Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium. Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. Recently, we reported that low doses of OTA promoted PCV2 replication in vitro and in vivo, but the underlying mechanism needed further investigation. The present studies further confirmed OTA-induced PCV2 replication promotion as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. Our studies also showed that OTA induced autophagy in PK-15 cells, as assessed by the markedly increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II, autophagy-related protein 5 (ATG5), and Beclin-1 and the accumulation of green fluorescent protein (GFP)-LC3 dots. OTA induced complete autophagic flux, which was detected by monitoring p62 degradation and LC3-II turnover using immunoblotting. Inhibition of autophagy by 3-methylademine (3-MA) and chloroquine (CQ) significantly attenuated OTA-induced PCV2 replication promotion. The observed phenomenon was further confirmed by the knock-down of ATG5 or Beclin-1 by specific siRNA. Further studies showed that N-acetyl-L-cysteine (NAC), an ROS scavenger could block autophagy induced by OTA, indicating that ROS may be involved in the regulation of OTA-induced autophagy. Furthermore, we observed significant increases in OTA concentrations in lung, spleen, kidney, liver and inguinal lymph nodes (ILN) and bronchial lymph nodes (BLN) of pigs fed 75 and 150 μg/kg OTA compared with controls in vivo. Administration of 75 μg/kg OTA significantly increased PCV2 replication and autophagy in the lung, spleen, kidney and BLN of pigs. Taken together, it could be concluded that OTA-induced autophagy in vitro and in vivo promotes PCV2 replication.

Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single standard circular DNA virus belonging to the genus Circovirus of the Circoviridae family. 1 PCV2 is the primary causative agent of several diseases that are collectively referred to as porcine circovirus-associated diseases (PCVAD). However, not all pigs infected with PCV2 develop PCVAD, and other factors, such as animal management, viral infections, immune stimulation and nutrition, have been suggested to be associated with the disease. 2 Ochratoxins are a group of mycotoxins produced principally by ubiquitous strains of Aspergillus and Penicillium. 3,4 Ochratoxin A (OTA), the most prevalent ochratoxin. 5 Owing to its natural origin, pervasiveness at low levels and chemical stability, OTA contamination persists throughout the feed supply and the food chain. 3,6 Our previous work suggests that OTA may be an important trigger of PCV2 replication and that the varying levels of OTA in pig feed may partially explain why the morbidity and severity of PCVAD vary among PCV2infected pig farms. 7 However, the underlying mechanism through which OTA promotes PCV2 replication requires further investigation.
Autophagy is a conserved catabolic intracellular process through which long-lived proteins and damaged organelles are degraded by the lysosomal machinery. 8,9 Autophagy not only plays an essential role in cellular homeostasis but also participates in numerous other physiological and pathological processes. 10 Normally, autophagy acts as a defense mechanism against viral infection. [11][12][13] Interestingly, several types of viruses exploited the autophagic processes to enhance their own replication. [13][14][15] Few data involving autophagy, OTA and viral infection have been reported to date.
In the present study, we investigated the role of autophagy in OTA-promoted PCV2 replication in vitro and in vivo, thereby developing potential novel antiviral strategies against PCV2 infection.

Results
Cytotoxic effects of OTA on PK-15 cells. To determine whether the effect of OTA on PCV2 replication results from OTA induction of cell toxicity or alteration in the energy supply of PK-15 cells, we examined the effects of OTA on cell viability using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and ATP assays. As shown in Figure 1a, the viability of PK-15 cells was not affected by OTA up to a concentration of 2 μM, but at concentrations of 5 and 10 μM, OTA significantly lowered (Po0.05) the cell viability of PK-15 cells. A similar reduction in intracellular ATP levels after treatment of the cells with OTA was observed ( Figure 1b). Thus, in subsequent experiments, OTA was used at concentrations between 0.01 and 2 μM.
