Porcine Kidney Epithelial Cells Research Articles

Genome-Wide PERV Inactivation in Pigs Using CRISPR/Cas9.

The shortage of organs for transplantation is probably the biggest unmet medical need. A potential problem with the clinical use of porcine xenografts is the risk that porcine endogenous retroviruses (PERVs) could infect human cells. In the past, we determined the PERV copy number in the porcine kidney epithelial cell line PK15 and in primary fibroblasts. Using CRISPR-Cas9, we disrupted the catalytic center of pol, which is essential for virus replication. Next, we isolated cells in which 100% of the PERV elements had been inactivated. This method enables the possibility of eradicating PERVs in vitro for application to pig-to-human xenotransplantation. Here we describe the methodological bases of this work.

Structural Insights into Escherichia coli Shiga Toxin (Stx) Glycosphingolipid Receptors of Porcine Renal Epithelial Cells and Inhibition of Stx-Mediated Cellular Injury Using Neoglycolipid-Spiked Glycovesicles.

Shiga toxin (Stx) producing Escherichia coli (STEC) cause the edema disease in pigs by releasing the swine-pathogenic Stx2e subtype as the key virulence factor. Stx2e targets endothelial cells of animal organs including the kidney harboring the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Since the involvement of renal epithelial cells in the edema disease is unknown, in this study, we analyzed the porcine kidney epithelial cell lines, LLC-PK1 and PK-15, regarding the presence of Stx-binding GSLs, their sensitivity towards Stx2e, and the inhibitory potential of Gb3- and Gb4-neoglycolipids, carrying phosphatidylethanolamine (PE) as the lipid anchor, towards Stx2e. Immunochemical and mass spectrometric analysis revealed various Gb3Cer and Gb4Cer lipoforms as the dominant Stx-binding GSLs in both LLC-PK1 and PK-15 cells. A dihexosylceramide with proposed Galα1-4Gal-sequence (Gal2Cer) was detected in PK-15 cells, whereas LLC-PK1 cells lacked this compound. Both cell lines were susceptible towards Stx2e with LLC-PK1 representing an extremely Stx2e-sensitive cell line. Gb3-PE and Gb4-PE applied as glycovesicles significantly reduced the cytotoxic activity of Stx2e towards LLC-PK1 cells, whereas only Gb4-PE exhibited some protection against Stx2e for PK-15 cells. This is the first report identifying Stx2e receptors of porcine kidney epithelial cells and providing first data on their Stx2e-mediated damage suggesting possible involvement in the edema disease.

Porcine IFITM1 is a host restriction factor that inhibits pseudorabies virus infection

Interferon-inducible transmembrane proteins (IFITMs) restrict infection by several viruses, such as influenza A virus, West Nile virus and dengue virus. It has not been determined whether porcine IFITMs (pIFITMs) inhibit infection by pseudorabies virus (PRV), an enveloped, double-stranded DNA virus, which is the etiological agent of Aujeszky’s disease in pigs. Here, we report that PRV infection elicited pIFITM1 expression in PK15 porcine kidney epithelial cells and 3D4/21 alveolar macrophages. pIFITM2 and pIFITM3 expression was only elevated in PK15 cells during PRV infection. Depletion of pIFITM1 using RNA interference, either in PK15 or in 3D4/21 cells, enhanced PRV infection while overexpression of pIFITM1 had the opposite effect. Knockdown of pIFITM2 and pIFITM3 did not influence PRV infection, suggesting that pIFITM2 and pIFITM3 are independent of PRV infection. PRV-induced pIFITM1 expression was dependent on the cGAS/STING/TBK1/IRF3 innate immune pathway and interferon-alpha receptor-1, suggesting that pIFITM1 is up-regulated by the type I interferon signaling pathway. The anti-PRV role of pIFITM1 was inhibited upon PRV entry. Our data demonstrate that pIFITM1 is a host restriction factor that inhibits PRV entry that may shed light on a strategy for prevention of PRV infection.

