Porcine Kidney Cell Line Research Articles

Phenethyl isothiocyanate induces cytotoxicity and apoptosis of porcine kidney cells through Mitochondrial ROS-associated ERS pathway

Glucosinolates (GLS) in cruciferous vegetables are anti-nutritional factors. Excessive or long-term intake of GLS-containing feed is harmful to animal health and may cause kidney damage. Phenethyl isothiocyanate (PEITC) is a GLS. In this study, we investigated the inhibitory effect of PEITC on a porcine kidney (PK-15) cell line and explored the mechanism of PEITC-induced apoptosis. We found that PEITC could affect cell viability and induce cell apoptosis after incubating cells for 24 h. High concentrations of PEITC can induce intracellular ROS accumulation, resulting in impaired mitochondrial function (decreased MMP, decreased ATP) and DNA damage (increased 8-OHdG), cytochrome c in mitochondria is released into the cytoplasm and activates mitochondrial pathway apoptosis-related proteins (Bcl-2 family and caspase-9, -3). Meanwhile, PEITC could induce intracellular Ca2+ accumulation, disrupt ER homeostasis, and activate the expression levels of three ER-resident transmembrane proteins orchestrating the UPR (PERK, IRE-1α and ATF6) and ER-related proteins (GRP78 and CHOP), thereby activating ERS-pathway apoptosis-related proteins (caspase-12, -7). Our results showed that low concentration (2.5 μM) of PEITC had no damaging effect on cells. In comparison, a high concentration (10 μM) of PEITC could induce cell damage in porcine kidney cells and induce apoptosis in PK-15 cells via the Mitochondrial ROS-associated ERS pathway.

Experimental infection of a porcine kidney cell line with hepatitis A virus

The hepatitis A virus (HAV) induces severe acute liver injury and is adapted to human and monkey cell lines but not other cells. In this study, the HAV was inoculated into porcine kidney (PK-15) cells to determine its infectivity in porcine cells. The growth pattern of the HAV in PK-15 cells was compared with its growth pattern in fetal rhesus kidney (FRhK-4) cells. The growth of HAV was less efficient in PK-15 cells. In conclusion, HAV replication was verified in PK-15 cells for the first time. Further investigations will be needed to identify the HAV-restrictive mechanisms in PK-15 cells.

A Novel Virus-Like Agent Originated From Genome Rearrangement of Porcine Circovirus Type 2 (PCV2) Enhances PCV2 Replication and Regulates Intracellular Redox Status In Vitro.

Genome rearrangement occurs to porcine circovirus type 2 (PCV2) during in vitro and in vivo infections, and a number of rearranged PCV2 genomes have been isolated and characterized. This study was conducted to investigate the role of the rearranged PCV2 (rPCV2) in PCV2 replication and the biological effect of rPCV2 in host cells. Two whole rPCV2 genome sequences (358 nt and 1125 nt in length) were synthesized and recombinant plasmids pBSK(+)-rPCV2 (pBSK(+)-1125 and pBSK(+)-358) were constructed. A novel virus-like agent (rPCV2-1125) was rescued by in vitro transfection of porcine kidney cell line (PK-15) and porcine alveolar macrophage 3D4/21 cells. The data indicate that rPCV2-1125 significantly enhanced PCV2 replication in vitro. Furthermore, rPCV2-1125 led to oxidative stress in host cells, as indicated by decreased intracellular glutathione (GSH) and total superoxide dismutase (SOD) activities, as well as increased malondialdehyde (MDA) levels. These results provide new insights into genome rearrangement of PCV2 and will contribute to future studies of PCV2 replication and associated mechanisms.

Comparative immunopathogenesis and biology of recently discovered porcine circoviruses.

