RT-qPCR for quantification of HEV-3ra RNA.

HEV-3ra genomic RNA was quantified using SensiFAST probe No-ROX one-step kit (Bioline USA Inc.) with primers and probe listed in Table 3, following a protocol described previously (85), with some modifications. The RT-qPCR assays were performed in a CFX96 real-time PCR system, C1000 thermal cycler (Bio-Rad Laboratories). A one-step RT-qPCR thermal cycling protocol was used as follows: 45°C for 10 min and 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. In vitro-transcribed HEV-3ra RNAs were used to generate a standard curve in each RT-qPCR run, which covered a quantification range from 4 × 10² to 4 × 10⁷ RNA copies per reaction. The total RNA extracted from each of the tissue samples was diluted at 250 ng/μL, thus allowing the use of 1 μg total RNA (4 μL)/reaction as recommended in the kit. The PCR quantification data were then used to calculate the viral RNA loads as genome copies per gram of tissue. Fold increase in viral loads in pregnant rabbits (HEV-P) compared to nonpregnant rabbits (HEV-NP) was calculated as follows: (HEV RNA copies in HEV-P − HEV RNA copies in HEV-NP)/HEV RNA copies in HEV-NP.

Oligonucleotide primers used in this study