Isolation of RNA, cDNA synthesis, PCR of the 5'-UTR, sequencing of PCR products and sequence analysis

The preserved and frozen M. ovinus were retrieved and placed in an autoclaved, chilled mortar. Liquid nitrogen was added and the samples were rapidly ground into powder. Next, total RNA from M. ovinus was extracted using the TaKaRa RNAiso Plus Kit (TaKaRa, Beijing, China, Code No. 9108) according to the manufacturer’s protocol. The precipitates were dissolved in 20 μL of RNase-free water in the final step. Next, cDNA was synthesized using the extracted RNA and according to the manufacturer’s protocol of the TaKaRa PrimeScript^™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China, Code No. 6210A). Subsequently, the 5′-UTR of BDV was amplified according to the manufacturer’s protocol of Premix Taq^™ (TaKaRa Taq^™ Version 2.0) (TaKaRa, Beijing, China, Code No. R004A) and using the KOD-Plus amplification enzyme (Toyobo Co. Ltd, Osaka, Japan). The amplified product was approximately 225 bp.

Each 50 μl PCR reaction mixture contained 25 μl of the 2× PCR solution for Premix Taq^™, 1 μl each of the forward and reverse primers (PBD1: 5'-TCGTGGTGAGATCCCTGAG-3'; PBD2: 5'-GCAGAGATTTTTTATACTAGCCTATRC-3' [21, 26]), 1 μl of the cDNA template, and distilled water.

The cycling conditions for the 5'-UTR amplification with primers PBD1 and PBD2 were as follows: initial denaturation at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 45 s; followed by final extension at 72 °C for 10 min.

The specific PCR amplification products were sequenced using an ABI PRISM^™ 3730XL DNA Analyzer (ABI, Carlsbad, America). The sequences were aligned with reference sequences (Table 1) downloaded from GenBank using MEGA 5.0 software. The sequencing results were analyzed using the BLAST online platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), as well as DNAStar and MEGA 5.0 molecular biology software, and were compared with the reference sequences (Table 1) downloaded from GenBank. The sequences were analyzed and a phylogenetic tree was constructed. The evolutionary history was inferred using the Neighbor-Joining method based on the Maximum Composite Likelihood method. The sequences obtained in this study were deposited in the GenBank database under the accession number {"type":"entrez-nucleotide","attrs":{"text":"MK322443","term_id":"1704641945","term_text":"MK322443"}}MK322443.