Tracking the evolution of the G1/RHDVb recombinant strains introduced from the Iberian Peninsula to the Azores islands, Portugal

Previous genetic characterization of rabbit haemorrhagic disease virus (RHDV) from Azores, Portugal, revealed the presence of genogroup 3-5 (G3-G5) like strains. These strains differed from the genogroup 1 (G1) strains circulating in mainland Portugal, suggesting an independent evolution of RHDV in Azores. More recently, the new variant RHDV (RHDVb) was detected in Azores. In mainland Portugal, current circulating strains resulted from recombination events between RHDVb and non-pathogenic or pathogenic G1 strains. To characterize the RHDVb strains from Azores, a ~2.5. kb fragment of the RHDV genome (nucleotide positions 4873-7323), including the complete sequence of the capsid gene VP60 (nucleotide positions 5305-7044), was amplified and sequenced. Samples were obtained from rabbits found dead in the field between December 2014 and March 2015 in the Azorean islands Flores, Graciosa, São Jorge, Terceira, Faial, Pico, São Miguel and Santa Maria. For VP60, the highest homology was found with Iberian RHDVb strains, while the upstream fragment revealed high similarity (~95%) with Iberian G1 strains. Phylogenetic reconstruction based either on VP60 or VP10 grouped the Azorean strains with Iberian RHDVb strains. For the fragment upstream of VP60, the Azorean strains grouped with G1. Our results show that the RHDVb strains circulating in Azores are G1/RHDVb recombinants and we hypothesize that such strains had their origin in Iberian strains. The geographic isolation of Azores suggests that arrival of RHDVb was man-mediated. A network analysis further allowed us to trace virus dispersion in Azores: from an initial outbreak in Graciosa, RHDVb spread to São Jorge and Faial, to Terceira, Flores and Santa Maria, and finally to Pico; dispersion to São Miguel occurred later from Terceira. As the consequences of the presence of G1/RHDVb strains in Azores are unpredictable, we suggest a continued monitoring and characterization of RHD outbreaks.This work is funded by National Funds through FCT – Foundation for Science and Technology under the Project FCT-ANR/BIA-BIC/0043/2012. FCT also supported the doctoral fellowships of AML, FN and AP (Refs.: SFRH/BD/78738/2011, SFRH/BD/81916/2011 and SFRH/BD/71252/2010), and the Investigator grant of JA (Ref.: IF/01396/2013). “Genomics Applied To Genetic Resources” co-financed by North Portugal Regional Operational Programme 2007/2013 (ON.2-O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) also supported the work.Peer Reviewe