Hypericum japonicum extract inhibited porcine epidemic diarrhea virus in vitro and in vivo

Abstract

Porcine epidemic diarrhea virus (PEDV) infection causes lethal watery diarrhea and high mortality in neonatal piglets, leading to huge economic losses in the global swine industry. Currently, the existing commercial vaccines cannot fully control PEDV, so it is urgent to develop effective antiviral agents to complement vaccine therapy. In the present study, we investigated the antiviral effect of Hypericum japonicum extract (HJ) against PEDV in vivo and in vitro. In in vitro assays, HJ could directly inactivate PEDV strains; moreover, it inhibited the proliferation of PEDV strains in Vero or IPI-FX cells at its non-cytotoxic concentrations. Time of addition assays revealed that HJ mainly inhibited PEDV at the later stages of the viral life cycle. In in vivo, compared with the model group, HJ could reduce the viral titers in the intestines of infected piglets, and improve their intestinal pathological, indicating that HJ could protect the newborn piglets from highly pathogenic PEDV variant infection. Furthermore, this effect may be related to the fact that HJ can not only directly inhibit viruses, but also regulate the structure of intestinal microbiota. In conclusion, our results indicate that Hypericum japonicum could inhibit PEDV replication in vitro and in vivo and might possess the potential to develop as the anti-PEDV drug.

Keywords: Hypericum japonicum; antiviral effect; intestinal microbiota; piglets; porcine epidemic diarrhea virus; watery diarrhea.

FIGURE 1

Cytotoxicity of HJ to Vero and IPI-FX cells in vitro. Viability of Vero cells treated with different concentrations of HJ (0.062–2 mg/mL) for 24 h (A) and 48 h (B), and the viability of IPI-FX cells treated with different concentrations of HJ (0.031–1 mg/mL) for 24 h (C) and 48 h (D) were measured by CCK-8 method. Meanwhile, the control normal DMEM or solvent DMEM with sterile water for 24 h and 48 h were detected. Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3. *, and p < 0.05 was considered statistically significant compared with the control group.

FIGURE 2

The PEDV N gene in infected Vero (A) or IPI-FX cells (B) was examined with real-time PCR using specific primers. Cells were treated with HJ throughout the experiment. The expression level of N mRNA was calculated in relation to the expression level of GAPDH at 24 hpi. Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3. *, and p < 0.05; **, and p < 0.01 were considered statistically significant compared with the model group.

FIGURE 3

The expression level of PEDV N protein in Vero (A) or IPI-FX (B) cells were examined by western blot assays. Cells were treated with HJ throughout the experiment. The expression level of PEDV N protein was calculated in relation to the expression level of GAPDH at 24 hpi. Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3. *, and p < 0.05 was considered statistically significant compared with the model group.

FIGURE 4

The viral titers of PEDV-CV777 (A) and PEDV-G2 (B) were determined by measuring TCID₅₀ with the end-point dilution methods. Cells were treated with HJ throughout the experiment, and samples were harvested for 24 h. Data are represented as mean ± SD, n = 3. *, and p < 0.05 was considered statistically significant compared with the model group, while ^#, and p < 0.05 was considered statistically significant compared with ribavirin group.

FIGURE 5

Inhibitory effects of HJ on IPI-FX cells infected with PEDV virus at different stages. (A) IPI-FX cells were treated with HJ at different times before or after infection. Double-headed red arrows indicate the presence of HJ, while the orange horizontal lines indicate the time of PEDV infection. The experiments are identified in the text by the numbers on the left (M0–M7). (B) The expression level of PEDV N protein in IPI-FX cells was examined by western blot assays. Cells were harvested for 12 h. The expression level of PEDV N protein was calculated in relation to the expression level of GAPDH. Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3. *, and p < 0.05 was considered statistically significant compared with M1, while ^#, and p < 0.05 was considered statistically significant compared with M2.

FIGURE 6

The effect of HJ on the survival rate of PEDV-challenged piglets was recorded daily. At 3 days of age, piglets were orally administered HJ or water twice a day. All piglets were orally challenged with 5 mL DMEM containing a total of l0⁵ PFU of PEDV-G2 solution on day 8.

FIGURE 7

Effect of HJ on the macroscopic and histological changes of piglets infected with PEDV. Piglets received HJ (1.28 g/kg) by oral administration for 6 days before PEDV-G2 (5 mL DMEM containing l0⁵ PFU) infection. The intestinal microscopic lesions of piglets in the model group (A) and the treated group (B) were recorded before sampled. The thin-wall intestinal tracts containing undigested food were indicated by blue arrows. The histological changes of intestine of piglets in the model group (C) and the treated group (D) were analyzed by hematoxylin-eosin (HE) staining. The damage and shedding of intestinal villi were indicated by red arrows. The height of villi (E) and crypt (F) in HE staining were measured to evaluate the growth and development of intestinal. Data are represented as mean ± SD, n = 3. *, and p < 0.05 was considered statistically significant compared with the model group.

FIGURE 8

Effect of HJ on the viral load of PEDV in the ileum of piglets. The presence of PEDV in ileum were examined with real-time PCR using specific primers. The expression levels of mRNA were calculated in relation to the expression level of GAPDH. Data are represented as mean ± SD, n = 7. *, and p < 0.05 was considered statistically significant compared with the model group.

FIGURE 9

Alpha-diversity and beta-diversity analysis on the intestinal microbiota of piglets. (A) Chao, (B) ACE, (C) Shannon, and (D) Simpson indexes calculated after rarefying to an equal number of sequence reads for all samples. Data are represented as box plots. (E) PCoA of the intestinal microbiota of 10-day-old piglets in different groups. The blue dots represented the model group, and the orange dots represented the treated group. (F) NMSD of the intestinal microbiota of 10-day-old piglets in different groups. Model represents piglets challenged with PEDV, and treated represents piglets received an oral administration of HJ before PEDV challenged. Dots of the same color indicate that they belong to the same group. The closer the dots were, the more similar the composition of the samples was, n = 7.

FIGURE 10

Average relative abundance of small intestinal microbial species at the phylum level. Model represents piglets challenged with PEDV, and treated represents piglets received an oral administration of HJ before PEDV challenged, n = 7.

FIGURE 11

Effects of HJ on intestinal microbiota and key microbiota at the species level. (A) Species composition heat map at the genus level for species clustering. The higher the yellow degree was, the lower the relative abundance of the species was; and the higher the red degree was, the higher the relative abundance of the species was. The contents of (B) Ligilactobacillus_agilis, (C) Weissella_cibaria, and (D) Lactobacillus_amylovorus were measured to evaluate the changes of intestinal microbiota. Data are presented as mean ± SD, n = 7.