Porcine circoviruses are important pathogens of production swine. Porcine circovirus type 1 (PCV1) is non-pathogenic, and discovered as a contaminant of a porcine kidney cell line, PK-15. The discovery of pathogenic variant, PCV2, occurred in the late 90s in association with post-weaning multi-systemic wasting disease syndrome (PMWS), which is characterized by wasting, respiratory signs and lymphadenopathy in weanling pigs. A new PCV type, designated as PCV3, was discovered in 2016, in pigs manifesting porcine dermatitis and nephropathy syndrome (PDNS), respiratory distress and reproductive failure. Pathological manifestations of PCV3 Infections include systemic inflammation, vasculitis and myocarditis. A fourth PCV type, PCV4, was identified in 2020 in pigs with PDNS, respiratory and enteric signs. All the pathogenic PCV types are detected in both healthy and morbid pigs. They cause chronic, systemic infections with various clinical manifestations. Dysregulation of the immune system homeostasis is a pivotal trigger for pathogenesis in porcine circoviral infections. While the study of PCV3 immunobiology is still in its infancy lessons learned from PCV2 and other circular replication-associated protein (Rep)-encoding single stranded (ss) (CRESS) DNA viruses can inform the field of exploration for PCV3. Viral interactions with the innate immune system, interference with dendritic cell function coupled with the direct loss of lymphocytes compromises both innate and adaptive immunity in PCV2 infections. Dysregulated immune responses leading to the establishment of a pro-inflammatory state, immune complex associated hypersensitivity, and the necrosis of lymphocytes and immune cells are key features of PCV3 immunopathogenesis. A critical overview of the comparative immunopathology of PCV2 and PCV3/4, and directions for future research in the field are presented in this review.

Bile acids LCA and CDCA inhibited porcine deltacoronavirus replication in vitro

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes gastroenteritis in pigs and no vaccines or antiviral drugs are available. Bile acids are active factors in intestines and influence the replication of enteric viruses. Currently, the role of bile acids on PDCoV replication is unknown. In this study, we tested the effects of different types of bile acids on the replication of PDCoV in cell culture. We found that physiological concentrations of bile acids chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) had antiviral activity against PDCoV in porcine kidney cell line (LLC-PK1) and porcine small intestinal epithelial cell line (IPEC-J2). In IPEC-J2 cells, CDCA and LCA inhibited PDCoV replication at post-entry stages by inducing the production of interferon (IFN)-λ3 and IFN-stimulated gene 15 (ISG15) via G protein-coupled receptor (GPCR). In summary, bile acids CDCA and LCA restricted PDCoV infection and LCA functioned through a GPCR-IFN-λ3-ISG15 signaling axis in IPEC-J2 cells. Our results may open new avenues for the development of antiviral drugs to treat PDCoV infection in pigs.

Replicative capacity of four porcine enteric coronaviruses in LLC-PK1 cells.

Enteric coronaviruses (CoVs) are major pathogens that cause diarrhea in piglets. To date, four porcine enteric CoVs have been identified: transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and HKU2-like porcine enteric alphacoronavirus (PEAV). In this study, we investigated the replicative capacity of these four enteric CoVs in LLC-PK1 cells, a porcine kidney cell line. The results showed that LLC-PK1 cells are susceptible to all four enteric CoVs, particularly to TGEV and PDCoV infections, indicating that LLC-PK1 cells can be applied to porcine enteric CoV research in vitro, particularly for coinfection studies.

Evaluation of Cell Lines for the Isolation of Foot-and-Mouth Disease Virus and Other Viruses Causing Vesicular Disease.

The most sensitive cell culture system for the isolation of foot-and-mouth disease virus (FMDV) is primary bovine thyroid (BTY) cells. However, BTY cells are seldom used because of the challenges associated with sourcing thyroids from FMDV-negative calves (particularly in FMD endemic countries), and the costs and time required to regularly prepare batches of cells. Two continuous cell lines, a fetal goat tongue cell line (ZZ-R 127) and a fetal porcine kidney cell line (LFBK-αVβ6), have been shown to be highly sensitive to FMDV. Here, we assessed the sensitivity of ZZ-R 127 and LFBK-αVβ6 cells relative to primary BTY cells by titrating a range of FMDV original samples and isolates. Both the ZZ-R 127 and LFBK-αVβ6 cells were susceptible to FMDV for >100 passages, and there were no significant differences in sensitivity relative to primary BTY cells. Notably, the LFBK-αVβ6 cell line was highly sensitive to the O/CATHAY porcine-adapted FMDV strain. These results support the use of ZZ-R 127 and LFBK-αVβ6 as sensitive alternatives to BTY cells for the isolation of FMDV, and highlight the use of LFBK-αVβ6 cells as an additional tool for the isolation of porcinophilic viruses.

Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct.

Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3-/- mice significantly attenuated gentamicin-induced AKI. A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

Phenylethyl isothiocyanate induces oxidative damage of porcine kidney cells mediated by reactive oxygen species.