Effects of porcine STC-1 on cell metabolism and mitochondrial function

Stanniocalcin (STC-1), a kind of glycoprotein hormone, was first found in fish and mainly regulates calcium/phosphorus metabolism in the body. To explore the biological function of the porcine STC-1 gene, the effects of changes in stanniocalcin expression on cellular metabolism and mitochondrial function were studied. A vector overexpressing the STC-1 gene and an siRNA silencer of the STC-1 gene were transfected into porcine kidney epithelial PK15 cells. After the STC-1 gene expression level was induced to change, STC-1 protein- and mitochondrial function-related proteins such as PMP70, OPA, DRP, Mfn and STC-1-related acetylated protein were detected by Western blotting. Cell apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS), and ATP were detected using flow cytometry methods. Transmission electron microscopy was used to observe the changes in mitochondrial structure and morphology. The results showed that overexpression of the STC-1 gene could significantly upregulate the levels of PMP70, OPA, DRP and Mfn. STC-1 gene expression, which could decrease the apoptosis rate and reactive oxygen species production to significantly increase the cell membrane potential and reduce the formation of intracellular ATP, which also affected the morphology and number of mitochondria. The results were reversed when the STC-1 gene expression was silenced. The results suggested that the porcine STC-1 gene is closely related to cell growth metabolism and mitochondrial function, which influence the mitochondrial function-related proteins. The present study is useful for further understanding STC-1 gene function and provides a theoretical basis for improving the production characteristics of domestic pigs.

Role of p53 in pseudorabies virus replication, pathogenicity, and host immune responses

As a key cellular transcription factor that plays a central role in cellular responses to a broad range of stress factors, p53 has generally been considered as a host cell restriction factor for various viral infections. However, the defined roles of p53 in pseudorabies virus (PRV) replication, pathogenesis, and host responses remain unclear. In the present study, we initially constructed a p53 overexpressing a porcine kidney epithelial cell line (PK-15) to detect the effect of p53 on PRV replication in vitro. The results show that viral glycoprotein B (gB) gene copies and the titers of virus were significantly higher in p53 overexpressing PK-15 cells than in PK-15 and p53 inhibitor treated p53 overexpressing PK-15 cells. A similar result was also found in the p53 inhibitor PFT-α-treated PK-15 cells. We then examined the effects of p53 on PRV infection in vivo by using p53-knockout (p53−/−) mice. The results show that p53 knockout not only led to significantly reduced rates of mortality but also to reduced viral replication and development of viral encephalitis in the brains of mice following intracranial inoculation. Furthermore, we examined the effect of p53 knockout on the expression of the reported host cell regulators of PRV replication in the brains of mice by using RNA sequencing. The results show that p53 knockout downregulated the interferon (IFN) regulator genes, chemokine genes, and antiviral genes after PRV infection. This finding suggests that p53 positively regulates viral replication and pathogenesis both in vitro and in vivo. These findings offer novel targets of intrinsic host cell immunity for PRV infection.

Disabling of nephrogenesis in porcine embryos via CRISPR/Cas9-mediated SIX1 and SIX4 gene targeting.

SIX1 and SIX4 genes play critical roles in kidney development. We evaluated the effect of these genes on pig kidney development by generating SIX1-/- and SIX1-/- /SIX4-/- pig foetuses using CRISPR/Cas9 and somatic cell nuclear transfer. We obtained 3 SIX1-/- foetuses and 16 SIX1-/- /SIX4-/- foetuses at different developmental stages. The SIX1-/- foetuses showed a migration block of the left kidney and a smaller size for both kidneys. The ureteric bud failed to form the normal branching and collecting system. Abnormal expressions of kidney development-related genes (downregulation of PAX2, PAX8, and BMP4 and upregulation of EYA1 and SALL1) were also observed in SIX1-/- foetal kidneys and confirmed in vitro in porcine kidney epithelial cells (PK15) following SIX1 gene deletion. The SIX1-/- /SIX4-/- foetuses exhibited more severe phenotypes, with most foetuses showing retarded development at early stages of gestation. The kidney developed only to the initial stage of metanephros formation. These results demonstrated that SIX1 and SIX4 are key genes for porcine metanephros development. The creation of kidney-deficient porcine foetuses provides a platform for generating human kidneys inside pigs using blastocyst complementation.

