The cytotoxic effects of RDV, RDV-N, and NHC towards Vero E6 cell were determined before the antiviral assay. As shown in Fig. 2, both of RDV and RDV-N exhibited low cytotoxicity against Vero cell line with the maximal non-toxic concentrations (MNTC) of 125 µmol/L, and NHC showed a higher cytotoxicity with MNTC of 31.25 µmol/L.
Figure 2. Cytotoxicity of RDV, RDV-N, and NHC in Vero E6 cells. Vero E6 cells were treated with indicated concentration of test compounds for 72 h, and cell viability was measured by CCK-8 assay. A The MNTC of RDV was 125 µmol/L. B The MNTC of RDV-N was 125 µmol/L. C The MNTC of NHC was 31.25 µmol/L.
PEDV infection of Vero cells can cause cytopathic effect (CPE). To determine whether the three nucleoside analogs possessed ant-PEDV activities, we investigated their effect on the morphological changes of PEDV-infected Vero E6 cells. As shown in Fig. 3, Vero E6 cells uninfected by PEDV were mainly in polygonal or irregular shape with clear outline (Fig. 3A), and not affected by 0.1% DMSO (Fig. 3B), while PEDV significantly caused cell fusion and the normal morphology of cells could not be observed (Fig. 3C). For the experiment groups, the PEDV-induced CPE was obviously blocked by the treatment with RDV, RDV-N, and NHC at the concentration of 0.5 μmol/L, 0.2 μmol/L, and 1.0 μmol/L, respectively (Fig. 3D-3F, Figures of high concentrations were not shown).
Figure 3. The effects of RDV, RDV-N, and NHC on PEDV-induced CPE. Vero E6 cells were seeded in 96-well plates for 24 h and washed thrice with serum-free medium. PEDV (0.01 MOI) was incubated with serial dilutions of test compounds at 37 ℃ for 1 h before infecting the Vero E6 cells. The mixture was incubated at 37 ℃ until the virus control group showed complete CPE. The characteristic morphological changes of PEDV-infected cells were photographed. A Non-infected cells. B Non-infected cells with 0.1% DMSO. C Cells infected with PEDV. D PEDV-infected cells with 0.5 μmol/L RDV. E PEDV-infected cells with 0.2 μmol/L RDV-N. F PEDV-infected cells with 1.0 μmol/L NHC (scale bar: 100 μm).
To measure the antiviral activities of the three compounds against PEDV, we used quantitative real-time RT-PCR (qRT-PCR) and TCID50 assay to determine the viral loads and titers in the infected cells, respectively. It was obvious to find that the viral genomic copies, as well as the viral titer, were decreased by all the three compounds in a dose-dependent manner at 48 h post infection (Fig. 4). Among them, RDV-N exhibited the strongest antiviral activity, and the virus replication was completely inhibited at the concentration of 2.0 μmol/L. Further, the EC50 values of them were determined at 72 h post infection using qRT-PCR. RDV-N had an EC50 value of 0.31 μmol/L which was twice active as its phospharamidate prodrug, RDV (EC50 = 0.74 μmol/L), and 3-folds more potent than NHC (EC50 = 1.17 μmol/L), shown in Fig. 5.
Figure 4. Reduction of PEDV replication by RDV, RDV-N, and NHC. PEDV (0.01 MOI) mixed with serial dilutions of test compounds was added to Vero E6 cells. After incubating at 37 ℃ for 48 h, cells and supernatants were harvested to determine viral loads (A-C) and titers (D-F) using qRT-PCR and TCID50 assay, respectively. A, D RDV completely inhibited PEDV replication at 7.8 μmol/L. B, E RDV-N completely inhibited PEDV replication at 2.0 μmol/L. C, F NHC completely inhibited PEDV replication at 15.6 μmol/L.
Figure 5. Dose-dependent curves of RDV, RDV-N, and NHC against PEDV. PEDV (0.01 MOI) mixed with serial dilutions of test compounds was added to Vero E6 cells. After incubating at 37 ℃ for 72 h, cells and supernatants were harvested to determine viral loads using qRT-PCR. The EC50 values were calculated from the dose response curve. A RDV had an EC50 of 0.74 μmol/L. B RDV-N had an EC50 of 0.31 μmol/L. C NHC had an EC50 of 1.17 μmol/L.
