Antiviral Effect of Lithium Chloride and Diammonium Glycyrrhizinate on Porcine Deltacoronavirus In Vitro

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging global swine virus that has a propensity for interspecies transmission. It was identified in Hong Kong in 2012. Given that neither specific antiviral drugs nor vaccines are available for newly emerging porcine deltacoronavirus, searching for effective antiviral drugs is a high priority. In this study, lithium chloride (LiCl) and diammonium glycyrrhizinate (DG), which are host-acting antivirals (HAAs), were tested against PDCoV. We found that LiCl and DG inhibited PDCoV replication in LLC-PK1 cells in a dose-dependent manner. The antiviral effects of LiCl and DG occurred at the early stage of PDCoV replication, and DG also inhibited virus attachment to the cells. Moreover, both drugs inhibited PDCoV-induced apoptosis in LLC-PK1 cells. This study suggests LiCl and DG as new drugs for the treatment of PDCoV infection.

Keywords: PDCoV; apoptosis; diammonium glycyrrhizinate; lithium chloride.

Figure 1

Cytotoxic effect of LiCl and DG treatment in LLC-PK1 cells. (A) Chemical structure of diammonium glycyrrhizinate (DG). Cells were treated with 0–100 mM LiCl (B) or 50–5000 μg/mL DG (C) for 36 h. The relative cell viability was evaluated by the CCK-8 Kit according to the manufacturer’s instructions. The data are expressed as the mean ± SD of three independent experiments. The dotted line indicates the 80% cytostatic concentration (CC₈₀).

Figure 2

Antiviral effect of LiCl on porcine deltacoronavirus (PDCoV) replication. (A) The relative viral RNA level was determined by real-time qPCR. (B) The viral titer (log₁₀ TCID₅₀/_mL) in LLC-PK1 cell lysates was calculated by the method of Reed and Muench. (C) At 24 h post-infection, PDCoV (MOI 0.05) replication in LLC-PK1 cells was determined by indirect immunofluorescence assay (IFA). Green fluorescence represents PDCoV replication, while blue fluorescence represents the nuclear distribution. (D) The fluorescence intensity in C was quantified with the software ImageJ. Quantification of PDCoV-infected cells from the IFA images is presented as percentage, taking 0 mM LiCl as 100%. Values represent the mean ± SD of three independent experiments; ns, not significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar, 250 μm.

Figure 3

Antiviral effect of DG on PDCoV replication. (A) The relative viral RNA level was determined by real-time qPCR. (B). The viral titer (log₁₀ TCID₅₀/_mL) in LLC-PK1 cell lysates was calculated by the method of Reed and Muench. (C) At 24 h post-infection, PDCoV (MOI 0.05) replication in LLC-PK1 cells was determined by IFA. Green fluorescence represents the PDCoV replication, while blue fluorescence represents the nuclear distribution. (D) The fluorescence intensity in C was quantified with the software ImageJ. Quantification of PDCoV infected cells from the IFA images is presented as percentage, taking 0 μg/mL DG as 100%. Values represent the mean ± SD of three independent experiments; ns, not significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar, 250 μm.

Figure 3

Antiviral effect of DG on PDCoV replication. (A) The relative viral RNA level was determined by real-time qPCR. (B). The viral titer (log₁₀ TCID₅₀/_mL) in LLC-PK1 cell lysates was calculated by the method of Reed and Muench. (C) At 24 h post-infection, PDCoV (MOI 0.05) replication in LLC-PK1 cells was determined by IFA. Green fluorescence represents the PDCoV replication, while blue fluorescence represents the nuclear distribution. (D) The fluorescence intensity in C was quantified with the software ImageJ. Quantification of PDCoV infected cells from the IFA images is presented as percentage, taking 0 μg/mL DG as 100%. Values represent the mean ± SD of three independent experiments; ns, not significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar, 250 μm.

Figure 4

Effect of LiCl and DG on different stages of the PDCoV replication cycle. (A). The levels of vRNA in cells treated with 60 mM LiCl during viral attachment and entry were determined by real-time qPCR. (B) Same experiment as in A but using DG at 1250 μg/mL. Values represent the mean ± SD of three independent experiments; ns, no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

The viricidal effect of LiCl and DG on PDCoV. (A) PDCoV virions were incubated with 60 mM LiCl at 37 °C or room temperature. The viral titers (log₁₀ TCID₅₀/_mL) were calculated by the method of Reed and Muench. (B) Same experiment as in A but using DG at 1250 μg/mL. Values represent the mean ± SD of three independent experiments, ns, no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Time-dependent effect of LiCl and DG on PDCoV replication. (A) Outline of the experimental set up. (B,C) LLC-PK1 cells were incubated with PDCoV (MOI 0.05) for 1 h, followed by treatment with 60 mM LiCl (B) or 1250 μg/mL DG (C) at the indicated time (hpi). The viral titer in LLC-PK1 cell lysates was calculated using the method of Reed and Muench. Values represent the mean ± SD of three independent experiments; ns, no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Time-dependent effect of LiCl and DG on PDCoV replication. (A) Outline of the experimental set up. (B,C) LLC-PK1 cells were incubated with PDCoV (MOI 0.05) for 1 h, followed by treatment with 60 mM LiCl (B) or 1250 μg/mL DG (C) at the indicated time (hpi). The viral titer in LLC-PK1 cell lysates was calculated using the method of Reed and Muench. Values represent the mean ± SD of three independent experiments; ns, no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7

Effect of LiCl and DG on cell apoptosis caused by PDCoV infection. The cells were incubated with PDCoV (MOI 0.05) for 1 h. Thereafter, the supernatant was removed, and DMEM supplemented with 60 mM LiCl or 1250 μg/mL DG was added. At 36 hpi, the rates of cell apoptosis were analyzed by flow cytometry.