OTA promotes PCV2 replication in PK-15 cells. To determine the effect of OTA on PCV2 replication, PK-15 cells were infected with PCV2 for 24 h and then incubated with OTA at concentrations between 0.01 and 2 μM for an additional 48 h. As shown in Figure 2, exposure of cells to the solvent control, dimethyl sulfoxide (DMSO), had no effect on PCV2 replication. However, treatment with 0.01, 0.1,1 or 2 μM OTA significantly (Po0.05) increased the cap protein level (Figure 2a OTA increases autophagosome formation in PK-15 cells. To determine whether OTA induces autophagy in PCV2-infected PK-15 cells, we examined the levels of autophagy marker proteins using Western blotting. LC3, which is necessary for the formation and maturation of the autophagosome, is a hallmark for assessing autophagy. 16,17 Our results showed that treatment with OTA led to a significant upregulation of LC3-II expression ( Figure 3a). We next examined the expression of ATG5, an essential protein that is associated with the formation of the autophagosomes membranes. 18 OTA-treated cells displayed significantly increased levels of ATG5 relative to control cells ( Figure 3a). We further measured the levels of Beclin-1, a protein involved in the early steps of the autophagy pathway. 19,20 Similar results were also observed in that OTA treatment induced the overexpression of Beclin-1 relative to control cells (Figure 3a). The maximal effects of OTA on the expression of autophagic markers were observed at an OTA concentration of 0.1 μM.
To further analyze whether the autophagy machinery was triggered by OTA treatment, PK-15 cells were transfected with green fluorescent protein-microtubule-associated protein 1 light-chain 3 (GFP-LC3), a specific marker of autophagic vesicles and autophagic activity. 21,22 As shown in Figure 3b, OTA treatment significantly increased the number of GFP-LC3 puncta compared with the control group, with the maximal effect observed at an OTA concentration of 0.1 μM. These findings indicate that OTA induces autophagy in PCV2infected PK-15 cells.
OTA treatment increases autophagic flux. To determine whether a complete autophagic flux is occurred by OTA treatment, we investigated the degradation of p62. [22][23][24] As shown in Figure 4a, the level of p62 protein in PK-15 cells decreased significantly with time during OTA treatment.
Since enhanced autophagosome accumulation could be caused either by increased autophagosome formation or by decreased autophagosome turnover, 25 the levels of LC3-II and p62 were measured in the presence of the lysosomal protease inhibitor CQ. 26 As demonstrated in Figure 4b, CQ treatment significantly increased the levels of LC3-II and p62 in OTA-treated PK-15 cells, suggesting that OTA treatment enhanced autophagic flux in the cells.
Pharmacological inhibition of autophagy reduces OTApromoted PCV2 replication in PK-15 cells. To evaluate the role of autophagy in OTA-promoted PCV2 replication in PK-15 cells, the effect of 3-MA, which inhibits autophagy by blocking the formation of autophagosomes, 27,28 on OTApromoted PCV2 replication was examined. Figure 5 shows that treatment with 3-MA reduced the level of cap protein (Figure 5a), viral titers (Figure 5b), PCV2 DNA copies ( Figure 5c) and the number of infected cells (Figure 5d), compared with the control group and that is attenuated the promotion of PCV2 replication by 0.1 μM OTA (Figures 5a-d).
Similarly, CQ, an inhibitor of autophagic flux, was utilized to inhibit the late stage of autophagy. 29 As demonstrated in Figure 6, CQ treatment significantly decreased the expression of cap protein (Figure 6a), viral titers (Figure 6b), PCV2 DNA copies ( Figure 6c) and the number of infected cells (Figure 6d) compared with the control group, it also decreased the promotion of PCV2 replication by 0.1 μM OTA (Figures 6b-e).
The results of these pharmacological experiments demonstrated that autophagy has a positive effect on OTA-promoted PCV2 replication.
