR = 5.61, 95%CI: 1.67–18.80) and large herd size (OR = 10.52, 95%CI: 2.46–44.91). This study estimated the disease’s seroprevalence in Egyptian dromedary camels, implying that camels could act as a BoHV-1 reservoir for transmission to other species.

Clinical status and endoscopy of the upper respiratory tract of dairy calves infected with Bovine viral diarrhea virus 2 and Bovine herpes virus 1 after vaccination and trace minerals injection

The objective was to compare clinical protection [evaluated through health scoring, endoscopy score of the upper respiratory tract (URT-ES), leukocyte count, viremia, and virus shedding in nasal secretions] following Bovine viral diarrhea virus 2 (BVDV2) and Bovine herpes virus 1 (BHV1) challenge among calves submitted to modified-live virus (MLV) booster vaccination (either intranasal or subcutaneous) concurrent with injectable trace minerals (ITM) or saline. Forty-eight dairy calves received an MLV intranasal (IN) vaccine containing BHV1, BRSV, and BPI3V and subcutaneous (SC) ITM (Se, Cu, Zn & Mn; ITM, n = 24) or saline (SAL, n = 24). Ten weeks later, calves received a second dose of ITM, or saline, according to previous groups and were randomly assigned to receive the same IN vaccine [ITM-IN (n = 12), SAL-IN (n = 12)] or a SC MLV vaccine containing BHV1, BRSV, BPI3V, BVDV1 & 2 [ITM-SC (n = 12), SAL-SC (n = 12)]. Additionally, 12 calves did not receive vaccine or treatment and served as a control group (UNVAC, n = 12). Forty-nine days after booster, calves were challenged with BVDV2; and seven days later with BHV1. Health scores indicated disease in UNVAC on days 6, 10 and 12 compared to the vaccinated groups. Unvaccinated calves had the highest URT-ES after BHV1 challenge. Calves that received SC booster had lower URT-ES after BHV1 challenge than UNVAC calves. Calves in ITM-IN had significantly lower URT-ES after BHV1 infection than SAL-IN and UNVAC calves. In conclusion, IN or SC MLV vaccination was similarly effective in protecting calves from BVDV2 + BHV1 challenges (reducing clinical and endoscopy scores, preventing leukopenia, and viremia), compared to unvaccinated calves. Endoscopic evaluation of the URT allowed visualization of the inflammation and damage at multiple depths in the URT caused by a serial BVDV2 + BHV1 challenge. Calves that received SC vaccination had significantly lower URT-ES after BHV1 challenge than the UNVAC calves. Administration of ITM concurrent with IN vaccination was associated with reduced URT inflammation after BVDV2 + BHV1 challenge.

DNA Damage Response Differentially Affects BoHV-1 Gene Transcription in Cell Type-Dependent Manners.

Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, is also a promising oncolytic virus. Recent studies have demonstrated that the virus infection induces DNA damage and DNA damage response (DDR), potentially accounting for virus infection-induced cell death and oncolytic effects. However, whether the global DDR network affects BoHV-1 productive infection remains to be elucidated. In this study, we show that global DDR induced by ultraviolet (UV) irradiation prior to BoHV-1 infection differentially affected transcription of immediate early (IE) genes, such as infected cell protein 0 (bICP0) and bICP22, in a cell-type-dependent manner. In addition, UV-induced DDR may affect the stabilization of viral protein levels, such as glycoprotein C (gC) and gD, because the variation in mRNA levels of gC and gD as a consequence of UV treatment were not in line with the variation in individual protein levels. The virus productive infection also affects UV-primed DDR signaling, as demonstrated by the alteration of phosphorylated histone H2AX (γH2AX) protein levels and γH2AX formation following virus infection. Taken together, for the first time, we evidenced the interplay between UV-primed global DDR and BoHV-1 productive infection. UV-primed global DDR differentially modulates the transcription of virus genes and stabilization of virus protein. Vice versa, the virus infection may affect UV-primed DDR signaling.

