Abstract

Objective: To present a comprehensive protocol for the processing of hepatitis E virus (HEV) infected samples and propagation of the virus in primary cell cultures. Methods: Hepatitis E was extracted from porcine liver and faecal samples following standard protocols. The virus was then allowed to attach in the presence of trypsin to primary cells that included porcine and bovine intestinal epithelial cells and macrophages over a period of up to 3 h. The virus was propagated by rotational passaging through the cell cultures. Propagation was confirmed by immunoblotting. Results: We developed a comprehensive protocol to propagate HEV in porcine cell model that includes (i) rotational culturing of the virus between porcine cell types, (ii) pre-incubation of infected cells for 210 min, (iii) use of a semi-complete cell culture medium supplemented with trypsin (0.33 µg/mL) and (iv) the use of simple immunoblot technique to detect the amplified virus based on the open reading frame 2/3. Conclusions: This protocol opens doors towards systematic analysis of the mechanisms that underlie the pathogenesis of HEV in vitro. Using our protocol, one can complete the propagation process within 6 to 9 d.

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