The Anti-bacterial and Anti-adherent Effects of Pentraxin-3 on Porcine Kidney Epithelial PK15 Cells Against Staphylococcus aureus Infection

Staphylococcus aureus is a common zoonotic bacterium for disease in pigs and human and causes health problem. The aim of the study is to investigate the potential of soluble pathogenic recognition protein pentraxin-3 (PTX3) on inhibiting proliferation, adhesion, and infection of S. aureus in porcine kidney epithelial PK15 cells (PK15 cells). PTX3 suppressed the growth of S. aureus and avoided it adhering to PK15 cells. PTX3 treatment also attenuated cell apoptosis induced by S. aureus in PK15 cells. The results suggest that PTX3 is able to interfere with the bacterial pathogenesis via inhibiting the growth of S. aureus and decreasing their adhesion to cells. Hence, PTX3 may replace the use of antibiotics and develop as a novel anti-microbial agent for disease prevention in piglets.

ITRAQ-based quantitative proteomic analysis reveals multiple effects of Emodin to Haemophilus parasuis

Haemophilus parasuis, a symbiotic bacteria of upper respiratory tract of swine, is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis. Emodin, exhibits antibacterial activity against H. parasuis, yet the action mode has not been fully understood. In present study, isobaric tag for relative and absolute quantification (iTRAQ) method was applied to analyze the global protein alteration of H. parasuis in response to 16μg/mL Emodin. In total, 338 proteins exhibiting significant differential expressions were identified. It was speculated that, through application of bioinformatics analysis to theses differentially expressed proteins, Emodin mainly inhibited some key proteins expression of ABC transport system, carbohydrate metabolism pathway and bacterial cell division by inhibiting the ribosome synthesis, resulting in the growth inhibition of H. parasuis. Remarkably, nine virulence-associated proteins were detected differently expressed, further experiments revealed that after treatment with Emodin, H. parasuis could be inhibited to adhere to and invade into porcine kidney epithelial cells (PK-15 line) and exhibited increased sensitivity to serum complement in a concentration-dependent manner. Phagocytosis assay showed Emodin also could enhance phagocytic activity of porcine alveolar macrophages PAM to H. parasuis. These results indicated that Emodin also can attenuate virulence of H. parasuis and reduce infection. Biological significanceThe Glässer's disease caused by H. parasuis has become a typical bacterial disease and cause serious economic loss to the swine industry around the world. Antibiotics are extensively used to control the infection, but increasing antibiotic resistance has been a severe problem. Hence, novel treatment agents are needed. So far, few antibacterial agents were reported that could control H. parasuis infection. In the present study, the state-of-the-art quantitative proteomic technology was applied to uncover underlying action mechanism of Emodin. This study extends understanding of antibacterial effect of Emodin to H. parasuis at molecular level and provides useful information for further investigations. Moreover, our results provide theoretical foundation for the practical application of Emodin.

Swine IRF3/IRF7 attenuates inflammatory responses through TLR4 signaling pathway.

To explore the role of IRF3/IRF7 during inflammatory responses, we investigated the effects of swine IRF3/IRF7 on TLR4 signaling pathway and inflammatory factors expression in porcine kidney epithelial PK15 cell lines. We successfully constructed eukaryotic vectors PB-IRF3 and PB-IRF7, transfected these vectors into PK15 cells and observed GFP under a fluorescence microscope. In addition, RT-PCR was also used to detect transfection efficiency. We found that IRF3/IRF7 was efficiently overexpressed in PK15 cells. Moreover, we evaluated the effects of IRF3/IRF7 on the TLR4 signaling pathway and inflammatory factors by RT-PCR. Transfected cells were treated with lipopolysaccharide (LPS) alone, or in combination with a TBK1 inhibitor (LiCl). We revealed that IRF3/IRF7 enhanced IFNα production, and decreased IL-6 mRNA expression. Blocking the TBK1 pathway, inhibited the changes in IFNα, but not IL-6 mRNA. This illustrated that IRF3/IRF7 enhanced IFNα production through TLR4/TBK1 signaling pathway and played an anti-inflammatory role, while IRF3/IRF7 decreased IL-6 expression independent of the TBK1 pathway. Trends in MyD88, TRAF6, TBK1 and NFκB mRNA variation were similar in all treatments. LPS increased MyD88, TRAF6, TBK1 and NFκB mRNA abundance in PBR3/PBR7 and PBv cells, while LiCl blocked the LPS-mediated effects. The levels of these four factors in PBR3/PBR7 cells were higher than those in PBv. These results demonstrated that IRF3/IRF7 regulated the inflammatory response through the TLR4 signaling pathway. Overexpression of swine IRF3/IRF7 in PK15 cells induced type I interferons production, and attenuated inflammatory responses through TLR4 signaling pathway.

