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

Zhai, Xiaofeng; Wang, Shilei; Zhu, Mengyan; He, Wei; Pan, Zhongzhou; Su, Shuo

The antiviral effects of LiCl and DG occur at the early stage of PDCoV replication. To further illustrate the effect of LiCl and DG on PDCoV infection, we performed time of addition experiments ( Figure 6A ). Therefore, LiCl or DG was added to infected LLC-PK1 cells at different times. In the '0 hpi' experiment, drugs and virus were added together to LLC-PK1 cells for 1 h. The cells were then washed, and the incubation was continued in the presence of the drugs. In the other experiments, LLC-PK1 cells were infected with PDCoV for 1 h, the cells were then washed, and the drugs were added at 1, 3, 6, 9, 12, 16, and 20 hpi. In all experiments, the cells were infected with PDCoV [ multiplicity of infection (MOI) = 0.05], and 60 mM LiCl or 1250 µg/mL DG was added. The viral contents were significantly reduced in cells treated with LiCl at 0 to 16 h, but no significant reduction was detected after 16 h ( Figure 6B ). For DG, the latest effective time point of drug treatment was 12 hpi ( Figure 6C ). Therefore, we can conclude that the antiviral effects of LiCl and DG occur at the early stages of PDCoV replication. 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. DG but not LiCl inhibits virus attachment to cells. To further explore in which step of the viral life cycle the drugs work, viral attachment and entry assays were performed in LLC-PK1 cells. There were no significant differences between mock-treated cells and cells treated with 60 mM LiCl in viral attachment or entry based on the mean relative viral mRNA levels ( Figure 4A ). In the entry experiments, there were no significant differences between mock-treated cells and cells treated with 1250 μg/mL DG in the mean relative viral mRNA levels. In viral attachment assays, DG treatment led to a significant reduction of viral mRNA levels compared to mock-treated cells ( Figure 4B ). DG but not LiCl inhibits virus attachment to cells. To further explore in which step of the viral life cycle the drugs work, viral attachment and entry assays were performed in LLC-PK1 cells. There were no significant differences between mock-treated cells and cells treated with 60 mM LiCl in viral attachment or entry based on the mean relative viral mRNA levels ( Figure 4A ). In the entry experiments, there were no significant differences between mock-treated cells and cells treated with 1250 μg/mL DG in the mean relative viral mRNA levels. In viral attachment assays, DG treatment led to a significant reduction of viral mRNA levels compared to mock-treated cells ( Figure 4B ). LiCl and DG have no viricidal effect on PDCoV virions. To understand whether LiCl and DG have a direct (viricidal) effect on virions, we incubated PDCoV (MOI 0.05) with 60 mM LiCl or 1250 μg/mL DG for 2 h at room temperature or 37 °C before infection of LLC-PK1 cells. The infectious titer was determined by TCID50 at 48 hours postinfection (hpi). We found that none of the drugs directly inhibited PDCoV infectivity ( Figure 5A,B) . Figure 6A ). Therefore, LiCl or DG was added to infected LLC-PK1 cells at different times. In the '0 hpi' expe