Viral RNA Extraction and Sequencing from Plasma

Plasma was separated from whole blood. Viral RNA was extracted from 140 μl of plasma by using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA, United States) according to the manufacturer’s instructions, and was then subjected to nested polymerase chain reaction (PCR) to amplify the fragments of the E1E2 (H77: 933–2060) and NS5B (H77: 8340–9233). The primers and conditions of PCR reaction were described previously [28 (link)]. The first PCR reaction was performed using One Step reverse transcription PCR (Takara, Dalian, China). The second PCR reaction was performed using 2×Taq PCR MasterMix (Tiangen, Beijing, China). PCR products were sent to ZIXIBIO Co. (Beijing, China) for sequencing using an ABI 3730XL automated DNA sequencer (Applied Biosystems, Carlsbad, USA).