Knockdown of autophagy-related genes (ATGs) reduces OTA-promoted PCV2 replication. To exclude any nonspecific effects in our pharmacological experiments, we further examined the effect of reducing the levels of the intracellular autophagy proteins ATG5 and Beclin-1 on the OTA induces autophagy via ROS induction. To assess the role of ROS in OTA-induced autophagy, we first determined whether ROS accumulate after the treatment of cells with OTA and whether the commonly used ROS inhibitor NAC inhibits ROS production. FACS analysis revealed an increase in the mean DCFH-DA fluorescence after the treatment of the cells with OTA ( Figure 9a). Similar results were observed in cells stained with DCFH-DA and examined by confocal microscopy. As shown in Figure 9b, DCFH-DA fluorescence significantly increased after OTA treatment, whereas NAC could significantly decrease OTA-induced ROS production (Figures 9a and b). As shown in Figure 9b, the majority of the green fluorescence (DCFH-DA staining) was localized in the mitochondria after OTA treatment, indicating that mitochondria are the major sources of intracellular ROS. To verify that OTA-induced autophagy is associated with ROS induction, the level of autophagy was determined in the presence or absence of NAC. As shown in Figures 9c and d, OTA significantly increased (Po0.05) the expression levels of LC3-II, ATG5 and Beclin-1 and the formation of GFP-LC3 puncta. However, pretreatment with NAC markedly attenuated the induction of autophagy by OTA. These results indicate that OTA-induced autophagy was mediated by ROS induction.
Alteration of autophagy does not affect cell viability. To assess whether the pharmacological alteration of autophagy with 3-MA and CQ or transfection with siRNA or NAC affected PCV2 replication by changing the cell viability, the effects of our pharmacological treatments or siRNA transfection or NAC on PK-15 cell viability were analyzed using the MTT assay. Statistical analyses showed no significant differences (P40.05) on the viability of cells treated with agents or siRNA transfection ( Figure 10).
OTA treatment in vivo. We further evaluated the effects of OTA on PCV2 infection in an animal model and determined whether autophagy can also be induced in vivo. As Figure 11a shows, no OTA was detected in the tissues from the control group, whereas OTA concentrations were significantly increased in the tissues of pigs fed an OTAcontaminating diet. The levels of OTA vary from 0.04 to 0.12 μg/g in the tissues of the animals in the 75 μg/kg OTA group and from 0.08 to 0.16 μg/g in the tissues of the animals in the 150 μg/kg OTA group. These concentrations are similar to concentrations of OTA that promoted PCV2 replication in the in vitro experiment. Next, we determined the PCV2 viral loads in the tissues of the three groups of pigs using qRT-PCR. Figure 11b shows that, compared with the pigs in the control group, significant increases in PCV2 DNA copies were found in the lungs, spleens, and kidneys of the pigs treated with 75 μg/kg OTA and in the bronchial lymph nodes (BLN) of the pigs treated with 75 and 150 μg/kg OTA. We also analyzed the PCV2 viral cap protein levels in the tissues of all the animals by Western blotting. We observed that the lungs, spleens, kidneys, and BLN of the pigs treated with 75 μg/kg OTA contained significantly higher expression levels of cap protein ( Figure 11e). These in vivo results are consistent with the in vitro data showing that relatively low doses of OTA could promote PCV2 replication.
Serum SOD activity and serum MDA content were measured following the treatment of the animals with OTA. As shown in Figures 11c and d, following the feeding of OTAcontaining diets, significant reductions in SOD activities compared with those in the control group were observed in the 75 μg/kg OTA group on day 42 and in the 150 μg/kg OTA group on days 28 and 42, whereas slight reductions in SOD activity were observed on days 14 and 28 in the 75 μg/kg OTA  Figure 11c). In addition, significant increases in MDA levels were observed in both OTA-fed groups on days 28 and 42, whereas slight increases were observed on day 14 ( Figure 11d). We also measured the autophagy levels in the tissues of OTA-fed animals by measuring the LC3-II/β-actin ratio in the tissues. Figure 11e shows that the LC3-II/β-actin ratio increased significantly in the lungs, spleens, and kidneys of pigs treated with 75 μg/kg OTA and in the BLN of pigs treated with 75 and 150 μg/kg OTA, compared with pigs that received basal diet. The LC3-II/β-actin ratios in the examined tissues were generally consistent with the viral infection levels in those tissues.
