Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Abstract

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral–host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c

“…Furthermore, a single nucleotide exchange leading to a unique restriction site could be successfully introduced into the recombinant virus genome. This demonstrates the suitability of the system for site-directed mutagenesis, which might be used in future studies for investigating the effect of single nucleotide mutations on viral fitness, e.g., mutation G1634R, which has been described to increase the replication efficiency in other HEV strains [22,23].…”

Hepatitis E Virus Infection: Circulation, Molecular Epidemiology, and Impact on Global Health

“…The most commonly used are the Sar-55-related genotype 1 clone [ 68 ], the genotype 3a and 3c Kernow-C1- [ 69 ], and 47832-related [ 70 ] clones, respectively, both of which contain insertions in the HVR, and the genotype 4 TW6196 clone [ 71 ]. A recent presentation of a novel in vitro method to produce high viral titers, allowing study of the full HEV replication cycle in cell culture, has additionally created confidence that we may overcome our limited understanding of HEV pathophysiology [ 72 ]. Although these systems are most commonly used in conventional cell culture systems, several animal infection models have been developed.…”

Hepatitis E Virus: How It Escapes Host Innate Immunity

“…These differences in the requirement for RIG-I, MDA-5, and MAVS could be due to the use of different cell lines. The host response of primary human hepatocytes (PHHs) to an HEV3–Kernow infection revealed the intrinsic expression of the pattern recognition receptors RIG-I, MDA-5, and TLR3, as well as downstream signaling molecules, including Myd88 and MAVS, showing that this model can trigger an innate immune signaling cascade [ 58 ]. The level of IRF7 mRNA in the livers of rhesus macaques was increased at the peak of HEV1-Sar55 infection [ 50 ], but was reduced when the same monkeys were re-infected [ 59 ].…”

“…Infecting HepG2 cells and PHHs with the same strain confirmed the increases in type III IFNs λ1 and λ2/3 at the mRNA and protein levels, but not those of type I IFNs [ 56 ]. Lastly, gene ontology enrichment analyses of the biological processes of infected PHHs showed that the IFN signaling pathway was among those with the highest ratio of significantly differentially regulated genes [ 58 ]. These results are in line with the microarray analyses of the chimpanzees’ liver response to an HEV1–Sar55 infection [ 65 ].…”

“…Similarly, infecting human lung epithelial A49 cells with HEV1–DQ459342 also increased ISG production, including ISG15 or IFIT1 [ 49 ]. HepG2 cells or PHHs infected with the HEV3–Kernow strain secreted type III IFNs that activated the transcription of multiple ISGs, including ISG15 and IFIT1 [ 56 , 58 ]. Human liver chimeric mice (homozygous uPA +/+ -SCID mice) infected with HEV1–Sar55 produced increased IFIT1, while the concentrations of TLR3, ISG15, and MAVS remained unchanged [ 71 ].…”