Human galectin-9 on the porcine cells affects the cytotoxic activity of M1-differentiated THP-1 cells through inducing a shift in M2-differentiated THP-1 cells.

In xenotransplantation, immune rejection by macrophages occurs rapidly and remains a major obstacle. Studies to control immune rejection in macrophages have been continuing to date. Recent studies have reported that human galectin-9 (hGal-9) can regulate the function of regulatory T cells (Treg), as well as cytotoxicity T cells (CTL) and natural killer cells (NK). Although the effect of hGal-9 on lymphocytes has been well studied, the relationship between hGal-9 and myeloid cells has been scarcely studied. To confirm the decreased cytotoxic activity effect by hGal-9 in M1-differentiated THP-1 cells, we established the hGal-9 expressing transgenic porcine cell line. hGal-9 siRNA was transfected to transgenic cells and recombinant hGal-9 (rhGal-9) was treated to co-culturing condition, and then, flow cytometry assay was conducted for analyzing the cytotoxic activity of M1-differentiated THP-1 cells. Related inflammatory cytokines (IL-1β, IL-10, TNF-α, IL-6, IL-12, IL-23, and TGF-β) and related enzymes (iNOS and Arginase 1) were analyzed by qPCR and Western blot assay. To identify the shift in M1/M2-differentiated THP-1 cells, expression levels of CCR7, CD163, iNOS, and Arginase 1 and population of M2 marker positive cells were analyzed. The expression levels of pro-inflammatory cytokines in M1-differentiated THP-1 cells co-cultured with hGal-9-expressing porcine kidney epithelial cells were decreased, but not in co-cultured THP-1 cells. However, the expression levels of anti-inflammatory cytokines were also increased in co-cultured M1-differentiated THP-1 cells. The cytotoxicity effect of M1-differentiated THP-1 cells on transgenic cells was decreased while the expression levels of anti-inflammatory cytokines and M2 macrophages-related molecules were increased. M2 differentiation program was turned on while M1 program was turned down by enhancing the phosphorylation levels of Akt and PI3K and the expression level of PPAR-γ. Due to these changes, differentiation of M2 program was enhanced in cells co-cultured with hGal-9. These data suggested that hGal-9 has a reduction in M1-differentiated THP-1 cell cytotoxic activity-related acute immune rejection in pig-to-human xenotransplantation in addition to its role in lymphoid lineage immune cell regulation.

ITRAQ-based Proteomic Analysis of Porcine Kidney Epithelial PK15 cells Infected with Pseudorabies virus

Pseudorabies virus (PRV) is one of the most important pathogens of swine, resulting in severe economic losses to the pig industry. To improve our understanding of the host responses to PRV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry to quantitatively identify the differentially expressed cellular proteins in PRV-infected PK15 cells. In total, relative quantitative data were identified for 4333 proteins in PRV and mock- infected PK15 cells, among which 466 cellular proteins were differentially expressed, including 234 upregulated proteins and 232 downregulated proteins. Bioinformatics analysis disclosed that most of these differentially expressed proteins were involved in metabolic processes, cellular growth and proliferation, endoplasmic reticulum (ER) stress response, cell adhesion and cytoskeleton. Moreover, expression levels of four representative proteins, beta-catenin, STAT1, GRB2 and PCNA, were further confirmed by western blot analysis. This is the first attempt to analyze the protein profile of PRV-infected PK15 cells using iTRAQ technology, and our findings may provide valuable information to help understand the host response to PRV infection.

