Differences in the Susceptibility of Human Tubular Epithelial Cells for Infection with Orthohantaviruses

Abstract

Diseases induced by infection with pathogenic orthohantaviruses are characterized by a pronounced organ-specific manifestation. Pathogenic Eurasian orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS) with often massive proteinuria. Therefore, the use of a relevant kidney cell culture would be favorable to analyze the underlying cellular mechanisms of orthohantavirus-induced acute kidney injury (AKI). We tested different human tubular epithelial cell lines for their suitability as an in vitro infection model. Permissiveness and replication kinetics of highly pathogenic Hantaan virus (HTNV) and non-/low-pathogenic Tula virus (TULV) were analyzed in tubular epithelial cell lines and compared to human primary tubular epithelial cells. Ana-lysis of the cell line HK-2 revealed the same results for viral replication, morphological and functional effects as observed for HTNV in primary cells. In contrast, the cell lines RPTEC/TERT1 and TH1 demonstrated only poor infection rates after inoculation with HTNV and are unusable as an infection model. While pathogenic HNTV infects primary tubular and HK-2 cells, non-/low-pathogenic TULV infects neither primary tubular cells nor the cell line HK-2. Our results show that permissiveness of renal cells varies between orthohantaviruses with differences in pathogenicity and that HK-2 cells demonstrate a suitable in vitro model to study viral tropism and pathogenesis of orthohantavirus-induced AKI.

Keywords: Hantaan virus; Tula virus; acute kidney injury; cell-to-cell contacts; kidney; orthohantavirus; pathogenesis; receptor; tubular epithelium.

Figure 1

Characterization of human tubular epithelial cell lines. HK-2, TH1, and RPTEC/TERT1 cells were (A) stained for the epithelial marker protein cytokeratin 18 (CK18). Scale bar: 100 µm. (B) Surface expression of orthohantaviral entry receptors integrin αvβ3 and CD55 was detected on viable cells by flow cytometry.

Figure 2

Infection of tubular epithelial cell lines with Hantaan virus (HTNV). Cells were inoculated with HTNV, and infection was monitored by detection of nucleocapsid protein (N protein) by (A) immunofluorescence and (B) Western blot analysis of cellular lysates and cell-free supernatants (SN). Shown is a representative image at six days post infection (dpi). Scale bar: 100 µm. (C) Percentage of infected cells was quantified by counting cells expressing N protein. Three independent experiments were performed. Mean and standard deviation (±SD) are shown.

Figure 3

Viability of HTNV-infected HK-2 cells. The viability of HTNV-infected cells was tested at six dpi. Infection was monitored by staining of N protein and more than 95% of cells were infected. Scale bar: 100 µm. The viability of uninfected cells was set to 100%. Three independent experiments were performed. Mean ± SD is shown.

Figure 4

Localization of the tight junction marker protein zonula occludens-1 (ZO-1) in HTNV-infected HK-2 cells. N protein and ZO-1 were stained at three dpi in HTNV-infected and uninfected HK-2 cells. Scale bar: 100 µm.

Figure 5

Motility of HTNV-infected human primary epithelial cells (HREpCs) and HK-2 cells. (A,C) Infection of cells was monitored by immunostaining of N protein. (B,D) Migration of 30 infected and uninfected cells was analyzed in each experiment. Experiments were performed at six dpi with more than 95% of cells being infected as demonstrated by detection of N protein expression via immunofluorescence. Scale bar: 100 µm. Three independent experiments were performed. Motility of uninfected cells was set to 100%. Shown is mean ± SD. *** p < 0.0001.

Figure 6

Adhesion of HTNV-infected HREpCs and HK-2 cells. Adhesion of uninfected and infected HREpCs and HK-2 cells was analyzed. Experiments were performed at six dpi with more than 95% of cells being infected as demonstrated by detection of N protein expression by immunofluorescence. Scale bar: 100 µm. Three independent experiments are performed. Adhered uninfected cells were set to 100%. Shown is mean ± SD. * p < 0.05; ** p < 0.005.

Figure 7

Infection of HK-2 cells and HREpCs with Tula virus (TULV). Cells were inoculated with TULV at an MOI of 1. Cells were analyzed for the expression of N protein by (A,C) immunofluorescence and (B,D) Western blot. TULV-infected Vero E6 cells served as positive control for the detection of N protein by (E) immunofluorescence staining and (F) Western blot analysis. Scale bar: 100 µm.