2.4. Necropsy and molecular diagnosis
Necropsies were carried out at the Nacional Institute of Agrarian and Veterinary Research, Portugal, and at the Veterinary Science College, Universidad de Córdoba and at the Instituto de Investigación en Recursos Cinegéticos (UCLM & CSIC), Spain.
Tissue samples from hares collected in Portugal, were analysed according to Duarte et al. (Duarte et al., 2014). The diploid targeted gene M000.5L/R is present in the MYXV and MYXV-like genomes, but not in other leporipoxviruses, such as Shope Fibroma Virus. For nucleic acid extraction, fresh samples of liver, spleen and palpebra were homogenised at 20% (w/v) with phosphate buffered saline (PBS) and clarified at 3000g for 5 min at 4ºC. Sera was diluted at 1:4 in PBS. Total DNA was extracted from 200μl of the clarified tissue supernatants and diluted serum samples, using the MagAttract 96 cador Pathogen Kit in a BioSprint 96 nucleic acid extractor (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.
Hare samples collected in Spain were analysed according to García-Bocanegra et al. (Bocanegra et al., 2019). At the Central Veterinary Laboratory in Algete (Spanish National Reference Animal Health Laboratory), total DNA was extracted with the MagAttract® 96 Cador® Pathogen Kit (QIAGEN, Germany) following the manufacturer’s instructions. For the detection of DNA of both the classical MYXV strains and the novel ha-MYXV isolate, a conserved region of the M071L or M000.5L/R gene was amplified by PCR or real time PCR, respectively, as previously described (Cavadini et al., 2010; Duarte et al., 2014; Dalton et al., 2019).Using TaKaRa LA Taq DNA polymerase (TaKaRa, Japan), a specific ha-MYXV PCR was carried out with primers M009L (Dalton et al., 2019).