Major Equine Herpesvirus ‐ 1 epizootic in Europe: Identification of a marker for epidemiological surveillance

Background: Equine herpesvirus type-1 (EHV-1) is an important threat to the equine industry, as illustrated by the ongoing outbreak of neurological disease that was initially reported during a large equestrian event in Valencia, Spain in 2021. Horses returning from this event to their training yards have contributed to the spread of the virus to nine other European countries and to Qatar. Objectives: To design a “tracking” marker in order to specifically follow the dissemination of the Valencia strain, in EHV-1 infected horses with no known epidemiological link with the Valencia outbreak. Study design: Strain isolation and genome sequences’ comparison. Methods: 67 nasopharyngeal swab samples from horses stationed in Valencia were analysed by EHV-1 qPCR and a rapid A/G/C2254 (ORF30) typing test. Positive samples were used for strain isolation in vitro, and Multilocus Sequence Typing (MLST). Results: 19/67 (28%) samples were EHV-1 positive (all 19 typed as A2254). Two strains (FR/Valencia1/2021 and FR/Valencia2/2021) were successfully isolated and characterised by MLST as belonging to clade 10. Analysis of the MLST ORF sequences revealed a mutation at position 713 of the ORF11 (A713G) in FR/Valencia1/2021 and FR/Valencia2/2021, when compared with reference strains Ab4 and V592. This A713G mutation was not present in 104 ORF11 sequences obtained from Genbank (strains from the UK, USA, China, Australia, Belgium, New-Zealand, Japan or India), or 131 and 14 ORF11 sequences from strains isolated in Ireland or France, respectively. This marker allowed subsequent confirmation of suspicious epidemiological links in EHV-1 cases. Main limitation: The limited number of ORF11 sequences available in some countries. Conclusions: Although the existence of this mutation in other field strains cannot be excluded, its absence in the 249 ORF11 sequences analysed, suggests that this SNP constitutes an interesting epidemiological marker to identify horses infected with the EHV-1 strain which was associated with the outbreak in Valencia.