key: cord-0705776-zyaeinse
authors: Xu, Zhichao; Liu, Yuan; Peng, Peng; Liu, Yufang; Huang, Meiyan; Ma, Yehuan; Xue, Chunyi; Cao, Yongchang
title: Aloe extract inhibits porcine epidemic diarrhea virus in vitro and in vivo
date: 2020-09-15
journal: Vet Microbiol
DOI: 10.1016/j.vetmic.2020.108849
sha: fc4b6d5c3b9c15116988f6a9f7b6bfb639cf678b
doc_id: 705776
cord_uid: zyaeinse

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.

as virus attachment and entry, and induction of neutralizing antibodies in vivo (Wen et al., 2018) . PEDV can cause severe watery diarrhea with subsequent dehydration in pigs of all ages, and a high mortality rate in 7-10-day-old suckling piglets Pijpers et al., 1993; Stevenson et al., 2013) . Since PED was first reported in Belgium in 1978, this disease spreaded soon to other regions in Europe and Asia (Pensaert and Bouck, 1978; Song and Park, 2012) . Recently, there have been devastating outbreaks of PEDV variants in China and United States, which is characterized by high death rates among infected suckling piglets, result in substantial economic losses to the swine industry (Sun et al., 2016; Vlasova et al., 2014) .

To combat the virus, great efforts have been made to prevent and control PEDV since the variants emerged. For virus infection prevention, the administration of vaccines is one of important tool. Currently a number of vaccines including killed, live-attenuated vaccine, recombinant vector vaccine, DNA vaccine and subunit vaccine are widely used to prevent PEDV infection (Collin et al., 2015; Lawrence and Bey, 2015; Lin et al., 2017a; Liu et al., 2012; Meng et al., 2013) . However, it still can not stop the PED outbreak, due to these vaccines no capable of inducing solid intestinal mucosal immunity (Wen et al., 2018) . Antiviral agents can be used in either a therapeutic or a prophylactic mode to compensate for vaccines. Previous studies have discovered a few natural compounds and compositions such as quercetin J o u r n a l P r e -p r o o f 7-rhamnoside (Choi et al., 2009) , 2-Deoxy-D-glucose , glycyrrhizin (Huan et al., 2017) , lithium chloride (Li et al., 2018a) and surfactin (Yuan et al., 2018) have antivral activities on PEDV. However, there are no effective commercial antiviral agents available to control PEDV infection until now.

It has been identified that natural products can be candidates as new generations of antiviral agents (Harvey, 2008) . Aloe has antiviral activity against a wide range of viruses. Research has proved that Aloe has significant inhibitory effect on influenza virus, pigeon paramyxovirus type 1, Herpes simplex virus type 1 (Dziewulska et al., 2017; Gansukh et al., 2018; Rezazadeh et al., 2016) . Antiviral effects are described not only for whole extracts of Aloe, but also for their isolated compounds. It was reported that the constituents of Aloe, such as quercetin, catechin hydrate, kaempferol, aloin and emodin have significant antiviral activity (Huang et al., 2019; Jang-Gi Choi, 2019; Li et al., 2014; Lin et al., 2008; Mohammad et al., 2019; Sydiskis et al., 1991) .

The antiviral activity could be caused by inducing antiviral genes, inhibition of viral enzymes and protein expression into mammalian cells (Huang et al., 2019; Jang-Gi Choi, 2019; Li et al., 2014; Lin et al., 2008) .

Although Aloe could serve as antiviral angent against many viruses, detailed information about the antiviral capacity against PEDV remain unclear. In this study, we investigated whether the Aloe extract (Ae) had the ability to inhibit PEDV infection. We first examined its potential to inhibit PEDV replication in vitro and determined the stages in the PEDV life cycle that could be blocked by Ae, and then extended our study in newborn piglets at a relatively safety concentration to see if Ae J o u r n a l P r e -p r o o f could inhibit PEDV infection in vivo. Our results showed that Ae inhibited PEDV infection both in vitro and in vivo, indicating that Ae can be potentially useful for the development of anti-PEDV therapeutics.

African green monkey kidney (Vero) cells were obtained from ATCC (ATCC number: CCL-81) (USA) and Porcine intestinal epithelial cell clone J2 (IPEC-J2) cells were obtained from Wen' s Foodstuffs Group Co, Ltd (Guangdong, China) . All cells were cultured in Dulbecco' s modified eagle medium (DMEM) (Hyclone, USA) supplemented with 100 U/mL penicillin, 100 U/mL streptomycin, and 10% fetal bovine serum (FBS) (BOVOGEN, Australia). The maintenance medium for PEDV propagation was DMEM supplemented with 7.5 μg/mL trypsin (Gibco, USA).