The aim of this study is to confirm the toxic effect of phenylethyl isothiocyanate (PEITC) on porcine kidney cells (PK-15) and explore the effect of oxidative damage mediated by reactive oxygen species (ROS) induced by PEITC in PK-15 cells. Porcine kidney cell line (PK-15) was treated with PEITC (2, 5, and 10 µM) for 24 hours, and the oxidative damage mediated by PEITC through ROS was investigated. The survival rate of PK-15 cells decreased in a dose-dependent manner after the treatment of PEITC in a dose-dependent manner. A high concentration of PEITC (10 µM) can change cell morphology, increase the content of malondialdehyde, ROS, and lactate dehydrogenase, and decrease the activity of SOD, CAT, GSH-PX, and GSH. PEITC has a toxic effect on PK-15 cells by inducing oxidative stress in PK-15 cells through the generation of ROS.

Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro.

Endoplasmic reticulum (ER) stress-mediated autophagy plays significant roles in replication and pathogenesis of many animal viruses. However, the relationship between ER stress, autophagy, and viral replication during in vivo and in vitro infection of classical swine fever virus (CSFV) remains unclear. In this study, we established a pig model for CSFV infection and found that viral loads of CSFV differed in 10 kinds of infected organs, and that the degree of tissue lesions was to some extent positively correlated with CSFV replication in vivo. Next, we found that CSFV infection not only induced ER stress and subsequently activated three unfolded protein responses (UPR) pathways including protein kinase R-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor-6 (ATF-6) pathways, but also triggered complete autophagy in main immune organs and partial nonimmune organs exhibiting severer pathological injuries and higher viral loads. However, only the IRE1 pathway and no autophagy were activated in some other nonimmune organs with slighter pathologies and lower viral loads. These results indicate a potential link between CSFV-induced ER stress and autophagy, both of which are associated with the CSFV replication in vivo. We further performed in vitro experiments and found that CSFV infection activates the PERK and IRE1 pathways and autophagy in cultured porcine kidney cell lines (PK-15) and macrophage cell lines (3D4/2), and pharmacological regulation of ER stress remarkably changed autophagic activities induced by CSFV, suggesting that CSFV-induced autophagy can be mediated by ER stress possibly via the PERK and IRE1 pathway. Furthermore, treatment with ER stress regulators significantly altered copy numbers of NS5B genes, expression of Npro proteins, and viral titers in CSFV-infected cells or in cells treated with autophagy regulators prior to CSFV infection, suggesting the requirement of ER stress-mediated autophagy for CSFV replication in vitro. Collectively, our data demonstrate that CSFV induces ER stress-mediated autophagy to sustain its replication in vivo and in vitro, which may be one of the potential strategies exploited by CSFV for immune evasion. This finding will provide new insights into mechanisms of replication and pathogenesis of CSFV, and development of new strategies for controlling CSF.

AUJESZKY’S DISEASE IN A DOG - CASE REPORT

This article reports on the occurrence and diagnosis of Aujeszky’s disease in a dog. The procedure for isolation and identification of Aujeszky’sdisease virus was described. A dog of unknown breed aged about two years died of Aujeszky’s disease after consuming animal offal (internal organs: lungs, spleen, kidneys) fed by the owner after slaughtering piglets and preparing meat for cooking. As early as 24 hours after consuming the offal, the dog manifested characteristic symptoms of Aujeszky’s disease, which were immediately recognized by the veterinarian. The death occurred within less than 24 hours upon first clinical signs of disease. Aujeszky’s disease virus was isolated and identified from brain and internal organ (lung and spleen) samples of the dog at the Department of Virology of the Scientific Veterinary Institute „Novi Sad“. Isolation and identification of the virus was performed on PK-15 porcine kidney cell line and using nested PCR technique.

Porcine circovirus type 2 induces a strong cytopathic effect in the serum-free culture cell line CPK-NS

When the adherent stable serum-free porcine kidney cell line CPK-NS were inoculated with porcine circovirus type 2 (PCV2) and passaged, viral titre concentration-dependent cell detachment was observed. The copy number of viral genes in supernatants of the infected CPK-NS cells decreased as cell detachment progressed. Furthermore, cell detachment was completely inhibited via neutralisation of the virus using antisera collected from PCV2-infected specific pathogen-free pigs. These results indicated that detachment of CPK-NS cells is a cytopathic effect (CPE) caused via infection with PCV2. Only a single round of cell passaging was required to observe clear a CPE when the inoculated viral titre was significantly high [≥104.5 median tissue culture infectious dose (TCID50)/mL]. Our study confirms that PCV2, which is normally non-cytopathogenic, is capable of inducing a distinct CPE in CPK-NS cells. Application of CPK-NS cells for detection of viruses may contribute towards the diagnosis and control of PCV2-mediated infectious diseases.

Nanoparticles for regenerative medicine.

