2.3 Molecular analysis
For nucleic acid extraction, fresh samples of eyelid were homogenized at 20% (w/v) with phosphate buffered saline and clarified at 3,000g for 5 min. Total nucleic acid were extracted from 200μl of the clarified supernatants, using the MagAttract 96 cador Pathogen Kit in a BioSprint extractor (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.
In the Scope of Project +Coelho, all the rabbits were tested for RHDV, RHDV2 and MYXV due to possible co-infections (Duarte et al., 2018; C. L. Carvalho et al., 2020). The rabbits were tested for rabbit haemorrhagic disease virus 2 (RHDV2) by the qPCR (Duarte et al., 2015), and for RHDV by conventional PCR (Tham et al., 1999),using the AgPath (Thermo Fisher Scientific) and One step RT-PCR kit (Qiagen, Hilden, Germany), respectively. MYXV was initial tested by the qPCR method described by (Duarte et al., 2014), which detects both MYXV and ha-MYXV virus, using the FastStart TaqMan Probe Master Kit (Roche, Germany).
The preliminary analysis for recombinat MYXV DNA was performed with the conventional PCR system 9E/9F described by Dalton et al. (Dalton et al., 2019), using the HighFidelity PCR Master Mix (Roche, Germany), according with manufacturer protocol.
Amplifications were carried out in a Bio-Rad CFX96™ Thermal Cycler (Bio-Rad Laboratories Srl, Redmond, USA).
The PCR products were visualized in 2% horizontal electrophoresis agarose gels, purified with the NZYGelpure kit (Nzytech, Portugal), and directly sequenced using the ABI Prism BigDye Terminator v3.1 Cycle sequencing kit on a 3130 Genetic Analyser (Applied Biosystems, Foster City, CA, U.S.A). Sequencing of the 859nt fragment within the 2.8Kb insert that includes the partial M064L and M061L gene, was performed with primers designed in this work, 1467F (5’- TTCATCGTTTATGGGAAAATCTATG-3’), 1819R (5’- GAGGGGACAGTTATGGATGTAC- 3’), 2028F (5’-AAGATGCGTCTGTGTAACAATCC-3’) and 2325R (5’- AACAATGTATACACTCATGACAGTAC-3’). The nucleotide sequences obtained were assembled using the Seqscape Software v2.7 (Applied Biosystems, Foster City, CA, USA), and submitted to GenBank.