The isolation and identification of PEDV GDS01 strain were reported previously by our laboratory (Hao et al., 2014; Li et al., 2018b) . Virus propagation was performed as previously described with some modifications (Wen et al., 2018) . Briefly, Vero cells were seeded into T175 flasks and cultured for 90% confluent. Fifty microliters of PEDV GDS01 strain together with 50 mL maintenance medium were added to flask after the cell monolayers were washed three times with sterile pH 7.4 1 × phosphate buffered saline (PBS). The virus-inoculated cells were cultured continuously at 37℃ in 5% CO2 to observe cytopathic effect (CPE). Around 1 day post-infection (d.p.i.), > 90% CPE was evidently observed in the inoculated cell monolayers, the flasks were J o u r n a l P r e -p r o o f twice frozen at -80℃ and thawed. The cells and supernatants were harvested together and stored at -80℃ until subsequent viral titers determination. Virus titers were determined using the Reed-Muench method (Muench, 1938) and expressed as tissue culture infective dose 50% (TCID50). Plaque forming unit (PFU) was determined according to ''PFU = 0.7 × TCID50'' as described before (Birgit et al., 2007) , and the multiplicity of infection (MOI) was calculated based on PFU.

The dried powder of Ae was purchased from Bioforte Biotechnology Co., Ltd.

(Shenzhen, China). One hundred milligram (mg) Ae was dissolved with 1 milliliter (mL) DMEM and filtered with 0.22 mm filters and then stored at -4℃ until use.

The cytotoxicity of Ae in Vero and IPEC-J2 cells were detected by using commercial Cell Counting Kit-8 (CCK-8) (Yeasen Biotech, China) in accordance with the manufacturer's instructions. Briefly, cells were seeded into 96-well plates and cultured for 90% confluent, and then incubated with DMEM or Ae (2-32 mg/mL) for 24 h and 48 h. Then 10 μL CCK-8 reagent and 90 μL DMEM were added to each well and incubated at 37℃ for 1 h. The CCK-8 signal was measured at an absorbance of OD450 nm. The relative viability of the cells was counted based on the expressions: cell survival rate (%) = [OD (sample)-OD (blank)/OD (control) -OD (blank)] × 100%.

Confluent Vero or IPEC-J2 cell monolayers in 12-well plate were inoculated with various concentrations of Ae (4-16 mg/mL) or the control normal DMEM for 1 h, J o u r n a l P r e -p r o o f followed by infection with PEDV at an MOI of 0.2 or 0.4 for 1 h, and then the viral inoculums was removed and fresh maintenance medium containing different concentrations of Ae was added. Twenty-four hours later, cells were fixed for indirect immunofluorescent assay as described below. Cells were collected at indicated time points (12 h, 24 h and 48 h), cell lysates were prepared and examined with Western Blot as described below or the viral titers in the cell lysates were determined by TCID50 analysis to calculate PFU as above described.

Immunofluorescence assay was conducted to observe PEDV-infected Vero or IPEC-J2 cells as described previously with some modifications (Xu et al., 2019) .

Briefly, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton X-100 for 15 min at room temperature. After blocked with 1% bovine serum albumin (BSA), cells were stained with anti-PEDV N polyclonal antibody (Wen' s Foodstuffs Group Co., Ltd, China) (1:1000) at 37℃ for 1 h. Cells were then washed with 1 × PBS and incubated with fluoresceinisothiocyanate (FITC) (1:500) or Cy3-labeled goat anti-mouse secondary antibody (KPL, USA)

(1:500) at 37℃ for 1 h. After three washes in 1 × PBS, cells were counter-stained with DAPI and observed with a fluorescence microscope (LEICA DMi8, Germany).

The whole-cell extracts were prepared with RIPA lysis buffer (Beyotime, China) supplemented with 1% protease inhibitors (Yataihengxin, China) and boiled with 6 × SDS loading buffer for 10 min before same volume of protein samples were J o u r n a l P r e -p r o o f fractionated by 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) electrophoresis, and the resolved proteins were transferred onto PVDF membranes (Millipore, USA). After blocking with 5% skim milk, the membranes were incubated for 1 h at room temperature with anti-PEDV N polyclonal antibody (Wen' s Foodstuffs Group Co., Ltd, China) (1:1000) or anti-GAPDH antibody (Proteintech Group, Inc., USA) (1:1000). The blots were subsequently incubated with HRP-conjugated goat anti-mouse IgG (1:8000) (DingGuoShengWu, China) for 1 h min at room temperature. The blots were visualized using the ECL reagent according to the manufacturer's instructions (CWBio, China).

