Viral bait construction

TBEV and LIV viral RNAs were extracted using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol, and were reverse-transcribed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland). Briefly, viral RNAs were incubated with random hexamer primers (60 µM) for 10 min at 65 °C in a volume of 11.4 µL, and then chilled on ice. Transcriptase reaction buffer (4 µL), Protector RNase Inhibitor (20 U), dNTP (1 mM of each), DTT (5 mM) and Transcriptor High Fidelity Reverse Transcriptase (22 U) were added to the template-primer mix. Reverse transcription and inactivation of reverse transcriptase were achieved by incubation for 30 min at 55 °C and 5 min at 85 °C, respectively. Each viral bait was amplified by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using the primers listed in Additional file 2. The primers contained a recombination sequence at the 5′ terminus, allowing insertion of each viral bait into a pDONR207 using a recombination cloning system (Gateway^® System; Invitrogen, Carlsbad, CA, USA). PCR was performed using 1 µL of Reverse Transcriptase (RT) product in high-fidelity buffer in a final volume of 50 µL, with 0.02 U/µL of Phusion Hot Start II DNA Polymerase, 200 µM of dNTPs and 0.3 µM of forward and reverse primers. Amplification was performed for 35 cycles as follows: 98 °C for 10 s, 56 °C for 15 s and 72 °C for 3 min.