Nanoparticles for regenerative medicine.

Host microRNA miR-1307 suppresses foot-and-mouth disease virus replication by promoting VP3 degradation and enhancing innate immune response

MicroRNAs (miRNAs) play important regulatory roles during interactions between virus pathogens and host cells, but whether and how they work in the case of foot-and-mouth disease virus (FMDV) is less understood. Based on a microarray-based miRNA profiling in the porcine kidney cell line PK-15, we identified 36 differentially expressed host miRNAs at the early stage of FMDV infection, among which miR-1307 was significantly induced. Functional characterization demonstrated that miR-1307 attenuated FMDV replication. Further experiments proved that miR-1307 specifically promoted the degradation of the viral structural protein VP3 indirectly through proteasome pathway. Moreover, innate immune signaling was activated and expression of immune responsive genes was significantly enhanced in the miR-1307-overexpressing clones. Together, our data demonstrated that miR-1307 suppresses FMDV replication by destabilizing VP3 and enhancing host immune response. Importantly, subcutaneous injection of miR-1307 agomir delayed the FMDV-induced lethality in suckling mice, exhibiting its therapeutic potential to control foot-and-mouth disease (FMD).

A miR-511-binding site SNP in the 3′UTR of IGF-1 gene is associated with proliferation and apoptosis of PK-15 cells

Insulin-like growth factor-1 (IGF-1) is a functional candidate gene for pig growth and development due to its crucial role in the growth axis of growth hormone-IGF-1. Considering that the 3' untranslated region (3'UTR) of gene may affect its expression, we analyzed the effect of a single-nucleotide polymorphism (SNP) (rs34142920, c.674C > T) on gene expression, cell proliferation, and apoptosis and the possible related molecular mechanisms in PK-15 cells. The SNP was found in the 3'UTR of IGF-1 in Bama Xiang pig in previous investigations. Results showed that the SNP was located at the target site binding to microRNA (miR-511). The 3'UTR of IGF-1 gene with C allele significantly downregulated the expression of IGF-1 gene compared with that of the gene with T allele by luciferase assay. miR-511 was transfected into porcine kidney cell line (PK-15 cells) to reveal its effects on cells and whether or not it targets IGF-1. The expression levels of IGF-1 at mRNA and protein levels were remarkably downregulated. miR-511 significantly inhibited cell proliferation and promoted cell apoptosis by downregulating the phosphorylation level of AKT and ERK1/2. This finding confirmed that miR-511 inhibits proliferation and promotes apoptosis by downregulating the IGF-1 in PK-15 cells.

Apoptosis Induction by Pseudorabies Virus via Oxidative Stress and Subsequent DNA Damage Signaling

Background: Pseudorabies virus (PRV) infection induces apoptosis in swine cells both in vitro and in vivo; however, the mechanism associated with host-cell signaling has not been studied. This study investigated the role of free radicals caused by cellular oxidative stress after viral infection and examined whether the DNA damage response plays an important role in PRV-induced apoptosis. Methods: Several apoptosis assays and western blotting confirmed PRV-induced apoptosis. PRV-mediated oxidative stress was evaluated by reactive oxygen species (ROS) assay. Results: Our results showed that PRV caused apoptosis in a porcine kidney cell line, PK15, and induced expressions of proapoptotic Bcl family proteins in a dose- and time-dependent manner. Expressions of specific DNA damage sensors and phosphorylation of histone H2AX were also significantly increased, which subsequently activated the expressions of checkpoint kinase 1/2 and proapoptotic p53. Caffeine, a known DNA damage inhibitor, was found to inhibit caspase-3 activation and protect cells from PRV-induced apoptosis. Additionally, the antioxidant N-acetyl-L-cysteine was shown to prevent the production of cellular ROS, protecting DNA from cleavage. Conclusions: Our results confirmed that oxidative stress and free radicals arising from PRV infection cause DNA damage, which consequently triggers apoptosis.

Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter.