IPEC-J2 cells were seeded on 12-well plates and cultured overnight before infection with PEDV at an MOI of 0.4 and were incubated for 1 h at 4°C to synchronize infection. The inoculum was removed; 1 mL of DMEM was added to each well; and samples were placed in a humidified incubator at 37°C. At various time points, a Ae solution was added to the wells or to the virus samples, to a final concentration of 16 mg/mL ( Fig 3A) . After 12 h, the mRNA and protein levels of N in the cells were measured by real-time PCR as described below and Western Blot as above described.

Cell lysates were collected and the viral titers were determined by a TCID50 assay to calculate PFU as above described.

Total viral RNA was prepared from IPEC-J2 cells after infection with PEDV using a RNeasy kit (Magen, China) per the manufacturer' instruction, and was treated with for 15 s. The final step was to obtain a melt curve for the PCR products to determine the specificity of the amplification. All samples were carried out in triplicate on the same plate, and the GAPDH gene was utilized as the reference gene. Expression levels of N genes were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples.

Determination of direct virion inactivation activity of Ae was performed as previously described with some modifications (Gao et al., 2014) . Briefly, PEDV GDS01 of 1 × 10 6 PFU was incubated with Ae at a concentrations of 16 mg/mL for 1 J o u r n a l P r e -p r o o f h or 3 h at 37°C. Following the treatment, viral infectivity was determined on Vero cells and the viral genome after Ae treatment was examined by RT-PCR using the specific primers as previously described (Zhang et al., 2016) . To investigate whether

Ae could degrade directly S1 protein, Ae at the concentration of 16 mg/mL was incubated with 100 μg purified S1 protein (Wen's Foodstuffs Group Co., Ltd, China) for 1 h or 3 h at 37°C, and then the mixtures were harvested to analysis by SDS-PAGE gel as above described and the gel was stained with Coomassie blue dye for analysis of specific bands.

The animal study was approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University (Guangdong, China) and animals were treated in accordance with the regulations and guidelines of this committee. The toxicity evaluation and anti-PEDV of Ae in vivo were performed as previously described with some modifications (Yuan et al., 2018) . Briefly, 36 BALB/c mice were procured from Yancheng Biological Technology Co, Ltd (Guangzhou, China) and were randomly divided into six groups (6 mice/group) and were housed in six separate rooms. On day 0, mice in group 1 were intragastrically administered with 0.5 mL DMEM and served as controls. Mice in group 2, 3, 4, 5 and 6 were intragastrically administered with 0.5 mL DMEM containing Ae at a dose of 5, 20, 100, 500 and 2500 mg/kg of body weight (bw), respectively. In the acute toxicity study, the death of mice was observed and recorded in 8 days. For subchronic toxicity assessment, after Ae administration, the body weight of each mouse was measured daily and all mice were necropsied at J o u r n a l P r e -p r o o f 11 28 days post inoculation (d.p.i.). At necropsy, blood samples from all mice were collected and blood cells detection were performed.

For the PEDV protection experiment, twelve 4-day-old crossbred conventional newborn piglets produced by Duroc × Landrace × Big White were procured from Wen' s Foodstuffs Group Co, Ltd (Guangdong, China) randomly divided into three groups (4 piglets/group) and were housed in three separate rooms. All pigs were maintained in our animal facility with a mixture of skim milk powder (Zhaoqing Bozheng Technology Co., Ltd, China) with warm water ad libitum for 1 day before the experimentation. Prior to inoculation, newborn piglets were confirmed negative for the major procine enteric viruses (PDCoV, PEDV, TGEV, PRoV) by testing the rectal swabs on day -1 as previously described . In addition, serum of each piglet was collected and a commercial kit (ALLRIGHT BIOTECHONOLOGY, China) was applied in order to detect PEDV-specific antibody according to the manufacturer' instruction. PEDV-specific antibodies were not detected in all sera samples (data not shown). Piglets in group 2 were orally administered Ae at a dose of 100 mg/kg of bw every 6 h before feeding. Piglets in group 1 and 3 were orally administered the same amount of DMEM at the same time. One day later, piglets in group 2 and 3 were orally challenged with 2 mL DMEM containing a total of 1 × 10 7 PFU of PEDV GDS01 strain. Piglets that died in the PEDV group were recorded and immediately necropsied and sampled. All piglets were sacrificed 48 h after infection, and the jejuna tissues of piglets were collected and examined by histopathology, immunohistochemical staining.

