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. 2020 Jan 21;117(3):1731-1741.
doi: 10.1073/pnas.1912307117. Epub 2020 Jan 2.

Robust Hepatitis E Virus Infection and Transcriptional Response in Human Hepatocytes

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Free PMC article

Robust Hepatitis E Virus Infection and Transcriptional Response in Human Hepatocytes

Daniel Todt et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Keywords: hepatitis E virus (HEV); humanized mice; infection; primary hepatocytes; transcriptomics.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Optimizing cell culture conditions for high-titer virus production. (A) Workflow of HEV particle production and infection of target cells. HepG2 cells or a subclone thereof, HepG2/C3A cells, were transfected with in vitro transcribed HEV p6_wt RNA. Each cell line was cultivated in either DMEM complete or a medium with lowered IgG levels (MEM low IgG FCS). After 7 d, extracellular and intracellular virus particles, resembling the enveloped and nonenveloped states of HEV, were harvested and used to inoculate both naïve cell lines cultivated in the 2 media. In total, 32 different conditions were tested. (B) Heat map displaying mean titers of different combinations of cell lines and culture media used for the production of viral particles (rows, labeling to the left) and infection of target cells (columns, labeling below) for both intracellularly and extracellularly harvested particles. Viral titers are expressed as mean log of FFU/mL of 3 independent biological replicates (n = 3). (C) Immunofluorescence staining of HEV ORF2-positive foci under optimized conditions. Representative example after production of intracellular virus in HepG2 cells cultivated in DMEM complete and infection of HepG2/C3A cultivated in MEM low IgG FCS. Cell nuclei are shown in blue (DAPI), and HEV ORF2-positive foci are shown in green (α-ORF2 pAb rabbit serum and α-rabbit mAb AF488 2ndary; 10× objective in widefield microscopy).
Fig. 2.
Fig. 2.
Production of infectious particles by trans-complementation of HEV reporter genomes. (A) Schematic representation of the workflow. HepG2 cells are transfected with equimolar amounts of either HEV p6_wt and p6_Gluc (Gaussia luciferase reporter) or p6_wt and p6_vGFP (venus green fluorescent protein) and incubated for 7 d. Assembled particles harboring either viral genome were purified from lysed cells and used to inoculate naïve HepG2/C3A. After an additional 7 d, infectivity was assessed via luminometer (relative light units [RLU]), immunofluorescence (IF), or ORF2 staining (FFU). (B) Infectivity of p6_Gluc trans-completed particles. Results of single experiments are shown as dots, and heights of bars indicate the mean of 3 independent biological replicates (RLU, linear y axis; n = 3 ± SD; *P < 0.05, ***P < 0.001, ANOVA followed by Dunnett’s corrected t test). RBV 25 µM was used to monitor effective replication in infected cells. (C) Infectivity of p6_vGFP trans-complemented virions was recorded either by microscopically counting vGFP-positive foci assessing infection events of trans-complemented particles only (green bars). Alternatively, ORF2 was stained with α-ORF2 pAb rabbit serum and a α-rabbit mAb AF568 (red bars) to assess p6_wt assembled viral particles. RBV 25 µM was used to monitor effective replication in infected cells (FFU, log y axis; n = 3 ± SD; *P < 0.05, ANOVA followed by Sidak’s corrected t test; dashed line, limit of quantification [LOQ]; titers below LOQ set to LOQ). (D) Representative fluorescence images of infection events following purification of assembled virions from lysed cells transfected with p6_wt only (Upper) or p6_wt and p6_vGFP cotransfected cells (Lower). VGFP-positive cells (first column) reflect infection of HepG2/C3A target cells with trans-complemented particles, while AF568-positive cells foci (second column) show p6_wt infection events in confocal microscopy. White arrows point to infection events with trans-complemented particles. (Scale bar, 20 µM.)
