Abstract
Myxoma virus (MYXV), the causative agent of myxomatosis, is endemic in wild populations of the European rabbit (Oryctolagus cuniculus). In 2018, the virus acquired four genes (M157L, M158L, M159L and M160L) from a yet unidentified source and gained the capacity to infect the Iberian hare (Lepus granatensis). The new viral strain, termed ha-MYXV (hare myxoma virus), continues to circulate within Iberian hare populations, causing a dramatic decline in numbers of this species. To analyse the genetic stability of the emergent ha-MYXV, following its host species jump, we sequenced the novel four-gene cassette in 106 samples collected over an 8-year period (2018-2025) from Iberian hares and wild and domestic rabbits throughout Spain. Samples were either collected as part of an active surveillance campaign or through passive sampling of diseased animals. Our results show that M157 and M158 are completely conserved in all samples, while M160 demonstrated occasional sporadic mutations. However, gene M159L, the C7L-like host range virulence factor, demonstrated simple sequence repeat (SSR) trinucleotide expansions and contractions in ~60% of samples analysed. Further analysis showed that ha-MYXV contains unique SSR in genes associated with early stop mutations. Sequencing of the ha-MYXV-specific SSR regions in field samples shows a large degree of variation. These findings indicate that the variable nature of SSR in the emerging virus contrasts with the stability of the other novel genes present in the ha-MYXV genome. This study highlights the need for continued surveillance in order to better understand virus evolution following a cross-species virus transmission event.