Optimized Hepatitis E Virus (HEV) Culture and Its Application to Measurements of HEV Infectivity

Abstract

Hepatitis E virus (HEV) is a major concern in public health worldwide. Infections with HEV genotypes 3, 4, or 7 can lead to chronic hepatitis while genotype 1 infections can trigger severe hepatitis in pregnant women. Infections with all genotypes can worsen chronic liver diseases. As virions are lipid-associated in blood and naked in feces, efficient methods of propagating HEV clinical strains in vitro and evaluating the infectivity of both HEV forms are needed. We evaluated the spread of clinical strains of HEV genotypes 1 (HEV1) and 3 (HEV3) by quantifying viral RNA in culture supernatants and cell lysates. Infectivity was determined by endpoint dilution and calculation of the tissue culture infectious dose 50 (TCID50). An enhanced HEV production could be obtained varying the composition of the medium, including fetal bovine serum (FBS) and dimethylsulfoxide (DMSO) content. This increased TCID50 from 10 to 100-fold and allowed us to quantify HEV1 infectivity. These optimized methods for propagating and measuring HEV infectivity could be applied to health safety processes and will be useful for testing new antiviral drugs.

Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

“…However, for decades, these cell culture systems did not allow replication of HEV at high levels. It is only recently that optimised cell models have been developed and high HEV viral loads detected in HepG2/C3A, A549, PLC/PRF/5 and Huh-7-Lunet BLR cells [24][25][26]. Increased viral replication has also been reported in hepatoma cells expressing HEV-1 ORF4 [27].…”

Harringtonine Inhibits Herpes Simplex Virus Type 1 Infection by Reducing Herpes Virus Entry Mediator Expression

“…About 2 h post-infection (hpi), the supernatant was removed, and the new DMEM containing 2% FBS was added, and the culture was maintained for 24 h. Then, 10-fold serially dilutions of the virus suspension were used to infect monolayer Vero cells in a 96-well plate for 72 h. Each dilution was tested with eight replicates in each experiment, and the wells were observed and scored for the presence or absence of cytopathic effect (CPE). The 50% tissue culture infectious dose (TCID 50 ) was calculated based on the Reed-Muench method (Capelli et al, 2020).…”

Progress in the Production of Virus-Like Particles for Vaccination against Hepatitis E Virus

“…For example, human hepatomaderived HepaRG and porcine hepatocyte-like PICM-19 cell lines have been shown to support HEV replication, and are useful for studying virus-host interactions and species barrier crossing, especially since HEV infection is a zoonosis in developed countries [61]. Capelli et al [62,63] showed that different HEV genotypes release more than 90% of the virus from the apical membrane after ORF1 encodes nonstructural proteins involved in viral replication [45,46]. A small multifunctional 13 kDa protein is expressed from ORF3, which facilitates HEV transport throughout the cell and acts as viroporin for the release of the infectious virus from the host cell [47,48].…”

“…For example, human hepatomaderived HepaRG and porcine hepatocyte-like PICM-19 cell lines have been shown to support HEV replication, and are useful for studying virus-host interactions and species barrier crossing, especially since HEV infection is a zoonosis in developed countries [61]. Capelli et al [62,63] showed that different HEV genotypes release more than 90% of the virus from the apical membrane after ), with a small multifunctional protein encoded by ORF3. Three different capsid proteins have been discovered in vitro during infection, i.e., gORF2-glycosylated, iORF2-infectious and cORF2-cleaved ORF2.…”

“…For example, human hepatoma-derived HepaRG and porcine hepatocyte-like PICM-19 cell lines have been shown to support HEV replication, and are useful for studying virus-host interactions and species barrier crossing, especially since HEV infection is a zoonosis in developed countries [61]. Capelli et al [62,63] showed that different HEV genotypes release more than 90% of the virus from the apical membrane after infecting polarized human hepatocellular carcinoma HepG2/C3A cells, suggesting the main route of release for infectious virions [62,63]. In recent years, the key steps of HEV's natural infectious cycle in vivo have been confirmed by employing polarized human stem-cell-derived, hepatocyte-like cells (HLCs).…”