Horses were subject to euthanasia as enforce by the Serbian legislation as follow, an intravenous injection of the anaesthetic Xilazin 2% was performed. The usual dose of Xilazin for anaesthesia is 1,1 mg/kg, so for a horse that weight 500 kg half a dose would be 12-15 ml of Xilazin 2% just for sedation. The horse became very quiet in several minutes. Then a mix of euthanizing substances (T61) was injected intravenously to the EIA infected horses. For a horse of 500 kg it should be a dose of 50 ml. After 20–30 s, the horse just kneeled down and then slowly lied to the side. There was no excitation, no nervous reactions. Once the death is confirmed by the official veterinarian, spleen from one EIA positive horse was collected and kept at − 80°c until use. Genomic DNA from collected spleen was extracted as previously described [4]. DNA libraries were prepared using the SureSelect XT Library preparation kit for Illumina Multiplexed sequencing Kit (Agilent Technologies, Santa Clara, CA, USA) as described previously [9] and subjected to multiplex deep sequencing on the MiSeq platform using the MiSeq Reagent Kit V2 (Illumina). Fastq format files from each viral genome were trimmed and paired reads were merged using the Geneious Prime sequencing data analysis software (Biomatters LTD, Auckland, New Zealand) [15]. Contigs were obtained by alignment to the reference genome EIAV F2 (GenBank accession number: JX480631.1). Phylogenetic reconstruction was carried out using the MEGA software version 7 [16]. Nucleotide and amino acid identities were calculated using MegAlign from DNA star software.

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