Detection and Characterisation of The ‘A’ Variant of Rabbit Haemorrhagic Disease (RHDVa, GI.1a) Over a Decade (2013–2023) in Italy

Since the 1980s, highly pathogenic and hepatotropic Lagovirus species have significantly impacted rabbit populations, including Rabbit Haemorrhagic Disease Virus (RHDV, genotype GI.1), its variant RHDVa (genotype GI.1a), and RHDV type 2 (RHDV2, genotype GI.2). Non‐pathogenic Lagovirus strains such as Rabbit Calicivirus (RCV, genotypes GI.3 and GI.4), which mainly replicate in the gastrointestinal tract, have also been identified. Recombination between pathogenic and non‐pathogenic strains, particularly at the junction between non‐structural (NS) and structural genome regions, is a major driver of the emergence of new variant strains. In the context of the widespread and predominant circulation of RHDV2, we analysed 51 RHDVa strains collected from domestic rabbit farms across Italy between 2013 and 2023 to assess their genetic and antigenic variability. Phylogenetic analyses were performed using full and partial genome sequences, and antigenic characterisation was conducted using a panel of RHDVa‐specific monoclonal antibodies (MAbs). Phylogenetic analysis showed no evidence of recombination involving the Italian RHDVa strains. Nucleotide and amino acid identities with the reference strain RHDVa_Pv97 ranged from 95% to 98% and 97%–99%, respectively, confirming high genetic stability. The antigenic study indicated overall stability, consistent with the genetic results, but the few mutants found allowed us to map at least two epitopes located on the outer loops of the VP60 protein, which constituted a large part of the virus surface. In particular, the substitution at position N414S leads to the loss of MAb 2B4 binding to the RHDV/Italy/FG2014_1 strain, and the N348S mutation, which is present in five strains, results in the acquisition of binding to the MAb 2A10. Despite the global predominance of RHDV2, RHDVa persists to be detected sporadically in various Italian regions, especially within commercial rabbit farms. Continuous molecular and antigenic monitoring of Lagovirus strains remains essential to detect potential co‐circulation and recombination events that could lead to the emergence of novel strains with distinct genomic and antigenic traits.