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

Abstract

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.

Investigation of an EHV-1 Outbreak in the United States Caused by a New H752 Genotype

“…The genetic characterization of the EHV-1 strain showed a new C 2254 (H 752 ) mutation of the ORF 30 gene, similar to the mutation reported in a recent outbreak from France [9]. The clinical signs reported from the French horses were respiratory signs (96.9%), pyrexia (70.3%), limb edema (59.4%) and lethargy (28.1%) [9]. In the performance horses from Pennsylvania, pyrexia (84%) was the predominant clinical sign.…”

“…Both the H 752 EHV-1 strain from France and Pennsylvania showed neuropathogenicity. Among the French horse population, two horses showed neurological signs of disease, which required euthanasia in one of them [9]. In the present outbreak, two adult horses outside the show barn had to be euthanized due to severe neurological deficits.…”

“…Since the full genome sequencing of two prototype EHV-1 strains, Ab4 (neuropathogenic) and V592 (non-neuropathogenic), the majority of diagnostic laboratories in the USA rely solely on allelic discrimination qPCR assays to detect EHV-1 [11][12][13][14]. Unfortunately, such a molecular approach may lead to missed EHV-1 cases as exemplified by the present outbreak and by a recently reported outbreak from France [9].…”

“…Until recently, asparagine (N) and aspartic acid (D) have been the only two amino acids identified at position 752 of the DNA polymerase gene of EHV-1 [7]. In 2020, a French group reported on the fortuitous identification and characterization of a new ORF 30 genotype H 752 associated with one specific outbreak that lasted several weeks in 2018 [9]. Similar to the Pennsylvania outbreak, the molecular profile of the EHV-1 strain, i.e., positive by a conventional qPCR gB method and negative by a qPCR ORF30 N/D 752 genotyping method, prompted further investigations.…”

“…Since asparagine (N) and aspartic acid (D) are considered the only two amino acids identified at position 752 of the DNA polymerase gene of EHV-1 [7], the majority of diagnostic laboratories in the USA are often only using allelic discrimination qPCR assays to detect EHV-1. Unfortunately, such a molecular approach may lead to missed EHV-1 cases as exemplified by the present outbreak and by a recently reported outbreak from France [9].…”