Evaluation of antiviral activity of Bacillus licheniformis-fermented products against porcine epidemic diarrhea virus

Abstract

Bacillus licheniformis (B. licheniformis) is commonly used as probiotic and its secondary metabolites are attractive anti-microbial candidate. In the present study, we aimed to evaluate the antiviral activity of crude extracts from B. licheniformis against porcine epidemic diarrhea virus (PEDV), a highly contagious enveloped porcine virus that has caused great economic loss in pigs. In vivo, PEDV-infected piglets supplemented with air-dried solid state fermentative cultivate containing B. licheniformis-fermented products (BLFP) showed milder clinical symptoms and decreased viral shedding. Importantly, no significant systemic pathological lesions and no reduction in average daily gain were noted in pigs supplemented with the BLFP, which suggests that it is safe for use in pigs. In vitro experiments revealed that while B. licheniformis crude extracts exhibited no toxicity in Vero cells, co-cultivation of B. licheniformis crude extracts with PEDV significantly reduced viral infection and replication. Summarized current results suggest that the B. licheniformis-fermented products could be a novel candidate food additive for reducing the impact of PED on the swine industry.

Keywords: Antiviral; B. licheniformis-fermented products; PEDV.

Fig. 1

Identification of the 2nd metabolites in standard substance surfactin derived from Bacillus subtilis (a) and fermentative sample derived from Bacillus licheniformis-fermented products (BLFP) (b) by liquid chromatography–mass spectrometry (LC–MS). Using the standard curve of surfactin derived from Bacillus subtilis (c), the 2nd metabolite at [M+Na]⁺m/z 1058 in Bacillus licheniformis-fermented products crude extract (d) was identified and quantified

Fig. 2

Clinical scoring of fecal consistency. Compared to PEDV-infected groups, the appearance and duration of typical diarrhea (score 2–3) were reduced in the PEDV + BLFP group

Fig. 3

Fecal shedding of PEDVPT-P6 detected in piglets fed < 5 kg/ton BLFP in each group (n = 5). The pattern of viral shedding in the PEDV + BLFP group was similar to but lower than that of the PEDV group during the study, with no significant difference detected. Changes in the mean values of genomic equivalents (GE)/mL are presented as log10 values ± SD. The viral RNA loads of inoculated groups and the control group were compared using a non-parametrical Kruskal–Wallis test

Fig. 4

Average daily gain of pigs in each group. All pigs were weighed weekly in order to evaluate their growth performance with or without BLFP as feed additives. No statically significant difference in the average daily gain was noted among all groups each week

Fig. 5

The 50% cytotoxicity concentration of crude extract derived from Bacillus licheniformis (BLFP) in Vero cells. Different concentrations of BLFP crude extract were added to Vero cells and incubated for 48 h. BLFP crude extract showed no cytotoxicity to Vero cells when evaluated using the AlamarBlue™ assay. Data are presented as the mean ± SD out of three test replicates

Fig. 6

Antiviral activity of crude extract derived from Bacillus licheniformis (BLFP) against PEDV infection, as well as the IC₅₀, was determined using the AlamarBlue™ assay. a PEDV-infected Vero cells treated with (left) and without (middle) 150 ppm BLFP crude extract. The right panel shows Vero cells without any treatment. Brown coloration indicates a positive PEDV S protein signal located in the cytoplasm of PEDV-infected cells. b Dose-dependent effect of BLFP crude extract against PEDV. The half-maximal inhibitory concentration (IC₅₀) of BLFP crude extract against PEDV in Vero cells is 0.07 ± 0.45 ppm. Data are presented as the mean ± SD out of three test replicates

Fig. 7

Time-of-addition assay of the BLFP crude extract. a Plot of the time-of-addition assays for BLFP crude extract treatment. 150 ppm of BLFP crude extract was added to PEDV-infected cells with various treatments. b Evaluation of antiviral activity of BLFP crude extract by AlamarBlue™ assay. c Detection of viral RNA of PEDV in the supernatants. Statistical analysis was performed by one-way ANOVA and followed by Tukey’s multiple comparisons test. a, b significant differences between groups are indicated with different letters (p < 0.05)

Fig. 8

Replication kinetics of PEDV in Vero cells treated with or without BLFP crude extract. a Extracellular viral titers in the supernatants of PEDV-infected Vero cells treated with and without BLFP crude extract were determined by viral titration in Vero cells using the Reed-Müench method and expressed as the 50% TCID₅₀/ml. b Extracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time reverse transcription (RT)-PCR. c Intracellular viral titers in PEDV-infected Vero cells treated with or without BLFP crude extract were determined by viral titration in Vero cells. d Intracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time RT-PCR. Statistical analysis was performed using Student’s t-test, and statistically significant differences were labeled with *(p < 0.05)