Aloe extract inhibits porcine epidemic diarrhea virus in vitro and in vivo

Abstract

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhoea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Currently there are no adequate control strategies against circulating PEDV variants, making an urgent need to exploit effect antiviral therapies to compensate for vaccines. Here, we report that Aloe extract can hamper completely the proliferation of PEDV at a non-cytotoxic concentration of 16 mg/mL determined by CCK-8 assay in Vero and IPEC-J2 cells in vitro. Furthermore, time course analysis indicated the extract exerted its inhibition at the late stage of the viral life cycle. Moreover, we also confirmed that the extract can inactivated PEDV directly but did not act on the viral genome and S1 protein. Importantly, the extract at a relatively safety concentration of 100 mg/kg of body weight, which was confirmed in mice, could reduce virus load and pathological change in intestinal tract of pigs and protect newborn piglets from lethal challenge with highly pathogenic PEDV variant GDS01 infection, indicating that Aloe extract efficiently inhibited PEDV infection in vivo. Collectively, our findings suggest that the aqueous extract from the Aloe could inhibit PEDV replication in vitro and in vivo and might be a good target for drug development against PEDV.

Keywords: Aloe extract; Antiviral activity; Porcine epidemic diarrhea virus (PEDV); Swine.

Fig. 1

The cytotoxicity of Ae to Vero and IPEC-J2 cells in vitro. Vero cells (A) or IPEC-J2 cells (B) were cultured with various concentrations of Ae (2-32 mg/mL) or the control normal DMEM for 24 h and 48 h prior to the CCK-8 assay. Results are representative of three independent experiments. Data are represented as mean ± SD, n = 8. ***stands for p < 0.001.

Fig. 2

Ae inhibits PEDV replication in vitro. Vero or IPEC-J2 cells were treated with various concentrations of Ae (4-16 mg/mL) or the control normal DMEM for 1 h, followed by infection with PEDV at an MOI of 0.2 or 0.4. After 1 h, the cells were re-treated with Ae of DMEM as control. (A) At 24 h postinoculation, an indirect immunofluorescence assay was performed. CPE and PEDV antigen were indicated by arrows. Vero or IPEC-J2 cells were treated as above described, at indicated time points (12 h, 24 h and 48 h), cell lysates were prepared and examined with Western Blot using anti-PEDV N polyclonal antibody (B) or the viral titers in the cell lysates were determined by TCID₅₀ analysis (C). Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3.

Fig. 3

Time-of-addition experiments. (A) IPEC-J2 cells or PEDV was treated with Ae at different times before and after infection. Double-headed red arrows indicate the presence of Ae. The experiments are identified in the text by the numbers on the left (M1-M7). Cells were harvested at 12 hpi. (B) The mRNA expression of N was examined with real-time PCR using specific primers. The expression level of mRNA were calculated in relation to the expression level of GAPDH. (C) The N protein of PEDV was detected by Western Blot using anti-PEDV N polyclonal antibody. (D) The viral titers in the culture medium were determined by TCID₅₀ analysis. Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3. **stands for p < 0.01, ***stands for p < 0.001.

Fig. 4

Ae inactivated PEDV directly but do not act on the viral genome and S1 protein. Incubating PEDV with indicated concentration (16 mg/mL) of Ae at 37 °C for 1 h or 3 h, then virus titer was determined in Vero cells (A), the whole genome sequences of the PEDV were amplified by RT-PCR (B) and the degradation of S1 protein at the indicated times after treatment with Ae was examined by SDS-PAGE analysis (C). Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3.

Fig. 5

The toxicity study of Ae in vivo. (A) Survival curves of mice were orally administered Ae in a single dose of 0, 4, 20, 100, 500, or 2500 mg/kg bw in 8 days. Subchronic toxicity evaluation of oral Ae with a single dose of 0, 4, 20, 100, 500, or 2500 mg/kg bw in mice. (B) The oral doses of Ae for the 6 groups of mice were 0, 4, 20, 100, 500, or 2500 mg/kg bw, respectively. Body weight was monitored and plotted. Mice were sacrificed at day 28, and white blood cells (WBC) (C), red blood cells (RBC) (D), platelets (PLT) (E) were detected. Data are represented as mean ± SD, n = 6. *stands for p < 0.05.

Fig. 6

Oral administration of Ae can protect piglets against PEDV infection in vivo. (A) Macroscopic picture of intestine from a control piglet at 2 d.p.i. (B-C) Macroscopic pictures of intestine from Ae + PEDV and DMEM + PEDV-challenged piglets at 2 d.p.i. Thin-walled intestinal tracts were indicated by arrows. (D) H&E-stained jejunum tissue section of a control piglet at 2 d.p.i. (E-F) H&E-stained jejunum tissue section of Ae + PEDV and DMEM + PEDV-challenged piglets at 2 d.p.i. Blunt intestinal villus was indicated by arrows. (G) Immunohistochemically stained jejunum tissue section of a control piglet at 2 d.p.i. (H-I) Immunohistochemically stained jejunum tissue section of Ae + PEDV and DMEM + PEDV-challenged piglets at 2 d.p.i.. (J) The mortality of newborn piglets in each group was recorded from 1 to 2 d.p.i.. Data are represented as mean ± SD, n = 4.