Nucleic acid detection and molecular traceability of equine herpesvirus 1 in imported horses

Equid alphaherpesvirus 1 is a major pathogen threatening the global equine industry, causing respiratory disease, abortion, and neurological disease.

In April 2024, during routine pathogen screening of a batch of imported quarantine horses from the Netherlands, four out of 20 nasal swab samples tested positive for EHV-1 nucleic acid.

The affected horses exhibited only mild respiratory signs. ORF30 SNP typing at nucleotide position 2254 and Sanger sequencing confirmed that all positive samples belonged to the N752 non-neuropathogenic genotype. As virus isolation was unsuccessful, the ORF30, ORF33, and ORF34 gene fragments were amplified and sequenced directly from the positive samples. The complete identity of these gene sequences across all four samples indicated that they were caused by the same strain. Bayesian phylogenetic analysis based on concatenated multi-sequence alignment revealed that all reference strains first divided into two distinct clades according to host range: Clade A encompassed Equus caballus, Equus ferus, and Equus asinus, whereas Clade B was strictly restricted to Equus caballus. The strain identified in this study (EHV-1/China/2024) was located within the broadly host-adapted Clade A and formed a monophyletic group with an Equus ferus-derived strain isolated in Ireland in 2017 (MZ318706) with high posterior probability, which subsequently clustered with a sister group comprising another Irish Equus ferus-derived strain and a UK Equus caballus-derived strain, collectively constituting a predominantly European-derived evolutionary cluster.

While the horses were imported from the Netherlands, the evolutionary background of the virus pointed to the Ireland/British Isles region, suggesting that the strain may have entered the Netherlands via the European equine trade network before being introduced. This study reveals the molecular characteristics and phylogenetic position of EHV-1 carried by imported horses and uncovers a geographic discordance between the evolutionary background of the virus and its direct country of origin, providing molecular evidence for understanding the international transmission dynamics of EHV-1 and pathogen surveillance in cross-border equine movements.