2.5. Sequencing of the Complete Genome of Phodopus Sungorus Papillomavirus Type 1

Three randomly selected PsuPV1 L1 gene–positive samples were first subjected to rolling circle amplification (RCA) using an Illustra TempliPhi 100 Amplification Kit (GE Healthcare Life Sciences, Little Chalfont, UK), following the manufacturer’s instructions. The PsuPV1 type-specific primer set (PsuPV1_LNGL1-F: 5′-CGTTGCTAAACAAACTAGGTGAC-3′ and PsuPV1_LNGL1-R: 5′-GATGTCCGGTTGTCCCTAC-3′), targeting the PsuPV1 L1 gene, was subsequently used to amplify their complete viral genomes, with the help of inverted long-range PCR in combination with a Platinum Taq DNA Polymerase High Fidelity Kit (Invitrogen, Carlsbad, CA, USA), as described previously [31].

Sanger sequencing of the PCR products obtained was performed by a primer-walking strategy, using 22 sequencing primers, as already described [31]. Sequences were constructed using the Vector NTI Advance v11.5.4 (Thermo Fisher Scientific, Waltham, MA, USA) and BioEdit Sequence Alignment Editor v7.2.6.1 (Ibis Therapeutics, Carlsbad, CA, USA) [35] software packages and compared with the PsuPV1 reference sequence (GenBank acc. no. HG939559), using the BLAST algorithm [33].