Overall, these results indicated that low doses of OTA could promote PCV2 replication and induce oxidative stress, and active autophagy in vivo.

Discussion
The presence of mycotoxins derived from naturally occurring fungal metabolites in feed and foodstuffs of humans and animals is unavoidable. Owing to their widespread presence in the environment, mycotoxins often co-exist with other infectious agents such as viruses, bacteria and other pathogens and may increase the susceptibility to these infectious agents. OTA is a natural contaminant of farm animal feeds worldwide and poses a potential threat to animal production. 30 Among farmed animals, pigs are particularly sensitive to OTA and are frequently exposed to this mycotoxin. PCV2 is an emerging viral disease that is a major concern for pig health and has significant economic impact on the swine industry, but the morbidity and severity of PCVAD vary among pig farms. 31,32 Numerous cofactors have been shown to contribute to the development of PCVAD. Our previous work suggests that OTA may be an important trigger of PCV2 replication and that the varying levels of OTA in pig feed may partly explain the difference in the morbidity and severity of PCVAD vary among pig farms. 7 However, the underlying mechanisms of OTA-promoted PCV2 replication require further investigation.
Autophagy is an evolutionarily ancient pathway that plays essential roles in host defenses against viral infection and cell survival; 33,34 for instance, it is involved in cellular defenses against herpes simplex virus and mosaic virus infections. 35,36 In contrast, many viruses exploit the autophagy machinery to enhance their own replication. 11,13,14 Mycotoxins have generally not been associated with autophagy. Recently, however, mycotoxins such as patulin, zearalenone and OTA have been linked to autophagy. 34,37,38 Based on recent findings concerning the interplay between mycotoxin, autophagy and viral infection, an autophagic mechanism appears to be involved in OTA-promoted PCV2 replication. In this study, we determined the role of autophagy in OTA-promoted PCV2 replication. Our results reveal a novel mechanism by which ROS-mediated autophagy induced by OTA contributes to PCV2 infection both in vitro and in vivo.
In the present study, the viability of PK-15 cells was significantly decreased by OTA at concentrations above 2 μM, consistent with previous studies. 39,40 As shown in Figure 1, no cell cytotoxicity or change in ATP contents was observed at OTA concentrations between 0.01 and 2 μM; however, PCV2 replication was significantly promoted  (Figure 2), and maximal promotion of PCV2 replication was observed at 0.1 μM OTA. The results further confirmed that low doses of OTA promote PCV2 replication in PK-15 cells 7 and that the promotion of PCV2 replication observed in these experiments was due to OTA exposure.
In the present study, we provide evidence that nontoxic concentrations of OTA induce autophagy in PCV2infected PK-15 cells through increased expression of LC3-II, ATG5, and Beclin-1. Consistently, a large number of cytoplasmic autophagosomes were observed by confocal fluorescence microscopy in OTA-treated PK-15 cells, further confirming that autophagy was induced by OTA. However, previous studies have demonstrated that the induction of autophagy involves not merely an increase in the formation of autophagosomes, but also an increase in autophagic flux. 26 P62 degradation is a method that is widely used to assess autophagic flux. 23,41 In our study, we found that the p62 expression level was significantly decreased in PCV2-infected PK-15 cells after the treatment with OTA over a prolonged period of time. We also measured the levels of LC3-II and p62 in the presence or absence of lysosome inhibitor CQ. Treatment with CQ could lead to a pH shift in the, thus inhibiting lysosomal acidification. Figure 4b shows that treatment of PK-15 cells with OTA decreased the p62 expression level in the cells and that treatment with CQ increased the amounts of LC3-II and p62 in OTA-treated cells, indicating that the accumulation of autophagosomes in OTA treated PCV2-infected PK-15 cells was due to the increased autophagy flux. Previous studies have shown that many viruses, including poliovirus, coxsackievirus B4, hepatitis C virus, dengue virus, and PCV2, utilize components of the autophagic pathway to facilitate their replication. 14,15,[42][43][44] However, the role of autophagy in OTA-promoted PCV2 replication has not been previously investigated.
