Interferon-induced immune signatures are associated with suppression of HEV infection in porcine cell culture models

Abstract

Hepatitis E virus genotype 3 (HEV-3) is a zoonotic pathogen with pigs representing the natural host. Although HEV-3 infections in humans are often self-limiting, severe or chronic cases can occur. In contrast, HEV-3 infections in pigs, the primary reservoir, remain asymptomatic. To assess the initial transcriptional response in porcine cells during HEV-3 infection and pave the way for mechanistic studies of species-specific virus-host interactions, we aimed to establish porcine cell culture models, including primary porcine hepatocytes (PPHs) and porcine cell lines. PPHs supported the full HEV-3 replication cycle while intrinsic immunity, driven by the interferon-stimulated gene (ISG) system, played a central role in restricting viral replication. JAK inhibition enhanced viral replication and suppressed ISG expression, highlighting the importance of IFN signalling in antiviral defense. Transcriptional profiling revealed a global modulation of host responses upon HEV infection, including pathways linked to immunity, inflammation, and metabolism. Porcine cell lines were permissive to HEV infection and treatment with recombinant porcine IFN-α subtypes induced a robust ISG response and effectively inhibited HEV replication in a dose-dependent manner. These findings establish porcine hepatocytes and cell lines as valuable tools to study HEV-host interactions, demonstrating the critical role of IFN-mediated intrinsic immunity in HEV restriction and highlighting subtype-specific antiviral effects of porcine IFN-α.

Keywords: Hepatitis E virus; cell culture models; innate immunity; natural host; pigs; zoonosis.

Figure 1.

Characterization of HEV infection in primary porcine hepatocytes. (A) Workflow for isolation, cultivation and infection of PPHs with HEV. (B) Representative immunofluorescence images of PPHs 3 d.p.i. with HEVcc^p6 ±JAK-I treatment. RBV treatment served as control. ORF2 = green; DAPI = blue; scale bar = 250 µm. (C) Quantification of progeny virus production kinetics in HEVcc^p6-infected PPHs ±JAK-I treatment including timepoints of 3, 5 and 7 d.p.i. RBV treatment served as control. Titres were determined by retitration on HepG2/C3a cells and quantified as FFU/mL ±SD from three independent donors. Dots represent values of individual donors and dashed line the LOQ. (D) Quantification of progeny virus production in HEVcc^p6-infected PPHs ±JAK-I or pIFN treatment 3 d.p.i. Titres were determined by retitration on HepG2/C3a cells and quantified as FFU/mL ±SD from three independent donors. Dots represent values of individual donors and dashed line the LOQ. (E) Expression of cell-type-specific genes in PPHs including hepatocyte, neuronal and lung markers, depicted as log₁₀ RPKM-values from three independent donors. (F) Expression of host factors known to be critical for HEV infection, depicted as log₁₀ RPKM-values. The significance of mean differences compared to untreated HEV-infected cells was tested using one-way ANOVA followed by Dunnett’s multiple comparisons test (C and D). p-Values <.05 (*), <.01 (**), <.001 (***), and <.0001 (****). p-Values >.05 were considered to be ns. Abbreviations: PPHs, primary porcine hepatocytes; HEVcc^p6, cell culture-derived HEV Kernow-C1/p6; JAK-I, JAK-inhibitor; RBV, ribavirin; d.p.i., days post-infection; ORF, open reading frame; FFU, focus forming units; LOQ, limit of quantification; pIFN, porcine IFN; RPKM, reads per kilobase per million; ALB, albumin; APOE, apolipoprotein E; APOA1, apolipoprotein A1; HLA-DRA, HLA class II histocompatibility antigen, DR alpha chain; HLA class II histocompatibility antigen, DO beta chain; PTPRC, protein tyrosine phosphatase receptor type C; SFTPA1, pulmonary surfactant-associated protein A1; SFTPB, pulmonary surfactant-associated protein B; MAP2, microtubule-associated protein 2; DCX, neuronal migration protein doublecortin; CTSL, cathepsin L; EGFR, epidermal growth factor receptor; ITGB1, integrin β1; ITGA3, integrin α3; YES1, Src-family kinase Yes1.

Figure 2.

Transcriptomic analysis of HEV-infected primary porcine hepatocytes. (A) Principal component analysis (PCA) of RNA-sequencing data from three independent donors of PPHs resulted in clustering of infected samples at 72 h. Clusters, depicted by ellipses, were assigned using the k-means algorithm applied to the PCA scores. The statistical robustness of the clustering was evaluated using a multivariate analysis of variance (MANOVA), which revealed a significant difference between the clusters (p-value = .0003). (B) Quantification of mapped ORF2 reads in HEVcc^p6-infected PPHs from three independent donors at 4 and 72 h. Connected dots represent values for individual donors. (C) Gene ontology (GO) analysis of HEVcc^p6-infected PPHs from three independent donors at 4 and 72 h. Dot size corresponds to the size ratio, colour to the z-score, and rim to significance (FDR ≤ 0.05). (D) Log₂ FC of DEGs in three independent donors 72 h.p.i. with HEVcc^p6. Dot size corresponds to FDR p-value and colour to the respective GO-term. (E) Volcano plots of differentially expressed genes in PPHs from three independent donors after 72 h of HEVcc^p6 infection compared to 72 h mock-infection. Core-ISGs are marked in dark blue. (F) Heatmap showing detailed deregulation of individual core-ISGs in PPHs from three independent donors at 4 and 72 h post-infection, depicted as log₂ FC over the corresponding mock-infected sample. Abbreviations: PPHs, primary porcine hepatocytes; ORF, open reading frame; HEVcc^p6, cell culture-derived HEV Kernow-C1/p6; JAK-I, JAK-inhibitor; h.p.i., hours post-infection; FC, fold-change.

