Search Results (19)

Here we discuss three veterinary alphaherpesviruses—pseudorabies virus, equid alphaherpesvirus 1, and bovine alphaherpesvirus 1—that were instrumental in uncovering the true extent of transcriptome complexity through long-read RNA sequencing, which earlier short-read approaches could not resolve. We focus on three major transcriptomic features whose discovery and characterization relied heavily on these viral models: (i) widespread transcriptional overlaps that complicate read assignment and may drive transcriptional interference; (ii) diverse transcript isoforms arising from alternative 5′ and 3′ transcript termini, as well as splicing; and (iii) non-coding RNAs clustered near replication origins that illuminate potential replication–transcription interactions on a shared nuclear template. Long-read viromics in these veterinary systems has additionally served as a stringent benchmark for transcript callers and annotation pipelines, because the extreme density of overlaps and co-terminal transcript families exposes errors that often go unnoticed in typical mammalian transcriptomes. This has made veterinary herpesvirus datasets disproportionately influential in shaping best practices for full-length isoform calling, transcript end mapping, and artifact-robust cDNA library handling. We also discuss animal gammaherpesviruses as proxies for human gammaherpesviruses, allowing experimental investigation of viral programs difficult to study in human infection. Finally, we describe pseudorabies virus applications as a retrograde transneuronal tracer. Full article

Comparative transcriptomics offers a powerful approach to elucidate host–virus interactions across related pathogens, yet systematic evaluations across species-matched cellular systems remain limited. We performed a cross-species RNA sequencing analysis of respective species’ cells infected with three alphaherpesviruses—herpes simplex virus 1 (HSV-1), bovine alphaherpesvirus 1 (BHV-1), and equid alphaherpesvirus 1 (EHV-1)—to dissect conserved and virus-specific transcriptional responses. We show that certain orthologous genes and orthologous pathways are differentially regulated upon infection among the three species like pathways related to translation rRNA processing and TNF-alpha signalling. We find that the earliest sampled timepoint of infection, 2 h post infection (hpi), shows the most commonly enriched pathways among the three species compared to later timepoints. At 6 h and 9 h post infection, BHV-1- and EHV-1 infections have more in common with each other in terms of enriched pathways than with HSV-1 infections. Moreover, we provide a comprehensive analysis of temporal viral gene expression for all three herpesviruses. Together, these findings provide a comparative framework for understanding alphaherpevirus–host interactions and reveal both conserved core responses and species-specific transcriptional signatures. This work establishes a foundation for identifying broadly acting antiviral targets as well as virus-specific vulnerabilities that may inform host-directed therapies and cross-species disease management. Full article

Monitoring infectious diseases is essential for safeguarding equine health and ensuring the sustainability of the horse industry. In 2019, the Royal Veterinary Association of the Netherlands (KNMvD) and Royal GD (GD Animal Health) launched SEIN (Surveillance of Equine Infectious diseases in the Netherlands), a voluntary surveillance system for laboratory-confirmed outbreaks of equid alphaherpesvirus 1 (EHV-1), equid alphaherpesvirus 4 (EHV-4), equine influenza virus (EIV), and Streptococcus equi subsp. equi. This retrospective study analyzed 364 confirmed outbreaks reported through SEIN between June 2019 and April 2023. S. equi was the most commonly reported pathogen overall (64%). Among outbreaks involving respiratory disease, S. equi accounted for 74% of cases, followed by EHV-4 (16%), EIV (6%), and EHV-1 (4%). The geographical distribution of outbreaks covered 80 of the 90 postal code regions (89%), and approximately half of all participating practices generated at least 1 alert. Vaccination data revealed low coverage against EHV-1/4, EIV, and S. equi among both affected horses and premises. Clinical signs overlapped between pathogens, but some were more pathogen-specific, e.g., coughing in EIV, and abscessation in S. equi. The SEIN system provided spatiotemporal information on confirmed outbreaks. These results underscore the importance of quick diagnostics and structured surveillance systems in guiding prevention strategies. Full article