Histological and Immunohistochemical staining were performed as previously described . Mann-Whitney accordingly.

To investigate the potential cytotoxicity of Ae, the relative viability rate of Vero and when the concentration was 32 mg/mL, indicating that the antiviral assays were carried out less than a concentration of 16 mg/mL.

Since determination the maximum concentration of Ae does not affect the cells viability, we further explored the influence of Ae on PEDV by IFA with PEDV N protein-specific polyclonal antibody. As shown in Fig 2A, Taken together, Ae could inhibit PEDV replication in vitro.

To determine the stage at which Ae exerts its anti-PEDV effect, Ae incubated with the virus or IPEC-J2 cells at different infection process. As shown in Fig 

To investigate whether Ae can directly inactivate the virus infectivity, we pretreated live PEDV with Ae at 37°C for 1 h or 3 h, and then tested the infectivity of the treated viruses. As shown in Fig 4A, protein of PEDV can be divided into S1 and S2, and S1 plays an important role in viral cell attachment (Lee et al., 2010; Li et al., 2017) . Ae was further incubated with S1 protein at 37°C for 1 h or 3 h, and then the mixtures were harvested for SDS-PAGE analysis. We found that Ae did not degrade S1 protein directly, as compared to the control (Fig 4C) . These data demonstrate that Ae inactivates PEDV virions directly had nothing to do with viral genome and S1 protein.

In an acute toxicity study, mice were orally administered Ae in a single dose of 0, 4, 20, 100, 500 or 2500 mg/kg of bw. No death of mice was observed in each dose group in 8 days (Fig. 5A) , indicating that there was no acute toxicity in mice after less than 2500 mg/kg of bw administration. For subchronic toxicity assessment, mice were orally administered Ae at different concentrations for 28 days. During this experiment, all mice lost weight and then gained weight in the first 4 days, but there was no siginificant difference in body weight between the Ae treatment groups and control group in 28 days ( Fig 5B) . Oral Ae did not affect white blood cells (WBC) (Fig. 5C) , red blood cells (RBC) (Fig. 5D ) in mice. However, the level of platelets (PLT) in the blood of the high-dose (500 or 2500 mg/kg) treatment groups was significantly increased (p<0.05) (Fig. 5E ), indicating high dose administration of Ae might increase blood viscosity. Therefore, oral dose was set to 100 mg/kg bw to explore whether Ae can protect piglets from PEDV challenge. As shown in Fig 6J, no piglets died in the mock and Ae treatment-PEDV challenge groups (4 piglets/group) in 2 J o u r n a l P r e -p r o o f days. In contrast, PEDV challenge without Ae treatment group (4 piglets/group), two piglets died in 2 days. To determine the gross pathological and histological changes in piglets after PEDV infection, all piglets from each group were necropsied at 2 d.p.i..

In the PEDV challenge without Ae treatment group, the small intestinal tract, where yellow watery contents accumulated, were transparent, thin-walled, and gas-distended ( Fig 6C) . No lesions or slight lesions were observed in the mock and Ae treatment-PEDV challenge groups (Fig 6A&B) . Microscopic lesions were also analysed. As shown in Fig 6D-F , abruption of jejunum villus was observed in the PEDV challenge without Ae treatment group, whereas the jejunum was normal in the mock and Ae treatment-PEDV challenge groups. Consistent with the histopathological results, more PEDV antigen was detected in the cytoplasm of the villous enterocytes of the PEDV challenge without Ae treatment piglets by immunohistochemical analysis (Fig 6G-I) . Taken together, these results indicate that

Ae could protect piglets against PEDV infection in vivo.

Since PEDV variants emerged in the swine industry, it has caused significant problems in the swine industry (Sun et al., 2016; Vlasova et al., 2014) . PEDV invasion of porcine intestinal epithelial cells causes enteritis in swine of all ages and is often fatal in neonatal piglets (Debouck and Pensaert, 1980; Ducatelle et al., 1981) .

Although vaccines are available for PEDV, they are not optimal in terms of safety and efficacy, and large-scale infections still occur (Yuan et al., 2018) . Antiviral drugs that J o u r n a l P r e -p r o o f are effective against PEDV are urgently needed to combat these threats. In the present study, we reported that Ae, a common plant extract, inhibited PEDV infection both in vitro and in vivo, might help to control PED in pig farm.