To determine the role of autophagy in OTA-promoted PCV2 replication, PK-15 cells were treated with 3-MA and CQ, which are pharmacological inhibitors of autophagy. 45,46 These compounds inhibited PCV2 replication and reduced the replication of PCV2 promoted by OTA, consistent with previous studies showing that PCV2 replication was significantly reduced by 3-MA 14 and that NDV replication was significantly reduced by CQ. 47 To further confirm the pharmacological data and exclude any non-specific effects of these pharmacological agents used in our experiments, we used target-specific RNA interference to examine the effect of reducing the levels of essential autophagy proteins ATG5 and Beclin-1 on the replication of PCV2 promoted by OTA. The ATG5 protein is necessary for the formation and maturation of autophagosome, 48,49 whereas Beclin-1 is an essential autophagy protein that regulates the initiation of autophagy and autophagosome-lysosome fusion by interacting with two protein complexes. Both proteins are necessary for autophagy to occur. 50,51 Depletion of ATG5 and Beclin-1 proteins dramatically suppressed the formation of OTA-induced autophagosomes and attenuated the promotion of PCV2 induced by OTA. The results are consistent with those of previous studies in which it was demonstrated that suppression of ATG5 and Beclin-1 with siRNAs reduced the replication of dengue virus, influenza A virus, and PCV2. 13,15,52 Accumulating evidence suggests that ROS and oxidative stress play a critical role in viral infection. 53, 54 We previously found that oxidative stress can enhance PCV2 infection and that OTA can induce oxidative stress and thereby promote  GFP-LC3 puncta formation were markedly attenuated when ROS formation was inhibited by NAC. Collectively, our data indicate that OTA triggers oxidative stress and thereby induces autophagy.
During the past few years, numerous in vitro studies have demonstrated the critical role of autophagy in virus infection. However, few studies have been conducted using in vivo models. To corroborate our in vitro results, we evaluated the effects of OTA on PCV2 infection in an animal model and determined whether oxidative stress and autophagy can also be induced in vivo. In the in vivo experiment, pigs were fed diets containing 75 or 150 μg/kg OTA. After such feeding, we observed OTA accumulation in various tissues. The highest concentration of OTA was found in lung tissue, followed by kidney, BLN, liver, inguinal lymph nodes (ILN), and spleen. This is apparently inconsistent with the fact that kidney is generally considered as the target organ for OTA toxicity. 60,61 However, similar results have also been reported in rats treated with low doses of OTA. 62 In our experiments, the ranges of OTA concentrations in tissues were 0.05-0.11 μg/g and 0.40-0.69 μg/kg in the animals in the 75 μg/kg OTA and 150 μg/kg OTA treatment groups, respectively. The observed levels of OTA in the tissues of the treated animals are consistent with the concentrations of OTA that promoted PCV2 replication in PK-15 cells in vitro. Our data further showed that treatment of pigs with 75 μg/kg OTA significantly increased PCV2 DNA copies and viral cap protein expression levels in various tissues, including lung, spleen, kidney, and BLN of pigs, a finding that is consistent with our in vitro results showing that relatively low doses of OTA can promote PCV2 replication.
In the present study, the oxidative stress status of pigs was examined. Increased levels of MDA in the serum and decreased serum SOD activities were observed following the treatment of the animals with OTA (Figures 11c and d). In addition, lapidated LC3-II was detected by western blotting in the tissues, and enhanced levels of LC3-II were observed in the lungs, spleens, and kidneys of pigs fed 75 μg/kg OTA and in the BLN of pigs fed with 75 or 150 μg/kg OTA. These results agree to some extent with the viral infectious levels measured in these animals.
