A Screening Study Identified Decitabine as an Inhibitor of Equid Herpesvirus 4 That Enhances the Innate Antiviral Response.

Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.

Immunostimulating Effect of Inactivated Parapoxvirus Ovis on the Serological Response to Equine Influenza Booster Vaccination.

Equine influenza virus (EIV) is responsible for recurring outbreaks that are detrimental to the equine industry. Vaccination is key for prevention, but the effectiveness and duration of protection provided by existing vaccines is often insufficient. In order to improve vaccine efficacy, we evaluated the benefit of immune stimulation with inactivated Parapoxvirus ovis (iPPVO) on the antibody response induced by a vaccine boost against EIV. A whole inactivated ISCOMatrix-adjuvanted equine influenza vaccine was administered alone (n = 10) or combined with iPPVO injections at D0, D2 and D4 post vaccination (n = 10) to adult horses that required a vaccine boost 6 months after the last immunization, as now recommended by the WOAH. Antibody levels were measured with the single radial haemolysis (SRH) assay at 1, 3 and 6 months post-vaccination. Results revealed that horses that received iPPVO had higher antibody levels than the control group injected with the EI vaccine alone. Although the vaccine used contains only a clade 1 and European lineage strain, the increase in protective antibodies was also observed against a clade 2 strain. Thus, immune stimulation with iPPVO, a substance already marketed as an immunostimulant, could be used to improve vaccination protocols in horses and potentially other species.

Equine Herpesvirus 1 Variant and New Marker for Epidemiologic Surveillance, Europe, 2021.

Equine herpesvirus 1 isolates from a 2021 outbreak of neurologic disease in Europe have a mutation, A713G, in open reading frame 11 not detected in 249 other sequences from equine herpesvirus 1 isolates. This single-nucleotide polymorphism could help identify horses infected with the virus strain linked to this outbreak.

Identification of a New Equid Herpesvirus 1 DNA Polymerase (ORF30) Genotype with the Isolation of a C2254/H752 Strain in French Horses Showing no Major Impact on the Strain Behaviour.

Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C2254 (H752) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as UL clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C2254 mutation. When cultured in vitro, the C2254 EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A2254 and G2254 EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C2254 strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C2254 strain and two other A/G2254 field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C2254 and A2254 strains were more sensitive to aciclovir than the G2254 strain, based on viral load measurement.

Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: Efficacy of decitabine and valganciclovir alone or in combination.

Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.

Further Evidence for in Utero Transmission of Equine Hepacivirus to Foals.

(1) Background: Equine hepacivirus (EqHV), also referred to as non-primate hepacivirus (NPHV), infects horses-and dogs in some instances-and is closely related to hepatitis C virus (HCV) that has infected up to 3% of the world's human population, causing an epidemic of liver cirrhosis and cancer. EqHV also chronically infects the liver of horses, but does not appear to cause serious liver damages. Previous studies have been looking to identify route(s) of EqHV transmission to and between horses. (2) Methods: In this retrospective study, we sought to evaluate the prevalence of vertical transmission taking place in utero with measuring by quantitative RT-PCR the amounts of EqHV genome in samples from 394 dead foals or fetuses, paired with the allantochorion whenever available. (3) Results: Detection of EqHV in three foals most likely resulted from a vertical transmission from the mares to the fetuses, consistent with the in utero transmission hypothesis. In support of this observation, the presence of EqHV genome was found for the first time in two of the allantochorions. (4) Conclusions: As seemingly benign viruses could turn deadly (e.g., Zika flavivirus) and EqHV happens to have infected a significant proportion of the world's horse herds, EqHV infectious cycle should be further clarified.

Screening and evaluation of antiviral compounds against Equid alpha-herpesviruses using an impedance-based cellular assay.

Equid alpha-herpesviruses (EHV) are responsible for different diseases in equine population. EHV-1 causes respiratory diseases, abortions and nervous disorders, EHV-4 causes respiratory diseases and sporadic abortion, while EHV-3 is responsible of equine coital exanthema. In view of the lack of efficacy of vaccines against EHV-1 and EHV-4 and in the absence of vaccines against EHV-3, the use of antiviral treatment is of great interest. In this study, we documented the interest of the Real-Time Cell Analysis (RTCA) technology to monitor the cytopathic effects induced by these viruses on equine dermal cells, and established the efficacy of this method to evaluate the antiviral effect of aciclovir (ACV) and ganciclovir (GCV). In addition, the RTCA technology has also been found appropriate for the high-throughput screening of small molecules against EHV, allowing the identification of spironolactone as a novel antiviral against EHV.

Equine PBMC Cytokines Profile after In Vitro α- and γ-EHV Infection: Efficacy of a Parapoxvirus Ovis Based-Immunomodulator Treatment.

Equine herpesviruses (EHV) infect horses early during life and the persistence of these viruses through establishment of latency represents a real risk. A better understanding of the immune response to EHV infection is necessary to improve our methods of prevention and decrease the risk of transmission. The objectives of this study were to characterise the cytokine gene expression profile of peripheral blood mononuclear cells (PBMC) after in vitro EHV-1, EHV-4, and EHV-2 infection and to determine the efficacy of inactivated Parapoxvirus ovis (iPPVO) against these 3 viruses. PBMC were isolated from 3 horses and infected in vitro with EHV-1, EHV-4, or EHV-2 in the presence or absence of iPPVO. In vitro culture of PBMC with EHV-1, EHV-4, and iPPVO induced a significant increase of IFN-α, IFN-ß, and IFN-γ gene expression. EHV-4 also triggered a significant increase of IL-6 and TNF-α mRNA. EHV-2 triggered a significant increase of IFN-α, IFN-ß, IFN-γ, IL-1ß, IL-6, and TNF-α mRNA. The presence of iPPVO induced an earlier and stronger expression of IFN-α, IFN-ß, and IFN-γ mRNA during EHV infection and reduced the inflammatory response induced by EHV-2. In conclusion, this study suggests that the presence of iPPVO potentiates the development of the immune response to in vitro EHV infection.

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids.

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Detection and quantitation of equid gammaherpesviruses (EHV-2, EHV-5) in nasal swabs using an accredited standardised quantitative PCR method.

Equid gammaherpesviruses-2 and -5 are involved in respiratory problems, with potential clinical manifestations such as nasal discharge, pharyngitis and swollen lymph nodes. These viruses are sometimes associated with a poor-performance syndrome, which may result in a significant and negative economic impact for the horse industry. The aim of the present study was to develop and validate quantitative PCR methods for the detection and quantitation of EHV-2 and EHV-5 in equine respiratory fluids. Two distinct tests were characterised: (a) for the qPCR alone and (b) for the whole method (extraction and qPCR) according to the standard model AFNOR XP U47-600-2 (viz., specificity, quantifiable sensibility, linearity, accuracy, range of application, trueness, precision, repeatability and precision of reproducibility). EHV-2 and EHV-5 detection were performed on nasal swabs collected from 172 horses, all of which exhibited clinical signs of respiratory disease. The data revealed a high rate of EHV-2/EHV-5 co-detection that was correlated significantly with age. Viral load of EHV-2 was significantly higher in young horses whereas viral load of EHV-5 was not significantly different with age.