HEV Genome Amplification Protocol

HEV primer design was based on gt3 genomes with accession numbers FJ705359 and FJ956757.1 [32 (link)]. cDNA of HEV0069 infected samples were used to generate seven overlapping amplicons of ~2000 bp length each (Figure S2). Subsequently, two or three 500 bp long amplicons, overlapping ~50–100 bp, per primary product were generated using nested primers to cover the full HEV genome (Table S1A,B). Each 50µL reaction contained 10 µL HEV cDNA, 0.4 µM primers, 1× PCR buffer (Qiagen), 1 mM MgCl₂ (Qiagen), 0.2 mM dNTP mix (Roche), and 2.5 U HotStar polymerase (Qiagen). For PCR, the conditions were 15 min 95 °C, 40 cycles of 1 min 95 °C, 1 min 50 °C, 3 min 72 °C, and 10 min 72 °C. 2 μL first PCR product served as template for the nested PCR. The PCR conditions were identical to first round PCR, with 40 cycles of 30 sec 95 °C, 30 sec 50 °C, and 1 min 72 °C. Amplicon 15 was amplified using an altered annealing temperature of 45 °C instead of 50 °C.
In order to amplify cDNA from serum and feces-derived HEV0122, additional primer pairs were adapted from Munoz-Chimeno et al. [33 (link)] as shown in Table S1C,D. PCR conditions were identical as described above, with minor modifications: 5 μL cDNA was used as input for the first round PCR, and 2.5 μL first PCR product served as template for nested PCR. The annealing temperatures during the PCR protocols were adjusted to match each primer set.