Figure 3.

Transcriptomic analysis of HEV-infected primary porcine hepatocytes treated with JAK-I. (A) Gene ontology (GO) analysis of HEVcc^p6-infected PPHs from one donor at 72 h ±HEV infection and ±JAK-I. Dot size corresponds to the size ratio, colour to the z-score, and rim to significance (FDR ≤ 0.05). (B) Volcano plots of differentially expressed genes in PPHs from one donor after 4 h compared to 72 h of HEVcc^p6 infection ±JAK-I treatment. Core-ISGs are marked in dark blue. (C) Heatmap showing detailed upregulation of individual core-ISGs in PPHs from one donor at 4 and 72 h post-infection ±JAK-I treatment, depicted as log₂ FC over untreated, mock-infected cells. Abbreviations: PPHs, primary porcine hepatocytes; HEVcc^p6, cell culture-derived HEV Kernow-C1/p6; JAK-I, JAK-inhibitor; FC, fold change.

Figure 4.

HEV infection, replication and progeny virus production in porcine cell lines. (A) Representative immunofluorescence images of NPTr, NSK and PK15 cells 5 d.p.i. with HEVcc^p6 ±JAK-I treatment. ORF2 = green; DAPI = blue; scale bar = 250 µM. (B) Quantification of HEVcc^p6 infection of NPTr, NSK and PK15 cells 5 d.p.i. ±JAK-I treatment. Depicted are mean FFU/mL ±SD from three independent experiments. Dots represent values of individual replicates. (C) Replication kinetics of HEV Kernow-C1/p6 strain subgenomic replicon in NPTr, NSK and PK15 cells ±JAK-I treatment. Depicted are mean RLU values normalized to 4 h.p.e. +SD from 5 (NPTr and PK15) and 3 (NSK) independent experiments. RBV treatment served as control. (D) Representative immunofluorescence images of NPTr, NSK and PK15 cells 7 d.p.e. with HEV Kernow-C1/p6 RNA ±JAK-I treatment. ORF2 = green; DAPI = blue; scale bar = 250 µm. (E) Viral titres of nonenveloped and enveloped HEVcc^p6 produced in NPTr, NSK and PK15 cells ±JAK-I treatment 3, 5 and 7 d.p.e. with HEV Kernow-C1/p6 RNA. Titres were determined by retitration on HepG2/C3a cells, depicted are mean FFU/mL ±SD from three independent experiments. Dots represent values of individual replicates. The significance of mean differences was tested using multiple t test (B) or two-way ANOVA followed by Šidák’s multiple comparisons test (E). p-Values <.05 (*), <.01 (**), <.001 (***), and <.0001 (****). p-Values >.05 were considered to be ns. Abbreviations: d.p.i., days post-infection; HEVcc^p6, cell culture-derived HEV Kernow-C1/p6; JAK-I, JAK-inhibitor; ORF, open reading frame; FFU, focus forming units; RLU, relative light units; h.p.e., hours post electroporation; d.p.e., days post electroporation.

Figure 5.

Effect of porcine IFN-α subtypes on HEV replication. (A) mRNA expression of IFIT1 and MX1 in pIFN-α (α1, α7, α8, α14) treated NSK cells electroporated with subgenomic HEV RNA (Kernow-C1/p6) 48 h.p.e., determined by RT-qPCR analysis. Cells were treated with 1000, 8 or 0.32 ng/mL of the respective pIFN-α with decreasing concentrations shown from left to right. Mean values of mRNA expression are shown as fold change compared to the untreated control consisting of electroporated cells without pIFN-α treatment. (B) Determination of the antiviral activity (IC₅₀) of porcine IFN-α subtypes (α1, α7, α8, α14) on HEV subgenomic reporter replicon (Kernow-C1/p6) in PK15, NPTr and NSK cells. Depicted are RLU values normalized to untreated control samples measured 48 h.p.e. IFN-α subtypes were titrated on PK15, NPTr and NSK cells after electroporation (25.6 pg/mL to 5000 ng/mL). IC₅₀ values are indicated for each pIFN-α subtype. Cell viability is indicated in grey. The depicted values are means ±SD of three independent experiments. Abbreviations: IC_50, inhibitory concentration 50, RLU, relative light units, h.p.e, hours post electroporation.