LLC-PK1 Cells Research Articles

Focal Adhesion Kinase (FAK) inhibition induces membrane accumulation of aquaporin2 (AQP2) through endocytosis inhibition and actin depolymerization in renal epithelial cells.

Cellular trafficking of the water channel aquaporin 2 (AQP2) is regulated by the actin cytoskeleton in collecting duct principal cells (PC) to maintain proper water balance in animals. Critical actin depolymerization/polymerization events are involved in both constitutive AQP2 recycling, and the pathway stimulated by vasopressin receptor signaling. Focal adhesion kinase (FAK) plays an important role in modulating the actin cytoskeleton through inhibiting small GTPases, and multiple studies have shown the involvement of FAK in insulin and cholesterol trafficking through actin regulation. To understand whether FAK contributes to water reabsorption by the kidney, we performed a series of in vitro experiments to examine the involvement of FAK and its signaling in mediating AQP2 trafficking in cultured renal epithelial cells. Our data showed that FAK inhibition by specific inhibitors caused membrane accumulation of AQP2 in AQP2expressing LLCPK1 cells by immunofluorescence staining. AQP2 membrane accumulation induced by FAK inhibition is associated with significantly reduced endocytosis of AQP2 via the clathrin-mediated endocytosis pathway. Moreover, AQP2 membrane accumulation induced by FAK inhibition also occurred in cells expressing the constitutive dephosphorylation mutant of AQP2, S256A. This was confirmed by immunoblotting using a specific antibody against phospho-serine 256 AQP2, supporting a phosphorylation independent mechanism. Finally, we demonstrated that inhibition of FAK caused reduced RhoA signaling and promoted F-actin depolymerization. In conclusion, our study identifies FAK signaling as a pathway that could provide a novel therapeutical avenue for AQP2 trafficking regulation in water balance disorders.

APC orchestrates microtubule dynamics by acting as a positive regulator of KIF2A and a negative regulator of CLASPs

Tumor suppressor protein Adenomatous polyposis coli protein (APC) is an EB-binding and microtubule (MT) plus end-tracking protein; however, how exactly APC regulates MT dynamics remains elusive. Here, we show that in LLC-PK1 cells, APC and KIF2A, an MT depolymerase, form a complex clustering at the cell edge and destabilize MTs at the MT plus ends. Further biochemical characterization and mutational analysis reveal key residues for the APC-KIF2A interaction. In addition, APC counteracts the major MT-stabilizer CLASPs at MT plus ends and promotes directional cell migration via modulating cell adhesion force. Reconstitution experiments demonstrate that APC potentiates KIF2A-induced MT catastrophes and antagonizes the stabilizing effect of CLASP2 in vitro. In summary, APC functions as a positive regulator of MT-destabilizer and a negative regulator of MT-stabilizer to orchestrate MT dynamics.

The GTP-tubulin cap is not the determinant of microtubule end stability in cells.

Microtubules are dynamic cytoskeletal polymers essential for cell division, motility, and intracellular transport. Microtubule dynamics are characterized by dynamic instability-the ability of individual microtubules to switch between phases of growth and shrinkage. Dynamic instability can be explained by the GTP-cap model, suggesting that a "cap" of GTP-tubulin subunits at the growing microtubule end has a stabilizing effect, protecting against microtubule catastrophe-the switch from growth to shrinkage. Although the GTP-cap is thought to protect the growing microtubule end, whether the GTP-cap size affects microtubule stability in cells is not known. Notably, microtubule end-binding proteins, EBs, recognize the nucleotide state of tubulin and display comet-like localization at growing microtubule ends, which can be used as a proxy for the GTP-cap. Here, we employ high spatiotemporal resolution imaging to compare the relationship between EB comet size and microtubule dynamics in interphase LLC-PK1 cells to that measured in vitro. Our data reveal that the GTP-cap size in cells scales with the microtubule growth rate in the same way as in vitro. However, we find that microtubule ends in cells can withstand transition to catastrophe even after the EB comet is lost. Thus, our findings suggest that the presence of the GTP-cap is not the determinant of microtubule end stability in cells.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method

BackgroundPorcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour.ResultsThis study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method.ConclusionThe developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Effective inhibition of PDCoV infection in chimeric APN gene-edited neonatal pigs.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are currently no effective measures for the prevention or control of PDCoV infection. Here, we have developed PK-15 cells, LLC-PK1 cells, and primary intestinal epithelial cells expressing chimeric APN, and viral challenge of these cells led to decreased PDCoV infection. Furthermore, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. These data show that chimeric APN is a promising strategy to combat PDCoV infection.

The angiotensin II/type 1 angiotensin II receptor pathway is implicated in the dysfunction of albumin endocytosis in renal proximal tubule epithelial cells induced by high glucose levels

It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10−7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.

Cyclophilin A promotes porcine deltacoronavirus replication by regulating autophagy via the Ras/AKT/NF-κB pathway

Porcine deltacoronavirus (PDCoV) is an important enteric coronavirus that has caused major worldwide economic losses in the pig industry. Previous studies have shown that cyclophilin A (CypA), a key player in aetiological agent infection, is involved in regulating viral infection. However, the role of CypA during PDCoV replication remains unknown. Therefore, in this study, the role of CypA in PDCoV replication was determined. The results demonstrated that PDCoV infection increased CypA expression in LLC-PK1 cells. CypA overexpression substantially promoted PDCoV replication. Proteomic analysis was subsequently used to assess changes in total protein expression levels after CypA overexpression. Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to further determine the mechanisms by which CypA affects viral replication. Proteomic analysis revealed that CypA protein overexpression significantly upregulated 75 differentially expressed proteins and significantly downregulated 172 differentially expressed proteins. The differentially expressed proteins were involved mainly in autophagy and activation of the host innate immune pathway. Subsequent experimental results revealed that the CypA protein promoted viral replication by reducing the levels of natural immune cytokines and mitigated the inhibitory effect of chloroquine (CQ) on viral replication. Further investigation revealed that CypA could activate the Ras/AKT/NF-κB pathway, mediate autophagy signalling and promote PDCoV replication. In summary, the findings of this study may help elucidate the role of CypA in PDCoV replication.

Serial cell culture passaging in vitro led to complete attenuation and changes in the characteristic features of a virulent porcine deltacoronavirus strain.

Porcine deltacoronavirus (PDCoV) is an important enteric coronavirus that has caused enormous economic losses in the pig industry worldwide. However, no commercial vaccine is currently available. Therefore, developing a safe and efficacious live-attenuated vaccine candidate is urgently needed. In this study, the PDCoV strain CH/XJYN/2016 was continuously passaged in LLC-PK cells until passage 240, and the virus growth kinetics in cell culture, pathogenicity in neonatal piglets, transcriptome differences after LLC-PK infection, changes in the functional characteristics of the spike (S) protein in the high- and low-passage strains, genetic variation of the virus genome, resistance to pepsin and acid, and protective effects of this strain when used as a live-attenuated vaccine were examined. The results of animal experiments demonstrated that the virulent PDCoV strain CH/XJYN/2016 was completely attenuated and not pathogenic in piglets following serial cell passage. Genome sequence analysis showed that amino acid mutations in nonstructural proteins were mainly concentrated in Nsp3, structural protein mutations were mainly concentrated in the S protein, and the N, M, and E genes were conserved. Transcriptome comparison revealed that compared with negative control cells, P10-infected LLC-PK cells had the most differentially expressed genes (DEGs), while P0 and P240 had the least number of DEGs. Analysis of trypsin dependence and related structural differences revealed that the P10 S protein interacted more strongly with trypsin and that the P120 S protein interacted more strongly with the APN receptor. Moreover, the infectivity of P240 was not affected by pepsin but was significantly decreased after exposure to low pH. