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

LiCl and DG cytotoxicity in LLC-PK1 cells. The cellular cytotoxic effect of LiCl and DG in LLC-PK1 cells was determined using the CCK8 kit. The structure of DG is shown in Figure 1A . The maximum LiCl and DG concentrations that resulted in a cell viability higher than 80% were 60 mM and 1250 μg/mL, respectively ( Figure 1B ,C). No differences in cell morphology compared to the mock-treated cells were observed (data not shown) at concentrations of 60 mM LiCl and 1250 μg/mL DG; therefore, 10-60 mM LiCl and 125-1250 μg/mL DG were used in subsequent antiviral assays. LiCl and DG inhibit PDCoV replication. Next, we investigated the effect of LiCl and DG on the replication of PDCoV in LLC-PK1 cells. PK-1 cells were infected with PDCoV at MOI 0.05 for 1 h and treated with 10-60 mM LiCl or 125-1250 μg/mL DG for 24 h. The relative mRNA expression was detected by real-time qPCR. Treatment with 10-60 mM LiCl or 125-1250 μg/mL DG inhibited viral mRNA levels significantly (Figures 2A and 3A) . Infectious virus loads in cell culture supernatants were determined by TCID50. We found that 10-60 mM LiCl or 125-1250 μg/mL DG significantly inhibited viral replication compared to mock-treated cells ( Figures 2B and 3B ). In IFA, the fluorescence intensity declined after treatment with 10-60 mM LiCl or 125-1250 μg/mL DG in a dosedependent manner ( Figures 2C and 3C ). The fluorescence intensity was quantified, showing that the number of infected cells decreased in a drug dose-dependent manner ( Figures 2D and 3D ). These results indicate that LiCl and DG inhibit PDCoV replication in a dose-dependent manner. 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 ).