Overall, these in vitro results reported here indicate that OTA-induced autophagy is mediated by oxidative stress and that it affects PCV2 replication. The increased levels of oxidative stress and LC3-II observed in the tissues of OTAtreated, PCV2-infected pigs indicate that oxidative stress and autophagy also occur in response to OTA treatment in vivo. These findings increase our knowledge of the pathogenesis of PCV2 and provide new insight into possible methods for the control of PCVAD. The wild-type PCV2 (PCV2NJ2002) used in the experiments was isolated originally from a kidney tissue sample of a pig with naturally occurring PMWS. The determination of PCV type was performed by sequencing (Invitrogen, Carlsbad, CA, USA). Stocks of PCV2 were generated from PK-15 cells infected with PCV2 as previously described. 55 To construct pEGFP-LC3B, the LC3B gene was amplified from PK-15 cells using primers (LC3F: 5′-GAAGATCTGGGCTGAGGAGACACAAGAG-3′; LC3R: 5′-CGGAATTCTCTCAGTTGGTAACATCCCTTT-3′) that were designed based on the sequence of LC3B (GenBank No. NM 001190290.1). The gene was then cloned into pEGFP-C1 to express LC3B fused with the EGFP protein at its N-terminus.  Virus infection and cell treatment. According to the requirements of individual experiments, PK-15 cells were infected with PCV2 at a multiplicity of infection of 1 at 37°C. Following a 1-h absorption period, the cell monolayers were rinsed with sterile phosphate-buffered saline, pH 7.2 (PBS) to remove unattached viruses and then incubated in the presence of fresh medium at 37°C for 24 h. Then the cells were then treated with various concentrations of OTA and incubated for an additional 48 h. For autophagy studies, PCV2-infected PK-15 cells were pretreated with 3-MA or CQ for 4 h at 37°C. The cells were then treated as described above and further incubated in fresh medium in the absence or presence of 3-MA and CQ at the same concentrations as used in the pretreatments or in medium containing a corresponding amount of the solvent DMSO(control), unless otherwise specified in the figure legends.
Cell viability assays. Cell viability was measured using the MTT assay according to the manufacturer's instructions. Briefly, PK-15 cells were cultured in 96-well plates at a density of 5 × 10 3 cells/well for 24 h and then subjected to various treatments for the specified periods. Following treatment, the culture medium was supplemented with 15 μl of MTT (5 mg/ml) for 4 h at 37°C. The supernatants were carefully aspirated and replaced with 150 μl DMSO to dissolve the precipitate. Absorbance was measured in a microplate reader spectrophotometer at a wavelength of 570 nm. All tests were performed three times.
Detection of cellular ATP levels. Cellular ATP levels were measured using a firefly luciferase-based ATP assay kit (Beyotime, Jiangsu Province, China) according to the manufacturer's instructions. Briefly, PK-15 cells were cultured in 24well plates at a density of 4 × 10 4 cells/well for 24 h. After treatment with various concentrations of OTA for 48 h, the cells were schizolyzed and centrifuged at 12 000 × g for 5 min. Then, 50 μl of each supernatant and 50 μl of ATP detection working dilution were mixed for 3 s prior to the measurement of luminescence 10 s in a GloMax 20/20 luminometer (Promega, Madison, USA). All assays were performed in triplicate.
Quantitative real-time PCR (qRT-PCR). Quantitative real-time PCR (qRT-PCR) was performed to determine the PCV2 DNA copies as previously described. 55 In brief, DNA was extracted using the TaKaRa DNA Mini kit (TaKaRa, China). The purified DNA was used as a template for PCR amplification. A pair of PCV2-specific primers (forward primer 5′-TAGTATTCAAAGGGCACAG-3′, reverse primer 5′-AAGGCTACCACAGTCAG-3′) was designed to amplify a 117-bp fragment from the PCV2 ORF2 gene. The qRT-PCR was performed using the ABI Prism Step One Plus detection system (Applied Biosystems, NY, USA). A recombinant pMD19 plasmid vector (TaKaRa) containing a PCV2 genome was used to construct a standard curve, and a TaKaRa SYBR green real-time PCR kit was used to measure the amount of viral DNA.
