A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3' end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R(2) value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy.

Myxoma virus (MYXV) and rabbit haemorrhagic disease virus 2 (RHDV2) are two major pathogens that affect the European rabbit ( Oryctolagus cuniculus ). Between August 2017 and August 2019, 1166 wild rabbits (971 legally hunted and 195 found dead) were tested by PCR‐based methods for MYXV and RHDV2 within the scope of an ongoing surveillance programme on wild leporids in Portugal. Despite never having been reported before and being considered a rare event, coinfection by RHDV2 and MYXV was detected in one juvenile wild rabbit found dead in the Évora district located in Alentejo. The relative frequency of coinfection in the group of diseased rabbits (found dead in the field) was 0.52 per cent (1/195). The positivity percentage of each single virus was much higher, namely, 14.36 per cent (28/195) for MYXV and 55.38 per cent (108/195) for RHDV2, within the 2 years of sample collection considered.

Myxomatosis was detected in Iberian hares ( Lepus granatensis ) in Portugal, October 2018, following its emergence in Spain 3 months earlier. Here, we describe the epidemiological, molecular and anatomo‐histopathological data of the first two cases. Myxoma virus DNA was detected in the eyelids, nose and perineal region in both hares. It was also detected in the lungs of hare 1 and in the spleen and liver of hare 2. The genomic insertion identified in strains from Spain was confirmed in both strains suggesting a common origin for the Iberian viruses. Gross lesions in hare 1 included palpebral oedema and conjunctival mucopurulent discharge, common in both forms of the disease in rabbits. Hare 2 presented eyelid thickening with small diffuse nodules. Histopathology of the eyelids showed extracellular myxoid matrix in hare 1 and purulent dermatitis in hare 2. Both animals exhibited good body condition, suggesting a short course of the disease and higher virulence of the virus towards the Iberian hare.