Abstract
Hepatitis E virus (HEV) infection in humans is primarily caused by genotypes within Paslahepevirus species balayani (HEV-A). Rocahepevirus species ratti (HEV-C1, otherwise known as rat HEV) can also infect humans. HEV grows poorly in cell culture. Recent studies have reported that hyper-confluent cell layers, amphotericin B, MgCl2, progesterone, and dimethyl sulfoxide (DMSO) increase HEV yield in vitro. Here, we describe an independent evaluation of the effectiveness of these modifications in improving the yield of HEV-A genotype 4 (HEV-A4) and HEV-C1 from clinical samples in PLC/PRF/5 cells. We found that amphotericin B, MgCl2, and DMSO increased HEV yield from high-viral-load patient stool samples, while progesterone was not effective. Yield of HEV-C1 was lower than HEV-A4 across all medium conditions, but was boosted by DMSO. HEV-A4 could be maintained for over 18 months in amphotericin B- and MgCl2-containing medium, with the demonstration of viral antigen in supernatants and infected cells. We also evaluated various protocols to remove pseudo-envelopes from cell culture-derived HEV. Treating cell culture supernatant with NP-40 was the most effective. Our findings identify key modifications that boost HEV growth in vitro and illustrate the importance of independent verification of such studies using diverse HEV variants and cell lines.
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