HPgV-1 Sequencing Protocol

HPgV-1 RNA was isolated from 200-µL plasma samples using the High Pure Viral RNA Kit according to the procedure described in the manual (Roche, Basel, Switzerland)). Then, the 5’-UTR (U36380:119-497) and E2 (U36380:950-1844) sequences were amplified by nested PCR; the PCR primers (supplement Table S1) and conditions were as reported in previous study [7 (link),9 (link)]. Owing to the high degree of conservation and amplification efficiency of the 5’-UTR, this region was used to evaluate the HPgV-1 infection rate.
The E2 region was used to determine the HPgV-1 genotype, as this sequence could analyze the different genotypes with the same consistency as the complete genome [7 (link),8 (link),9 (link)]. The first PCR reaction was performed using One Step reverse transcription PCR (Takara, Dalian, China) and the second using 2 × Taq PCR MasterMix (Tiangen, Beijing, China). The PCR products were firstly detected by agarose gel (1.0%) electrophoresis and visualized under ultraviolet (UV) illumination for the presence of an 895-nucleotide band and then purified by using a DNA purification kit (Tiangen, Beijing, China); subsequently, the purified products were sent to Shenzhen Invitrogen Biotechnology Co., Ltd. (Shenzhen, Guangdong, China) for sequencing by using an ABI 3730XL automated DNA sequencer (Applied Biosystems, Carlsbad, CA, USA).