Indirect immunofluorescence assay. PCV2-infected cells were detected using an indirect immunofluorescence assay (IFA) as previously described. PK-15 cells were washed three times with PBS containing 0.1% Tween-20 (PBST) and fixed in 4% paraformaldehyde for 20 min at room temperature (RT). After three washes in PBST, the cells were permeabilized with 0.1% Triton X-100 and then incubated in PBST containing 1% bovine serum albumin (BSA) for 1 h at 37°C to block nonspecific binding. The cells were then incubated with porcine anti-PCV2 antibody (UnivBiotech, Shanghai, China) diluted in PBST containing 1% BSA (PBSTB; 1:50) for 1 h at 37°C, and after three washes with PBST, FITC-conjugated rabbit anti-pig antibody (Sigma; diluted 1:100 in PBST) was added, and the cells were incubated for 1 h at 37°C. The cells were again washed three times with PBST, and examined under a fluorescence microscope. Cells positive for PCV2 viral antigens were counted in six fields of view.
Quantification of virus titer. PCV2-infected PK-15 cells grown in six-well plates were pretreated with DMSO or chemicals (3-MA or CQ) for 4 h, or transfected with ssiRNA, siATG5 or siBeclin-1 for 5 h. The cells were then treated with OTA for an additional 48 h. Samples of the culture supernatant samples were collected, and the cells were subjected to three freeze-thaw cycles. Total virus yield (intracellular and extracellular viruses) was determined by inoculating confluent PK-15 cells in 96-well culture plates with 10-fold dilutions of the culture supernatants. After 72 h of incubation, the viral antigen was detected by IFA as described above. Viral titers were calculated using the Reed-Muench method and expressed as TCID 50 /ml.
Cell lysis and western blotting analysis. PK-15 cells cultured in six-well plates were scraped from the plates at the specific times indicated in the figures and collected in cell lysis buffer containing protease inhibitor (Beyotime, Haimen, China) on ice. The cell lysates were sonicated using a Sonics VCX105 sonicator and centrifuged at 12 000 rpm for 20 min at 4°C. Protein concentration was determined using the BCA protein assay kit (Beyotime). Equal amounts of protein samples were diluted in 5 × SDS-PAGE loading buffer and heated at 95°C for 5 min. The samples were separated on 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. After blocking for 1 h at RT in Tris-buffered saline containing 5% nonfat milk powder and 0.1% Tween 20, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then washed and incubated in secondary antibody at RT for 1 h. Images of the immunoblots were acquired using an EU-88 image scanner (Seiko Epson Corp.). Quantification of protein blots was performed using the Image-Pro Plus 6.0 software (Media Cybernetics).
Confocal fluorescence microscopy. Confocal fluorescence microscopy was used for the analysis of LC3 expression after OTA treatment. Specifically, PK-15 cells grown on coverslips to 40-50% confluence were transfected with the plasmid GFP-LC3 using X-tremeGENE HP DNA transfection reagent (Roche, Indianapolis, USA) according to the manufacturer's guidelines. The cells were then infected with PCV2 for 24 h and inoculated with OTA for an additional 48 h as described above. The intracellular localization of LC3 was visualized using a Nikon C1-si confocal fluorescence microscope (Nikon Instruments, Inc.).