Fig. 3.
Fig. 3.
Introduction of the replication enhancing single nucleotide variant G1634R increases titers. (A) Kinetics of the production of intracellular (Left) and extracellular (Right) infectious particles assessed as FFU/mL (log y axes) over 9 d (linear x axes). Light green lines with inverted triangles represent the production in p6_wt HEV RNA transfected HepG2 cells, while dark green lines with triangles display the kinetics in the p6_G1634R mutant transfected cells (dashed line, LOQ; n = 2 ± SD; n.d., not detected). (B) Number of HEV ORF2-positive cells (green areas) in p6_wt (Left) or p6_G1634 mutant (Right) HEV RNA transfected cultures monitored over time (1 representative IF picture evaluated per construct and day; mean of 2 independent biological replicates; n = 2 ± SD). (C) Titer of infectious particles represented as FFU/mL (log y axes) harvested from lysed cells (Left) or supernatant (Right) of p6_wt (light green bars) or p6_G1634R (dark green bars) RNA transfected cells. Titers of single experiments are presented as white dots, and bars display the mean of 3 independent biological experiments (dashed line, LOQ; n = 3 ± SD, *P < 0.05 in a ratio paired t test, n.s., not significant). (D) Titer of infectious particles represented as FFU/mL (log y axes) harvested from lysed cells (Left) or supernatant (Right) of 83-2_wt (light red bars) or 83-2_G1634R (dark red bars) RNA transfected cells. Titers of single experiments are presented as white dots, and bars display the mean of 3 independent biological experiments (dashed line, LOQ; titers below LOQ set to LOQ; n = 3 ± SD, *P < 0.05 in a ratio paired t test, n.s., not significant). (E) Fluorescence pictures of RNA in situ hybridization of HEV p6_G1634R ORF2-positive strand subgenomic RNA. HepG2 cells were either mock-transfected (Left) or transfected with HEV p6_G1634R virus and left untreated (Middle) or treated with RBV (Right). (Scale bars, 20 µm.)
Fig. 4.
Fig. 4.
HEVcc establish high-titer infections in humanized liver chimeric mice. Humanized mice were inoculated intraperitoneally with either intracellular cell culture-derived HEV p6_wt (Upper; 5 mice) or p6_G1634R (Lower; 4 mice). HEV RNA (log y axes) was periodically measured in plasma (purple lines) and feces (orange lines). LOQ, limit of quantification.
Fig. 5.
Fig. 5.
HEVcc establish productive infections in primary human and porcine hepatocytes. (A) Schematic representation of the PHH experiment. PHH were plated on 6-well plates and inoculated with intracellular-derived HEV p6_G1634R (MOI = 1) with or without the administration of RBV 25 µM. After 4, 8, 12, 24, 48, and 186 h p.i., cell lysates were harvested and analyzed via qRT-PCR and RNAseq. The PHH were additionally used for IF staining and FFU count 168 h p.i. (B) Replication of HEV RNA in PHH was monitored via qPCR (log y axis) for 7 d (categorical x axis). Black solid line represents the course of HEV RNA in infected, untreated cells, while the gray solid line depicts the course in infected but RBV 25 µM-treated cells. Dotted lines exemplify mock-infected cells. Triangles and inverted triangles mark the mean of 2 technical replications (n = 2 ± SD). (C) Representative fluorescence images of HEVcc-infected PHH stained with α-ORF2 pAb rabbit serum and α-rabbit mAb AF488 2ndary (widefield microscopy). Administration of RBV served as control. (D) Newly produced viral particles (intracellular, Upper; extracellular, Lower) were recovered from productively HEVcc-infected, lysed PHH and used to inoculate naïve HepG2/C3A target cells. Assembly and infectivity were assessed by counting ORF2-positive foci (titer; log y axes). Titers of single experiments are presented as white dots, and green bars display the mean of 3 independent titrations (dashed line, LOQ; titers below LOQ set to LOQ; n = 3 ± SD). (E) Representative fluorescence images of HEVcc-infected PPH stained with α-ORF2 pAb rabbit serum and α-rabbit mAb AF488 2ndary (widefield microscopy). Administration of RBV served as control. (F) Newly produced viral particles (intracellular, Left; extracellular, Right) were recovered from productively HEVcc-infected, lysed PPH and used to inoculate naïve HepG2/C3A target cells. Assembly and infectivity were assessed by counting ORF2-positive foci (titer; log y axes). Titers are presented as green bars (dashed line, LOQ; titers below LOQ set to LOQ; error bars indicate SD of titration assay).