Two Glycosyltransferase Genes of Haemophilus parasuis SC096 Implicated in Lipooligosaccharide Biosynthesis, Serum Resistance, Adherence, and Invasion

Haemophilus parasuis is a common opportunistic pathogen known for its ability to colonize healthy piglets and causes Glässer's disease. The lipooligosaccharide (LOS) of H. parasuis is a potential virulence-associated factor. In this study, two putative glycosyltransferases that might be involved in LOS synthesis in H. parasuis SC096 were identified (lgtB and lex-1). Mutants were constructed to investigate the roles of the lgtB and lex-1 genes. The LOS from the ΔlgtB or Δlex-1 mutant showed truncated structure on silver-stained SDS-PAGE gel compared to the wild-type strain. The ΔlgtB and Δlex-1 mutants were significantly more sensitive to 50% porcine serum, displaying 15.0 and 54.46% survival rates, respectively. Complementation of the lex-1 mutant restored the serum-resistant phenotype. Additionally, the ΔlgtB and Δlex-1 strains showed impaired ability to adhere to and invade porcine kidney epithelial cells (PK-15). The above results suggested that the lgtB and lex-1 genes of the H. parasuis SC096 strain participated in LOS synthesis and were involved in serum resistance, adhesion and invasion.

MiR-124 attenuates Japanese encephalitis virus replication by targeting DNM2

BackgroundJapanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifier hosts of JEV. Emerging evidence indicates that host microRNAs (miRNAs) play key roles in modulating viral infection and pathogenesis. However, mechanistic studies delineating the roles of miRNAs in regulating host-JEV interactions remain scarce.ResultsIn this study, we demonstrated that miR-124 inhibited JEV replication in porcine kidney epithelial PK15 cells. Furthermore, using bioinformatics tools, we identified dynamin2 (DNM2), a GTPase responsible for vesicle scission, as a target of miR-124. Small interfering RNA (siRNA) depletion studies inicated that dynamin2 was required for efficient JEV replication. We also demonstrated that upregulation of miR-124 expression corresponded to decreased expression of its target, DNM2, in the JEV-infected PK15 cells.ConclusionsOverall, these results suggest the importance of miR-124 in modulating JEV replication and provide a scientific basis for using cellular miRNAs in anti-JEV therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0562-y) contains supplementary material, which is available to authorized users.

New Carbocyclic Amino Acid Derivatives Inhibit Infection Caused by Highly Pathogenic Influenza A Virus Strain (H5N1).

New amino acid derivatives with carbocycles of adamantine and quinaldic acid were synthesized and their in vitro antiviral activity against influenza A/H5N1 virus was evaluated. Experiments on cultured embryonic porcine kidney epithelial cells showed that amino acid derivatives suppressed viral replication. Tret-butyloxycarbonyl-DL-methionylsulfonyl-1-adamantayl ethylamine and benzyloxycarbonyl-L-trypthophanyl-1-adamantayl ethylamine compounds demonstrated high activity in all in vitro experiments. Moreover, some compounds showed virucidal activity against influenza A/H5N1 virus.

Porcine reproductive and respiratory syndrome virus non-structural protein Nsp2TF down-regulates Swine Leukocyte Antigen class I (SLA class I) expression

Abstract Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is arguably the most economically-important global swine pathogen. The virus typically induces sub-optimal immune responses especially a gradual increase of cellular immune-mediated (CMI) responses in the host. In this study, we demonstrated that PRRSV reduced SLA-I surface expression on porcine alveolar macrophages, monocyte-derived dendritic cells and a porcine kidney epithelial cell line, which may partly explain for the impaired CMI responses observed against PRRSV in pigs. To identify the PRRSV proteins responsible for SLA-I down-regulation, we screened all 22 PRRSV proteins by transient transfection assays. Our results indicated that Nsp1α, Nsp2TF and GP3 significantly down-regulated SLA-I surface expression of which Nsp2TF showed the greatest effect. To further validate whether Nsp2TF directly contributes to the PRRSV-induced SLA-I down-regulation, a panel of mutant viruses were generated with abolished Nsp2TF expression by disrupting −2 ribosomal frameshifting elements essential for the Nsp2TF protein synthesis as well as by introducing stop codons in TF domain of the protein. The infections with these mutant PRRSV strains fully restored the SLA-I surface expression in the infected cells. Additionally we demonstrated that the last 68 amino acids of TF domain are critical for this function. Our study for the first time uncovered an immune-modulatory function of PRRSV Nsp2TF in down-regulating SLA-I expression. Further studies are warranted to generate Nsp2TF-knockout vaccine strains and investigate their efficacies in inducing better CMI responses against the virus in pigs.