Moreover, we used an indirect immuno-fluorescence assay (IFA) to further confirm the anti-PEDV capacity of RDV-N. The viral protein expression level could be regarded as an indicator of viral quantity. From Fig. 6B and 6C, it can be found that the fluorescence intensity was significantly decreased in the presence of 0.2 μmol/L RDV-N, which strongly supported the potent antiviral activity of RDV-N.
Figure 6. Immunofluorescent imaging of PEDV-infected Vero E6 cells. Vero E6 cells precultured in 24-well plates for 24 h were infected with PEDV (0.01 MOI) in the presence or absence of 0.2 µmol/L RDV-N. DMSO was used as the blank control. The mixture was incubated for 48 h followed by immunofluorescence imaging. A Blank control. B virus control. C 0.2 µmol/L RDV-N (scale bar: 100 μm).
The pharmacokinetics of RDV-N was studied in ICR mice. As a nucleoside, RDV-N displayed poor water solubility and low lipophilicity. Predictably, this nucleoside demonstrated a low bioavailability (15.7%) in mice (Table 1). After oral administration, RDV-N rapidly reached the maximal plasma concentration within 1 h, and was eliminated quickly, similar to the data of intravenous administration. As an antiviral nucleoside, the active form of RDV-N was the nucleoside triphosphate (NTP) metabolite that mainly resided in tissues, and could hardly be detected in plasma, so we analyzed the in vitro metabolism of RDV-N and RDV in Vero E6 cells. As shown in Fig. 7, the two compounds were found to be metabolized to the corresponding triphosphate, and RDV-N gave a higher level than RDV (11.1 ± 2.5 vs 5.5 ± 1.8 pmol/million cells), which was consistent with the in vitro antiviral activities. For the acute toxicity study, the result showed that oral administration of RDV-N at the maximal dose of 1000 mg/kg did not cause animal death or marked toxic reactions.
Administration Dose (mg/kg) T1/2 (h) Tmax (h) Cmax (ng/mL) AUClast (h × ng/mL) F (%) PO 50 1.20 0.83 1826 4556 15.7 IV 25 1.02 – – 14, 513 –
Table 1. The pharmacokinetics of RDV-N in mice.
For RNA viruses, RdRp is the specific target of nucleoside antivirals, and the sequence similarity across different viruses is predictive for the antiviral spectrum of this kind of antivirals. Therefore, we performed an RdRp sequence similarity analysis among the six porcine coronaviruses (PEDV, PDCoV, TGEV, PRCV, PHEV, and SADS). The result indicated that PEDV (two strains: PEDV-CV777 and PEDV-SDSX16) shared a > 70% percent identity with SADS, TGEV, and PRCV, and a 60% percent identity with PHEV, but for porcine deltacoronavirus (PDCoV), the identity was only ~49%, shown in Fig. 8A.
Figure 8. Similarity analysis in the RdRp of porcine coronaviruses. A Sequence similarity in the RdRp of the porcine coronaviruses (PEDV, PDCoV, TGEV, PRCV, PHEV, and SADS). PEDV shared a 49.4% to 78.7% percent identity with the other five viruses. B Genetic relatedness of the porcine coronaviruses RdRp proteins shown by phylogenetic analysis. PEDV was most similar to SADS, followed by TGEV/PRCV, PHEV and PDCoV. Among them, PDCoV harbored a natural Leu at the 483 position. C Amino acid multiple sequence alignments for each RdRp functional motif (A-G) of the porcine coronaviruses. The functional domain sequences of the two PEDV strains were 100% identical. The viruses, including PEDV, PRCV, TGECV, and SADS exhibited a high sequence similarity (> 90%) in Motif B and C.
It was reported that a Leu at 483 residue of deltacoronavirus RdRp, may be associated with partial resistance to RDV (Brown et al. 2019). Accordingly, we analyzed the 483 residue of the RdRps of the six porcine coronaviruses. It was found that only PDCoV harbored a Leu at the 483 position, where was a Phe for the other viruses (Fig. 8B). The coronavirus RdRps contain four highly conserved sequence motifs (A-D) that are involved in the catalytic process, and the functions of Motif B (substrate binding) and Motif C (positioning of the template-primer and facilitating the nucleotide incorporation) are well understood (Xu et al. 2003). We mapped the amino acid residues of these functional domains of the six porcine coronaviruses. The result indicated that the functional domain sequences (Motif A-G) of the two PEDV strains were 100% identical, and the viruses, including PRCV, TGEV, and SADS shared a high similarity (> 90%) in Motif B and C with PEDV (Fig. 8C).