Intracellular redox state has been suggested to have various effects on the replication of different viruses within host cells. The aim of the present study was to investigate the influence of reactive oxygen species (ROS) on replication of porcine circovirus type 2 (PCV2), in PK15 cells. Following PCV2 infection there was a time-dependent increase in ROS. Antioxidant N-acetyl-l-cysteine treatment of cells resulted in lower ROS levels and lower PCV2 replication. In contrast, treatment by buthionine sulfoximine (BSO), a GSH synthesis inhibitor, resulted in elevation of ROS levels and increased PCV2 replication. Furthermore, inhibiting the activity of NF-κB, a redox-responsive transcription factor, suppressed BSO-mediated increase of PCV2 replication, indicating that increased PCV2 replication likely occurs via ROS activation of NF-κB. Taken together, our results indicate that the generation of ROS during PCV2 infection is involved in its replication and this progression is associated with the alteration in NF-κB activity induced by ROS.
Ochratoxin A (OTA), a worldwide mycotoxin found in food and feeds, is a potent nephrotoxin in animals and humans. Porcine circovirus-associated disease (PCVAD), including porcine dermatitis and nephropathy syndrome, is a worldwide swine disease. To date, little is known concerning the relationship between OTA and porcine circovirus type 2 (PCV2), the primary causative agent of PCVAD. The effects of OTA on PCV2 replication and their mechanisms were investigated in vitro and in vivo. The results in vitro showed that low doses of OTA significantly increased PCV2 DNA copies and the number of infected cells. Maximum effects were observed at 0.05 μg/ml OTA. The results in vivo showed that PCV2 replication was significantly increased in serum and tissues of pigs fed 75 μg/kg OTA compared with the control group and pigs fed 150 μg/kg OTA. In addition, low doses of OTA significantly depleted reduced glutathione and mRNA expression of NF-E2-related factor 2 and γ-glutamylcysteine synthetase; increased reactive oxygen species, oxidants, and malondialdehyde; and induced p38 and ERK1/2 phosphorylation in PK15 cells. Adding N-acetyl-L-cysteine reversed the changes induced by OTA. Knockdown of p38 and ERK1/2 by their respective specific siRNAs or inhibition of p38 and ERK1/2 phosphorylation by their respective inhibitors (SB203580 and U0126) eliminated the increase in PCV2 replication induced by OTA. These data indicate that low doses of OTA promoted PCV2 replication in vitro and in vivo via the oxidative stress-mediated p38/ERK1/2 MAPK signaling pathway. This suggests that low doses of OTA are potentially harmful to animals, as they enhance virus replication, and partly explains why the morbidity and severity of PCVAD vary significantly in different pig farms.
With the purpose to explore the mechanisms associated with the intestinal toxicity of Ochratoxin A (OTA), an intestinal porcine epithelial cell line (IPEC-J2) was applied in this study as in vitro models for intestinal epithelium. The results confirmed that OTA induced IPEC-J2 cell toxicity by MTT assay and apoptosis by Hoechst 33258 staining and flow cytometer analysis. We also observed that OTA induced the mitochondrial reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) opening by confocal microscopy. Western blot showed that OTA induced cytochrome c (cyt-c) release and caspase-3 activation, which could be suppressed by inhibition of mPTP opening with cyclosporin A. Treatment with Mito-TEMPO, the mitochondria-targeted ROS scavenger, blocked OTA-induced mitochondrial ROS generation and mPTP opening and prevented cyt-c release, caspase-3 activation, and apoptosis in IPEC-J2 cells.
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