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

Equine herpesvirus 1 (EHV-1), EHV-4, EHV-8, and EHV-9, are classified within the subfamily Alphaherpesvirinae and are recognized as causative agents of respiratory, urogenital, and neurological disorders in horses. These viruses, collectively referred to as αEHVs, exhibits both unique and shared characteristics in terms of host interaction, pathogenesis, epidemiology, and immune evasion, which arise from both the identities and discrepancies among respective genomic homologs. The genomic architecture of αEHVs is similar to other members of the same subfamily, such as well-known HSV-1, VZV, and PRV. However, research on the molecular mechanisms underlying αEHV infection and immune response remains significantly less advanced compared to studies on human, porcine, and bovine herpesviruses. This paper systematically describes the genomic structure, function, and genetic similarities of αEHVs and conducts a comparative analysis of selected αEHVs through pairwise sequence alignments of nucleotides and amino acids. This review offers an extensive synthesis of the current understanding related to the study of αEHVs, highlighting the challenges and potential solutions for future research endeavors. Full article

Equid alphaherpesvirus type 8 (EHV-8) is a contagious pathogen that causes reproductive disorders, respiratory diseases, and viral encephalitis in equids, resulting in significant economic losses for the global horse and donkey industries. Currently, there are no approved antiviral drugs or vaccines available for EHV-8 control. In this study, we investigated the antiviral efficacy of celastrol against EHV-8 both in vitro and in vivo. Our results demonstrated that celastrol significantly inhibited EHV-8 infection in Rabbit kidney (RK-13) and equine dermal cells (NBL-6) in a dose-dependent manner. Mechanistic studies revealed that celastrol interfered with viral replication at multiple stages of the infection cycle. Furthermore, we found that celastrol induced an antiviral interferon response through activation of the Nrf2/HO-1 signaling pathway. Importantly, celastrol treatment significantly reduced EHV-8 replication and ameliorated lung pathology in a mouse model. These findings suggest that celastrol may represent a promising therapeutic agent for the treatment of EHV-8 infections. Full article

Equid alphaherpesvirus 1 (EHV-1) has been linked to the emergence of neurological disorders, with the horse racing industry experiencing significant impacts from outbreaks of equine herpesvirus myeloencephalopathy (EHM). Building robust immune memory before pathogen exposure enables rapid recognition and elimination, preventing infection. This is crucial for effectively managing EHV-1. Removing neuropathogenic factors and immune evasion genes to develop live attenuated vaccines appears to be a successful strategy for EHV-1 vaccines. We created mutant viruses without ORF38 and ORF37/38 and validated their neuropathogenicity and immunogenicity in hamsters. The ∆ORF38 strain caused brain tissue damage at high doses, whereas the ∆ORF37/38 strain did not. Dexamethasone was used to confirm latent herpesvirus infection and reactivation. Dexamethasone injection increased viral DNA load in the brains of hamsters infected with the parental and ∆ORF38 strains, but not in those infected with the ∆ORF37/38 strain. Immunizing hamsters intranasally with the ∆ORF37/38 strain as a live vaccine produced a stronger immune response compared to the ∆ORF38 strain at the same dose. The hamsters demonstrated effective protection against a lethal challenge with the parental strain. This suggests that the deletion of ORF37/38 may effectively inhibit latent viral infection, reduce the neuropathogenicity of EHV-1, and induce a protective immune response. Full article

Background: Equid alphaherpesvirus 1 (EHV-1) is a highly contagious respiratory tract pathogen of horses, and infection may be followed by myeloencephalopathy or abortion. Surveillance and early detection have focused on PCR assays using less tolerated nasal swabs. Here, we assess non-invasive non-contact sampling techniques as surveillance tools in naturally equid gammaherpesvirus 2-shedding horses as surrogates for EHV-1. Methods: Horses were individually housed for 10 h periods on 2 consecutive days. Sampling included nasal swabs, nostril wipes, environmental swabs, droplet-catching devices, and air sampling. The latter was completed via two strategies: a combined air sample collected while going from horse to horse and a collective air sample collected at a stationary central point for 6 h. Samples were screened through quantitative PCR and digital PCR. Results: Nine horses on day 1 and 11 horses on day 2 were positive for EHV-1; overall, 90.9% of the nostril wipes, 81.8% of the environmental surfaces, and 90.9% of the droplet-catching devices were found to be positive. Quantitative analysis showed that the mean DNA copies detection per cm² of nostril wipe sampled concentration (4.3 × 10⁵ per day) was significantly (p < 0.05) comparable to that of nasal swabs (3.6 × 10⁵ per day) followed by environmental swabs (4.3 × 10⁵ per day) and droplet catchers (3.5 × 10³ per day), respectively. Overall, 100% of the air samples collected were positive on both qPCR and dPCR. In individual air samples, a mean concentration of 1.0 × 10⁴ copies of DNA were detected in per m³ air sampled per day, while in the collective air samples, the mean concentration was 1.1 × 10³. Conclusions: Environmental samples look promising in replacing direct contact sampling. Environmental and air sampling could become efficient surveillance tools at equestrian events; however, it needs threshold calculations for minimum detection levels. Full article

