Replication of Hepatitis E Virus (HEV) in Primary Human-Derived Monocytes and Macrophages In Vitro

Abstract

HEV is the most causative agent of acute viral hepatitis globally. HEV causes acute, chronic, and extrahepatic manifestations. Chronic HEV infection develops in immunocompromised patients such as organ transplant patients, HIV-infected patients, and leukemic patients. The source of chronic HEV infection is not known. Also, the source of extrahepatic manifestations associated with HEV infection is still unclear. Hepatotropic viruses such as HCV and HBV replicate in peripheral blood mononuclear cells (PBMCs) and these cells become a source of chronic reactivation of the infections in allograft organ transplant patients. Herein, we reported that PBMCs and bone marrow-derived macrophages (BMDMs), isolated from healthy donors (n = 3), are susceptible to HEV in vitro. Human monocytes (HMOs), human macrophages (HMACs), and human BMDMs were challenged with HEV-1 and HEV-3 viruses. HEV RNA was measured by qPCR, the marker of the intermediate replicative form (ds-RNA) was assessed by immunofluorescence, and HEV capsid protein was assessed by flow cytometry and ELISA. HEV infection was successfully established in primary HMOs, HMACs, and human BMDMs, but not in the corresponding cells of murine origin. Intermediate replicative form (ds RNA) was detected in HMOs and HMACs challenged with HEV. The HEV load was increased over time, and the HEV capsid protein was detected intracellularly in the HEV-infected cells and accumulated extracellularly over time, confirming that HEV completes the life cycle inside these cells. The HEV particles produced from the infected BMDMs were infectious to naive HMOs in vitro. The HEV viral load was comparable in HEV-1- and HEV-3-infected cells, but HEV-1 induced more inflammatory responses. In conclusion, HMOs, HMACs, and human BMDMs are permissive to HEV infection and these cells could be the source of chronic and recurrent infection, especially in immunocompromised patients. Replication of HEV in human BMDMs could be related to hematological disorders associated with extrahepatic manifestations.

Keywords: HEV infection; PBMCs; bone marrow; capsid protein; chronic; ds-RNA; extrahepatic; hematological disorders; immune response.

Figure 1

Characterization of murine monocytes and macrophages and infection with HEV inoculums (A) The isolated murine monocytes were analyzed by flow cytometry to check for purity, and isolated cells were stained with anti-CD14 and anti-CD11b (left). After differentiation into macrophages, the cells expressed a high level of F4/80 (right). Infection of murine monocytes (MMOs red) and murine macrophages (MMACs-blue) with HEV-1 (dotted line) and HEV-3 (solid line) inoculums. Intracellular HEV RNA (B) and extracellular HEV RNA (C) were quantified in the supernatants by qPCR. Huh7.5 (black) challenged with HEV-3 was used as a positive control. LOQ: limit of quantification. Depicted are the mean values of three independent experiments  ±  SEM.

Figure 2

Characterization of human monocytes and macrophages and infection with HEV inoculums (A) The isolated human monocytes were analyzed by flow cytometry to check for purity, and isolated cells were stained with anti-CD14 and anti-CD16 (left). After differentiation into macrophages, the cells expressed high levels of the intracellular marker CD68 (right). (B) Infection of human monocytes (HMOs) with HEV-1 (black) and HEV-3 (red) inoculums. HMOs challenged with HEV-1 were treated with ribavirin (RBV-50 um) (blue). Extracellular HEV RNA was quantified in the supernatants by qPCR. (C) Infection of human macrophages (HMACs) with HEV-1 (black) and HEV-3 (red) inoculums. HMACs challenged with HEV-3 were treated with ribavirin (RBV-50 um) (blue). Extracellular HEV RNA was quantified in the supernatants by qPCR. LOQ: limit of quantification. Depicted are the mean values of three independent experiments  ±  SEM.

Figure 3

Detection of the HEV capsid protein and ds-RNA in HMOs and HMACs challenged with HEV inoculums (A) Representative gating strategy showing the expression of HEV ORF2 Ag in HMOs and HMACs infected with HEV. (left): HMOs infected with HEV-1. (Middle): HMACs infected with HEV-3, and (right): mock infected cells. Red histograms represent cells stained with the secondary A488 conjugated anti-mouse antibodies alone; blue histograms represent cells stained by mouse anti-HEV-ORF2 followed by A488-conjugated anti-mouse antibody. (B) Representatives showing HMOs (upper panel) or HMACs (lower panel) either uninfected (left side) or infected with HEV preparations (right side) were fixed and stained with anti-ds-RNA (J2 Ab) (red). DAPI was used for nuclear staining (blue) (Scale bars, 20 µm). Representative showing infection of HMOs with HEV-1 and infection of HMACs with HEV-3. (C) Supernatants collected from HEV-1 (solid line) and/or HEV-3 (dotted line)-infected HMOs (red) and HMACs (blue) were tested for HEV ORF2 Ag by ELISA, C.O is the cut off. Depicted are the mean values of three independent experiments  ± SEM.

Figure 4

Infection of human BMDMs with HEV inoculums (A) Schematic flow showing differentiation of macrophages from bone marrow and preparation of cell lysate for infectivity assay (B) Infection of human BMDMs with HEV-1 (black) and HEV-3 (red) inoculums. Extracellular HEV RNA was quantified in the supernatants by qPCR. LOQ: limit of quantification. Depicted are the mean values of three independent experiments ± SEM. (C) HMOs were inoculated with cell lysate derived from BMDM on day 15 pi that contained HEV-1. HEV RNA (black; IU/mL, left Y-axis) and HEV ORF2 Ag (red; A_450/630/C.O., right axis) were measured at different time points after infection. LOQ: limit of quantification. C.O.: cut-off.

Figure 5

Immune response generated by HMACs after infection with HEV inoculums. Supernatants, collected from uninfected and HEV-infected HMACs on day 12 pi, were tested for TNF-α (A), MCP-1 (B), IL1-β (C), IFN-γ (D), IL-6 (E), and IL-12 (F) by ELISA. Depicted are the mean values of 3–5 independent experiments  ± SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 as determined by unpaired two-tailed Student’s t tests. * black compared HEV infected vs. uninfected. * red compared HEV-1-infected HMAC vs. HEV-3-infected HMACs.