Comparative transcriptome analysis of MDBK cells reveals that BoIFN-γ augmented host immune responses to bovine herpesvirus 1 infection.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle. Ιnterferon-gamma (IFN-γ) is a pleiotropic cytokine with antiviral activity that modulates the innate and adaptive immune responses. In this study, we prepared high-purity bovine interferon gamma (BoIFN-γ) dimer protein using prokaryotic expression system and affinity chromatography. We subsequently investigated the effect of BoIFN-γ on BoHV-1 infection in Madin-Darby bovine kidney (MDBK) cells. The results showed that BoIFN-γ pre-treament not only decreased the production of BoHV-1 but also reduced the cytopathic effect of the virus. Differential gene expression profiles of BoHV-1 infected MDBK cells were then analysed through high-throughput RNA sequencing. The data showed that BoIFN-γ pre-treatment reduced lipid metabolism disorder and DNA damage caused by BoHV-1 infection. Furthermore, BoIFN-γ treatment upregulated the transcription of interferon regulatory transcription factors (IRF1 and GBP5) and interferon-stimulated genes (ISGs) of MDBK cells. Additionally, BoIFN-γ promotes expression of cellular protein involved in complement activation and coagulation cascades response as well as antigen processing and presentation process, while BoHV-1 infection dramatically downregulates transcription of these immune components including C3, C1r, C1s, PLAT, ITGB2, PROCR, BoLA, CD74, B2M, PA28, BoLA-DRA, and TAPBP. Collectively, our findings revealed that BoIFN-γ pre-treatment can improve host resistance to BoHV-1 infection and regulate transcription or expression of host protein associated with cellular metabolism and innate immune response. This provides insights into the development of prophylactic agents for prevention and control of BoHV-1 infection.

A Cross-sectional study on the transmission dynamics of Bovine Herpesvirus-1 infection in the farms located in the Thrace Region of Turkey

In this study, detection of BHV-1 infections in cattle by using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) and investigation of the risk factors associated with the spread of the virus within the herd were aimed. Three dairy farms were selected in the Thrace district, Marmara region, Turkey, and two visits to these farms. A questionnaire was prepared for the farmers. On the first visit, 4 (14.81%) of 27 animals on Farm 1, 6 (28.57%) of 21 animals in farm 2, and 3 (7.31%) of 41 animals in Farm 3 were found to be seropositive for BHV-1. On the second visit, 6 (25%) of 24 animals in Farm 1, 6 (35.29%) of 17 animals in Farm 2, and 6 (20%) of 30 animals in Farm 3 were found to be seropositive for BHV-1. Thirteen (14.6%) of 89 blood samples were seropositive at the first sampling and 18 (25.35%) of 71 blood samples in the second sampling. Two calves belonging to 7 cows that were found to be seropositive at the first visit, and the calves of four of 10 mothers found to be seropositive at the second visit were also found seropositive. BHV-1 DNA was detected in only 3 of the 43 milk samples taken during the first and second visits (2 on the first visit, one on the second visit), and 13 of 160 nasal swabs (8 on the first visit, five on the second visit). All the cows with three milk samples whose BHV-1 DNA was detected by PCR were determined as seropositive. Three of the eight cattle with BHV-1 DNA detected in nasal swabs on the first visit, and 4 of the five cattle with BHV-1 DNA detected in nasal swabs on the second visit were detected seropositive by ELISA. The statistical analysis has shown that the association between age, sex, and BHV-1 seropositivity was statistically significant. The BHV-1 seroprevalence was increased in animals on the second visit in all farms. The preventive measurements need to be applied in Turkey to control BHV-1 infections in cattle.

DDIT3 antagonizes innate immune response to promote bovine alphaherpesvirus 1 replication via the DDIT3-SQSTM1-STING pathway

ABSTRACT DNA damage-inducible transcript 3 (DDIT3), a transcription factor, is typically involved in virus replication control. We are the first to report that DDIT3 promotes the replication of bovine viral diarrhea virus, an RNA virus, by inhibiting innate immunity. However, whether the DDIT3 gene participates in DNA virus replication by regulating innate immunity remains unclear. This study reported that DDIT3 suppressed the innate immune response caused by DNA viruses to promote bovine herpesvirus 1 (BoHV-1) replication. After BoHV-1 infection of Madin-Darby bovine kidney (MDBK) cells, upregulated expression of DDIT3 induced SQSTM1-mediated autophagy and promoted STING degradation. Overexpression of the SQSTM1 protein effectively reduced STING protein levels, whereas SQSTM1 knockdown increased STING protein levels. Coimmunoprecipitation experiments and confocal laser scanning microscopy revealed that the SQSTM1 protein interacts with and colocalizes with STING. Knockdown of SQSTM1 expression in DDIT3-overexpressing cell lines restored STING protein levels. Moreover, a dual-luciferase reporter assay revealed that DDIT3 directly binds to the bovine SQSTM1 promoter and induces SQSTM1 transcription. Overexpression of SQSTM1 promoted BoHV-1 replication by inhibiting IFN-β and IFN-stimulated genes (ISGs) production; silencing of SQSTM1 promoted the expression of IFN-β and ISGs to inhibit BoHV-1 replication. In conclusion, DDIT3 targets STING via SQSTM1-mediated autophagy to promote BoHV-1 replication. These results suggest a novel mechanism by which DDIT3 regulates DNA virus replication by targeting innate immunity. DDIT3 antagonizes the innate immune response to promote bovine alphaherpesvirus 1 replication via the DDIT3-SQSTM1-STING pathway.