(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics in vitro; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligible effect. The sgRNAs targeting ORF30 showed the strongest effect on the suppression of EHV-1 replication, with a reduction in viral genomic copy numbers and infectious progeny virus output. Next-generation sequencing identified variants with deletions in the specific cleavage site of selective sgRNAs. Moreover, we evaluated the combination between different sgRNAs and found that the dual combination of sgRNAs targeting ORF30 and ORF7 significantly suppressed viral replication to lower levels compared to the use of a single sgRNA, suggesting a synergic effect; (4) Conclusion: data demonstrate that sgRNA-guided CRISPR/Cas9 can be used to inhibit EHV-1 replication in vitro, indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1. Full article

As evidenced by sero-epidemiological studies, infections of horses with the tick-borne encephalitis virus (TBEV) occur frequently in TBEV-endemic areas. However, there are only very few reports of clinical cases. A possible underreporting may be due to a variety of diagnostic challenges. In this study, ELISA and neutralization tests were applied to serum samples. Brain tissue samples were investigated for the presence of nucleic acids of TBEV, Equid alphaherpesvirus 1, Borna disease virus 1, West Nile and Usutu viruses, rustrela virus, as well as Eastern, Western, and Venezuelan equine encephalitis viruses with RT-qPCR, RT-PCR, and qPCR, respectively. TBEV-specific amplification products were subjected to Sanger sequencing. In addition, a direct fluorescent antibody test for rabies was performed. Clinical and patho-histological findings are reported. Using specific RT-qPCR and RT-PCR assays, TBEV nucleic acids were demonstrated in brain tissue samples. Sequencing revealed the Western (formerly Central) European subtype of TBEV as the etiological agent. A high titer of TBEV-specific neutralizing antibodies was found in the serum. RNAscope in situ hybridization revealed TBEV RNA confined to neuronal cell bodies and processes. No other pathogens or nucleic acids thereof could be detected. Diagnostic procedures need to be carried out early after the onset of neurological signs to allow for a final etiological diagnosis of acute TBEV infections in horses. Full article

Equid alphaherpesvirus type 8 (EqHV-8) is the causative agent of severe respiratory disease, abortions, and neurological syndromes in equines and has resulted in huge economic losses to the donkey industry. Currently, there exist no therapeutic molecules for controlling EqHV-8 infection. We evaluated the potential antiviral activity of cobalt protoporphyrin (CoPP) against EqHV-8 infection. Our results demonstrated that CoPP inhibited EqHV-8 infection in susceptible cells and mouse models. Furthermore, CoPP blocked the replication of EqHV-8 via HO-1 (heme oxygenase-1) mediated type I interferon (IFN) response. In conclusion, our data suggested that CoPP could serve as a novel potential molecule to develop an effective therapeutic strategy for EqHV-8 prevention and control. Full article

Mitochondria are key cellular organelles responsible for many essential functions, including ATP production, ion homeostasis and apoptosis induction. Recent studies indicate their significant role during viral infection. In the present study, we examined the effects of equine herpesvirus type 1 (EHV-1) infection on the morphology and mitochondrial function in primary murine neurons in vitro. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). The organization of the mitochondrial network during EHV-1 infection was assessed by immunofluorescence. To access mitochondrial function, we analyzed reactive oxygen species (ROS) production, mitophagy, mitochondrial inner-membrane potential, mitochondrial mass, and mitochondrial genes’ expression. Changes in mitochondria morphology during infection suggested importance of their perinuclear localization for EHV-1 replication. Despite these changes, mitochondrial functions were preserved. For all tested EHV-1 strains, the similarities in the increased fold expression were detected only for COX18, Sod2, and Tspo. For non-neuropathogenic strains (Jan-E and Rac-H), we detected mainly changes in the expression of genes related to mitochondrial morphology and transport. The results indicate that mitochondria play an important role during EHV-1 replication in cultured neurons and undergo specific morphological and functional modifications. Full article