RNA interference. ATG5-specific and Beclin-1-specific siRNAs were designed using the sequence of Sus scrofa ATG5 mRNA (GenBank Accession No. NM_001037152.1) and Beclin-1 mRNA (GenBank Accession No. NM_ 001044530.1), respectively, and Invitrogen BlockiT RNAi designer. Control siRNA sequences were obtained from published material. 63 The ATG5-specific siRNA sequence was 5′-GCUUCGAGAUGUGUGGUUUtt-3′, the Beclin-1-specific siRNA sequence was 5′-CCUGGAUCGUGUUACCAUUtt-3′, and the sequence of control siRNA was 5′-UUCUCCGAACGUGUCACGUtt-3′. The three double-stranded RNAs were synthesized by Invitrogen. Duplex RNAs were resuspended in DEPC water to obtain 20 μM solutions prior to use. PK-15 cells in DMEM 8% FBS without antibiotics were seeded in six-well plates at a density of 1 × 10 5 cells/well and incubated for 24 h at 37°C. When the cells were 30-50% confluent, siRNA was introduced using the X-treameGENE siRNA transfection agent (Roche) according to the protocol provided by the manufacturer. Transfection reagent (2.5 μl) and 0.5 μg siRNA were added to each well, and the plates were incubated for 5 h. The cells were then washed with DMEM and cultured in DMEM + 4% FBS until further treatments.
Measurement of intracellular ROS. For the determination of ROS, PK-15 cells were cultured in six-well plates at a density of 1 × 10 5 cells/well. Intracellular ROS levels were measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Beyotime) as previously described. Briefly, after removing the culture medium, the cells were incubated with DCFH-DA (10 μM) at 37°C for 30 min. The ROS level was determined by fluorescence microscopy and flow cytometry.
Animal experiments. OTA extracts obtained from in vitro cultures of Aspergillus ochraceus strain (No. 3.4411) were added to the pig basal diet to provide the diets containing 70 and 150 μg/kg OTA.
The experiment was conducted at a 300-sow pig farm. Sixty weaning piglets (age 6 weeks) were selected for the experiment. The average of PCV2 DNA copies in these animals, as measured by real-time PCR, was 10 3 -10 5 due to natural infection. It was also determined by PCR that the piglets were negative for swine fever virus, swine influenza virus, porcine parvovirus, and porcine reproductive and respiratory syndrome virus. The experiments were conducted in accordance with the standards of the European Guidelines for Animal Welfare and were approved by the Committee for the Care and Use of Experimental Animals of the Nanjing Agricultural University (Animal Ethics Number: SYXK (Su) 2011-0036).
Twenty-seven piglets with an approximately equal body weights of 10.5 kg were selected, the average of PCV2 DNA copies in each of these animals measured by real-time PCR was 10 3 .37 The piglets were randomly divided into three groups, each group included three replicates, with 3 piglets per replicate. Group I, the control group, received a basal diet, groups II and III received the basal diet containing 75 and 150 μg/kg OTA, respectively, for 42 days. The piglets were housed in different rooms under biosafety conditions with ad libitum access to water and feed during the experiment. At the end of the experiment, the piglets were killed, and tissue samples of liver, kidney, spleen, lung, ILN, and BLN were collected. The animal's serum was analyzed for SOD activity and MDA content. Tissue samples were analyzed for OTA concentration and PCV2 DNA copies and the proteins were extracted for western blotting analysis.
Determination of OTA concentration by LC-MS/MS. OTA was extracted from tissues using the method as previously described. Briefly, 1 ml of tissue homogenates was mixed with 5 ml of a methanol/water (80/20) mixture. After vortexing for 2 min, the mixture was sonicated for 1 h, and centrifuged for 15 min at 3300 × g. The supernatant fluid was transferred to a 10-ml centrifuge tube and dried under nitrogen gas at 50°C. The residue was reconstituted in 500 μl acetonitrile/ water (20/80, v/v) containing 10 mmol/l ammounium acetate, and the resulting solution was passed through a nylon membrance (0.22 μm) prior to LC-MS/MS.
One milligram of OTA was dissolved in 5 ml of ethanol to yield a stock solution of 0.5 mg/ml OTA, which was stored at − 20°C in the dark. Standard solutions of OTA were prepared by diluting the stock solution with a mixture of acetonitrile/water