The premise of antiviral test is the cytotoxicity analysis of drug, since the cell toxicity of the drugs may influence the judgment of the antiviral effect of drug in vitro.

The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-lium bromide] and CCK-8 assays are common used to determinate the cytotoxicity of drug (Gao et al., 2014; Ting et al., 2018) . In the study, we chose more sensitive CCK-8 assay to determine the cytotoxicity to Vero and IPEC-J2 cells of Ae, due to these cell types are commonly used to study PEDV (Lin et al., 2017a; Lin et al., 2017b) . We found that the concentrations of 2-16 mg/mL of Ae were not cytotoxicity in Vero and IPEC-J2

cells, but the cell viability decreased to 20% when the concentration was 32 mg/mL, indicating that antiviral assays were carried out less than a concentration of 16 mg/mL.

In addition, the result demonstrated there exist some compositions in Ae have cytotoxicity, indicating that the toxic ingredients of Ae need to determine and remove.

Plants and plant-derived compounds are exploited extensively as candidates for new antiviral agents due to they produce few side effects and high availabilities and low cost (Hakobyan et al., 2016) . Aloe vera belongs to Liliaceae family among the succulent plants, which has got various properties such as immunomodulatory,

anti-inflammatory and antiviral in nature (Manigandan et al., 2015; Sudarshan et al., 2014) . Aloe or its constituents have significant inhibitory effect on influenza virus, pigeon paramyxovirus type 1, Herpes simplex virus type 1, Japanese encephalitis J o u r n a l P r e -p r o o f virus, enveloped viruses and hepatitis B virus (Dziewulska et al., 2017; Gansukh et al., 2018; Lin et al., 2008; Mohammad et al., 2019; Rezazadeh et al., 2016; Sydiskis et al., 1991) . In our experiment, we found that Ae could inhibit PEDV infection in Vero and IPEC-J2 cells in vitro. It is known that one of problems of antiviral drugs is resistance, which due to some antiviral drugs can not completely kill virus, then the surviving virus particles will mutate to resist the antiviral drugs (Leen et al., 2014) . RNA viruses are more likely to produce resistance than DNA viruses, due to the mutation rate of RNA viruses is higher than DNA viruses (Sanjuan et al., 2010) . well as the synthesis of capsid protein, reducing viral progeny. We also found that Ae could directly inhibit PEDV infection activity but had no effect on virus genome without the presence of cells. In addition, although S1 protein related to virus attachment and entry (Wen et al., 2018) , Ae could not degrade S1 protein in vitro,

indicating that Ae can directly inactivate the virus infectivity, but no effect on virus nucleic and virus invasion, consistent with the results of time-of-addition assay. It was reported that emodin from Aloe are directly virucidal to enveloped viruses and are related to the partially disruption of viral envelopes (Sydiskis et al., 1991) , indicating J o u r n a l P r e -p r o o f 20 that the direct inactivation PEDV by Ae might relate to emodin's destruction of the virus envelope.

For a antiviral drug candidate, safety is the top concern. Acute and subacute toxicity are the two important index for drug safety evaluation (Vakili et al., 2017) , which can help to determine the scope and the time of the drug use, reduce side effects. In this study, we fed Ae to mice and determined a safe dose of 100 mg/kg of bw by recording the deaths, weight changes and blood cell counts of Ae-treatmented mice. This will provide a reference for the proper use of drugs in pigs. It was reported that PEDV only infects pigs and has most harmful to 7-10-day-old suckling piglets with a high mortality rate Pijpers et al., 1993; Stevenson et al., 2013) . We fed Ae at a safe dose of 100 mg/kg of bw to newborn piglets then challenged with a lethal dose of PEDV, which confirmed that Ae can significantly protect piglets against PEDV infection in vivo, consistant with the results of in vitro experiments, indicating that Ae can be used as one of choice for prevention and control PEDV in pig farm.

Although we confirmed that Ae could inhibit PEDV in vivo, slight diarrhea (data not shown) was found in Ae-treatmented piglets, which might be related with Aloe vera ingredient emodin, which used as a laxative (Chen et al., 2012) 

Conflict of Interest: The authors declare that they have no conflict interest.

Ethical approval: The animal study was supervised by the Institutional Animal Care and Use Committee of Sun Yat-sen University (IACUC DD-18-0111) and used in accordance with regulation and guidelines of this committee.

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.

Vero were detected. Data are represented as mean ± SD, n=6. *stands for p < 0.05. 

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Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1. Virology. 489, 252-268. 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

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