Pigs are commonly used as an animal model to evaluate the toxic effects of exogenous compounds. Cytochrome P450 1A1 (CYP1A1) metabolizes numerous exogenous compounds and is abundantly expressed in the liver, kidneys, and intestines. The high amino acid similarity between human and porcine CYP1A1 indicates that they probably have the same metabolic characteristics. Therefore, understanding the regulatory mechanism of CYP1A1 expression in pigs is particularly important for predicting the toxicology and metabolic kinetics of exogenous chemicals. Currently, the transcriptional regulation of porcine CYP1A1 has rarely been studied, especially regarding basal transcription. In this study, we first confirmed that the key regulatory elements of porcine CYP1A1 basal transactivation are in the proximal promoter region using promoter truncation analysis via a dual luciferase assay in a porcine kidney cell line LLC-PK1. Two overlapping cis-elements, the xenobiotic response element (XRE) and GC box, in this proximal region potentially play key roles in the basal transactivation of porcine CYP1A1. Furthermore, using electrophoretic mobility shift assay and chromatin immunoprecipitation, the GC box binding protein Sp1 was confirmed to bind to the proximal promoter of porcine CYP1A1, instead of AhR, the XRE binding protein. In LLC-PK1 cells, by knocking down either Sp1 or AhR, the expression of porcine CYP1A1 at the mRNA level and protein level was significantly downregulated, suggesting both proteins are important for porcine CYP1A1 expression. However, promoter activity analysis in LLC-PK1 cells treated with an AhR agonist and antagonist confirmed that AhR does not participate in the basal regulation of porcine CYP1A1 at the proximal promoter. In conclusion, our study revealed that the proximal promoter is the key regulatory region for porcine CYP1A1 basal expression. Although AhR plays an important role in the transactivation of porcine CYP1A1 expression, the key determinant transcription factor for its basal transactivation is Sp1 at the proximal promoter of porcine CYP1A1.

Genetic variation analysis of PCV1 strains isolated from Guangxi Province of China in 2015

BackgroundPorcine circovirus type 1 (PCV1) was discovered in 1974 as a contaminant of a porcine kidney (PK-15) cell line and was generally accepted to be nonpathogenic. But recently it was shown to cause lesions in experimentally infected pig fetuses. Serological evidence and genetic studies suggested that PCV1 was widespread in domestic pigs. Thus, the molecular epidemiology and genetic variation of PCV1 are still necessary to understand.ResultsHere 247 tissue samples were collected from piglets in Guangxi Province, China and performed whole-genome sequencing of the PCV1 genome. Thirteen PCV1 strains were sequenced from the samples. Similarity analysis showed that there were 97.8% to 99.6% nucleotide similarity to each other and 97.1% to 99.8% nucleotide similarity to the 40 reference strains. Besides, based on sequence analysis, we found one putative recombinant virus named GXdx84 strain contained the open-reading frame 1 (ORF1) of PCV1 and the ORF2 of PCV2d-2, which was consistent with the results of phylogenetic analysis that compared PCV1 and PCV2 strains. Variation analysis of the amino acids of the capsid protein revealed that the GXyl224 strain, which encoded 235 amino acids, had two amino acids more than other strains. This is the first study to report that a cap gene mutation resulted in lengthening of in the gene sequence.ConclusionsThese data contribute to the understanding of PCV1 evolution and molecular epidemiology that will facilitate programs for its control and prevention.

Multiplatform next-generation sequencing identifies novel RNA molecules and transcript isoforms of the endogenous retrovirus isolated from cultured cells.

In this study, we applied short- and long-read RNA sequencing techniques, as well as PCR analysis to investigate the transcriptome of the porcine endogenous retrovirus (PERV) expressed from cultured porcine kidney cell line PK-15. This analysis has revealed six novel transcripts and eight transcript isoforms, including five length and three splice variants. We were able to establish whether a deletion in a transcript is the result of the splicing of mRNAs or of genomic deletion in one of the PERV clones. Additionally, we re-annotated the formerly identified RNA molecules. Our analysis revealed a higher complexity of PERV transcriptome than it was earlier believed.

MiR-194b-3p partially inhibits Streptococcus equi subsp. zooepidemicus adherence to PK15 cells

MicroRNAs are increasingly reported implicated in the host cell response to bacterial pathogens. In order to investigate whether miR-194b-3p regulates the adherence of Streptococcus equi subsp. Zooepidemicus (SeZ) to porcine kidney cell line PK15, the miR-194b-3p agomir and antagomir were transfected into PK15 cells respectively and the adherence rate of SeZ to each was determined. Adherence rate of SeZ C55138 was significantly decreased when miR-194b-3p agomir was transfected in PK15, while that of miR-194b-3p antagomir evaluated. These results confirmed that miR-194b-3p markedly inhibit the adherence of SeZ C55138 to PK15 cells. In addition, miR-194b-3p indeed regulated the expression level of CD44 in PK15 cells by targeting CD44 3’ UTR, and this interaction was involved in adhesion process. This study contributes to understanding the mechanism of the crosstalk between SeZ and PK15 cells.