293 cells express both epithelial as well as mesenchymal cell adhesion molecules.

The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells.

Identification and Analysis of Novel Viral and Host Dysregulated MicroRNAs in Variant Pseudorabies Virus-Infected PK15 Cells.

Pseudorabies (PR) is one of the most devastating diseases in the pig industry. To identify changes in microRNA (miRNA) expression and post-transcriptional regulatory responses to PRV infection in porcine kidney epithelial (PK15) cells, we sequenced a small RNA (sRNA) library prepared from infected PK15 cells and compared it to a library prepared from uninfected cells using Illumina deep sequencing. Here we found 25 novel viral miRNAs by high-throughput sequencing and 20 of these miRNAs were confirmed through stem-loop RT-qPCR. Intriguingly, unlike the usual miRNAs encoded by the α-herpesviruses, which are found clustered in the large latency transcript (LLT), these novel viral miRNAs are throughout the PRV genome like β-herpesviruses. Viral miRNAs are predicted to target multiple genes and form a complex regulatory network. GO analysis on host targets of viral miRNAs were involved in complex cellular processes, including the metabolic pathway, biological regulation, stimulus response, signaling process and immune response. Moreover, 13 host miRNAs were expressed with significant difference after infection with PRV: 8 miRNAs were up-regulated and 5 miRNAs were down-regulated, which may affect viral replication in host cell. Our results provided new insight into the characteristic of miRNAs in response to PRV infection, which is significant for further study of these miRNAs function.

Genome-wide inactivation of porcine endogenous retroviruses (PERVs).

The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.

Changes in cellular microRNA expression induced by porcine circovirus type 2-encoded proteins.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which leads to serious economic losses in the pig industry worldwide. While the molecular basis of PCV2 replication and pathogenicity remains elusive, it is increasingly apparent that the microRNA (miRNA) pathway plays a key role in controlling virus-host interactions, in addition to a wide range of cellular processes. Here, we employed Solexa deep sequencing technology to determine which cellular miRNAs were differentially regulated after expression of each of three PCV2-encoded open reading frames (ORFs) in porcine kidney epithelial (PK15) cells. We identified 51 ORF1-regulated miRNAs, 74 ORF2-regulated miRNAs, and 32 ORF3-regulated miRNAs that differed in abundance compared to the control. Gene ontology analysis of the putative targets of these miRNAs identified transcriptional regulation as the most significantly enriched biological process, while KEGG pathway analysis revealed significant enrichment for several pathways including MAPK signaling, which is activated during PCV2 infection. Among the potential target genes of ORF-regulated miRNAs, two genes encoding proteins that are known to interact with PCV2-encoded proteins, zinc finger protein 265 (ZNF265) and regulator of G protein signaling 16 (RGS16), were selected for further analysis. We provide evidence that ZNF265 and RGS16 are direct targets of miR-139-5p and let-7e, respectively, which are both down-regulated by ORF2. Our data will initiate further studies to elucidate the roles of ORF-regulated cellular miRNAs in PCV2-host interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0172-5) contains supplementary material, which is available to authorized users.

NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells.

The gain-of-function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop resulting in renal loss of NaCl in the absence of mutations in renal Na(+) and Cl(-) ion transporters. The molecular mechanisms underlying this clinical phenotype are incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC-PK1) cells, the functional cross-talk of CaR-A843E with the Na(+):K(+):2Cl(-) co-transporter, NKCC2, which provides NaCl reabsorption in the TAL. The expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC-PK1 cells. However, the steady-state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR-A843E compared with the control wild-type CaR (CaR WT)-transfected cells. Of note, low-Cl(-)-dependent NKCC2 activation was also strongly inhibited upon the expression of CaR-A843E mutant. The use of either P450 ω-hydroxylase (CYP4)- or phospholipase A2 (PLA2)-blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites. The data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5.