A Cytopathic Effect-Based Tissue Culture Method for HCoV-OC43 Titration Using TMPRSS2-Expressing VeroE6 Cells

Abstract

Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID₅₀) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID₅₀ assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID₅₀ assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID₅₀ assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.

Keywords: 50% tissue culture infectious dose assay; HCoV-OC43; TMPRSS2; cytopathic effect; human coronavirus; titration.

FIG 1

CPE evaluation after high-titer HCoV-OC43 infection. First, HCT-8, VeroE6, and VeroE6/TMPRSS2 cells were seeded in 96-well plates and infected with high-titer (2.0 × 10⁵ TCID₅₀/ml) HCoV-OC43 samples, and their CPEs were evaluated from 1 to 5 days after infection. CPE was observed with an inverted light microscope (Olympus IX71; Olympus, Tokyo, Japan). VeroE6/TMPRSS2 cells were cultured in DMEM supplemented with 1%, 2%, 3%, 4%, or 5% fetal bovine serum after virus inoculation. Scale bar, 100 μm.

FIG 2

CPE evaluation after medium-titer HCoV-OC43 infection. First, HCT-8, VeroE6, and VeroE6/TMPRSS2 cells were seeded in 96-well plates and infected with medium-titer (2.0 × 10³ TCID₅₀/ml) HCoV-OC43 samples, and their CPEs were evaluated from 1 to 5 days after infection. CPE was observed with an inverted light microscope (Olympus IX71; Olympus, Tokyo, Japan). VeroE6/TMPRSS2 cells were cultured in DMEM supplemented with 1%, 2%, 3%, 4%, or 5% fetal bovine serum after virus inoculation. Scale bar, 100 μm.

FIG 3

CPE evaluation after low-titer HCoV-OC43 infection. First, HCT-8, VeroE6, and VeroE6/TMPRSS2 cells were seeded in 96-well plates and infected with low-titer (2.0 × 10¹ TCID₅₀/ml) HCoV-OC43 samples, and their CPEs were evaluated from 1 to 5 days after infection. CPE was observed with an inverted light microscope (Olympus IX71; Olympus, Tokyo, Japan). VeroE6/TMPRSS2 cells were cultured in DMEM supplemented with 1%, 2%, 3%, 4%, or 5% fetal bovine serum after virus inoculation. Scale bar, 100 μm.

FIG 4

Viral kinetics of HCoV-OC43. HCT-8, VeroE6, and VeroE6/TMPRSS2 cells were seeded in 12-well plates and infected with a specific titer of HCoV-OC43 (1.0 × 10² TCID₅₀/ml). The viral titer in the culture supernatant of each well was measured from 1 to 6 days after infection. VeroE6/TMPRSS2 cells were cultured in DMEM supplemented with 2% or 5% fetal bovine serum after virus inoculation. For each measurement, three independent experiments were performed, and the results are expressed as mean ± standard error values.

FIG 5

Comparison between CPE-based and IPA-based titer measurements. HCoV-OC43 sample solutions with titers of 2.0 × 10⁵, 2.0 × 10³, and 2.0 × 10¹ TCID₅₀/ml were prepared as high-titer, medium-titer, and low-titer virus samples, respectively. High-titer (A), medium-titer (B), and low-titer (C) HCoV-OC43 samples were titrated with CPE evaluation and the IPA method. HCT-8, VeroE6, and VeroE6/TMPRSS2 cells were used for viral titration, and VeroE6/TMPRSS2 cells were cultured in DMEM supplemented with 2% or 5% fetal bovine serum during the titration. For each measurement, four independent experiments were performed, and the results are expressed as mean ± standard error values. The reference titer value, which is the titer measured with IPA using HCT-8 cells, is indicated by the dotted red line on each panel.