Fig. 6.
Fig. 6.
Total RNAseq of HEVcc-infected PHH reveals a replication-specific increase of distinct HEV genome transcripts. (A) Total RNA extracted from HEVcc p6_G1634R-infected PHH (compare Fig. 5) of one representative donor were supplied to Illumina RNAseq, and HEV RNA abundance (log y axis) was monitored over time (categorical x axis). Black solid line and dots represent the course of HEV RNA in infected, untreated cells, while the gray solid line and dots depict the course in infected but RBV 25 µM treated cells. Dashed lines and dots exemplify mock-infected cells. (B) Normalized coverage of mapped reads (linear y axes) along the HEV genome (linear x axes) in HEVcc-infected (Right) and mock-infected PHH (Left). Hepatocytes were either treated with RBV 25 µM (Lower) or left untreated (Upper ). Increments of purple lines indicate the change in coverage over the monitored time. Below the plots, a schematic of the HEV p6 genome acknowledges the positioning of the 3 ORF and the S17 insertion. (C) Genomic stability of the introduced G1634R variant over the time of the experiment (categorical y axis) depicted as frequency of amino acid residues (linear x axes) under RBV 25 µM treatment and untreated conditions. White bars represent the arginine variant, and gray bars depict frequency of the glycine variant.
Fig. 7.
Fig. 7.
Transcriptional responses in PHH to HEVcc infection. (A) Heat map of normalized transcript expression (reads per kilobase per million base pairs mapped [RPKM]) of biomarker of adult hepatocytes and PRRs as well as SM in HEVcc-infected or mock-infected cells treated with RBV 25 µM or left untreated over time. (B) Venn diagram of significant DEGs at each monitored time point in HEVcc-infected PHH compared to uninfected PHH. (C) Total number of significant DEG (linear y axes) up- (light orange bars) or down-regulated (light green bars) over time (categorical x axes) in HEVcc-infected PHH compared to either uninfected hepatocytes (Left) or infected but RBV 25 µM treated cells (Right). Fractions of previously described IRGs are colored darker. (D) Venn diagram of significant up-regulated DEG (red line, white fill) and IRG (red line, red fill) in HEVcc-infected PHH compared to uninfected PHH and significant down-regulated DEG (blue line, white fill) and IRG (blue line, blue fill) in HEVcc-infected PHH compared to infected but RBV-treated PHH. (EG) Representation of analysis of significant enriched (Bonferroni corrected P value < 0.05, dashed line, upper linear y axis) pathways in HEVcc-infected PHH compared to uninfected PHH. Pathways are ordered according to significance with color of bars representing time point of enrichment: (E) pink, 48 h p.i., (F) orange, 24 h p.i., and (G) cyan, 168 h p.i. Open circles depict the number of regulated genes as ratio of the total number of genes assigned to the respective pathway (lower linear x axis). (H and I) Comparison of DEG to genes previously identified in HEV-infected chimpanzees (31). (H) Venn diagram displaying 29 DEG overlapping in both studies; 5 genes differentially regulated in HEV-infected chimpanzees were not significantly regulated in HEV-infected PHH (marked with asterisks). (I) All 34 DEG were plotted that were regulated on average at least 1.2-fold in the mentioned study in alphabetical order. Color code represents the fold change (FC) of the expression in HEVcc-infected PHH compared to uninfected cells (log2FC) at different time points.

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