Equid alphaherpesvirus-1 (EHV-1) is one of the main pathogens in horses, responsible for respiratory diseases, ocular diseases, abortions, neonatal foal death and neurological complications such as equine herpesvirus myeloencephalopathy (EHM). Current vaccines reduce the excretion and dissemination of the virus and, therefore, the extent of an epizooty. While their efficacy against EHV-1-induced abortion in pregnant mares and the decreased occurrence of an abortion storm in the field have been reported, their potential efficacy against the neurological form of disease remains undocumented. No antiviral treatment against EHV-1 is marketed and recommended to date. This study aimed to measure the protection induced by valganciclovir (VGCV), the prodrug of ganciclovir, in Welsh mountain ponies experimentally infected with an EHV-1 ORF30-C₂₂₅₄ strain. Four ponies were administered VGCV immediately prior to experimental EHV-1 infection, while another four ponies received a placebo. The treatment consisted in 6.5 mg/kg body weight of valganciclovir administered orally three times the first day and twice daily for 13 days. Clinical signs of disease, virus shedding and viraemia were measured for up to 3 weeks. The severity of the cumulative clinical score was significantly reduced in the treated group when compared with the control group. Shedding of infectious EHV-1 was significantly reduced in the treated group when compared with the control group between Day + 1 (D + 1) and D + 12. Viraemia was significantly reduced in the treated group when compared with the control group. Seroconversion was measured in all the ponies included in the study, irrespective of the treatment received. Oral administration of valganciclovir induced no noticeable side effect but reduced clinical signs of disease, infectious virus shedding and viraemia in ponies experimentally infected with the EHV-1 C₂₂₅₄ variant. Full article

Equid alphaherpesvirus 1 (EHV-1) causes respiratory diseases, abortion, and neurological disorders in horses. Recently, the oncolytic potential of this virus and its possible use in anticancer therapy has been reported, but its influence on cytoskeleton was not evaluated yet. In the following study, we have examined disruptions in actin cytoskeleton of glioblastoma multiforme in vitro model—A172 cell line, caused by EHV-1 infection. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). Immunofluorescent labelling, confocal microscopy, real-time cell growth analysis and Oris^TM cell migration assay revealed disturbed migration of A172 cells infected with the EHV-1, probably due to rearrangement of actin cytoskeleton and the absence of cell projections. All tested strains caused disruption of the actin network and general depolymerization of microfilaments. The qPCR results confirmed the effective replication of EHV-1. Thus, we have demonstrated, for the first time, that EHV-1 infection leads to inhibition of proliferation and migration in A172 cells, which might be promising for new immunotherapy treatment. Full article

Viral contamination of edible bivalves is a major food safety issue. We studied the virucidal effect of a cold atmospheric plasma (CAP) source on two virologically different surrogate viruses [a double-stranded DNA virus (Equid alphaherpesvirus 1, EHV-1), and a single-stranded RNA virus (Bovine coronavirus, BCoV)] suspended in Dulbecco’s Modified Eagle’s Medium (DMEM). A 15 min exposure effectuated a statistically significant immediate reduction in intact BCoV viruses by 2.8 (ozone-dominated plasma, “low power”) or 2.3 log cycles (nitrate-dominated, “high power”) of the initial viral load. The immediate effect of CAP on EHV-1 was less pronounced, with “low power” CAP yielding a 1.4 and “high power” a 1.0 log reduction. We observed a decline in glucose contents in DMEM, which was most probably caused by a Maillard reaction with the amino acids in DMEM. With respect to the application of the virucidal CAP treatment in oyster production, we investigated whether salt water could be sanitized. CAP treatment entailed a significant decline in pH, below the limits acceptable for holding oysters. In oyster slurry (a surrogate for live oysters), CAP exposure resulted in an increase in total nitrogen, and, to a lower extent, in nitrate and nitrite; this was most probably caused by absorption of nitrate from the plasma gas cloud. We could not observe a change in colour, indicative for binding of NO_x to haemocyanin, although this would be a reasonable assumption. Further studies are necessary to explore in which form this additional nitrogen is deposited in oyster flesh. Full article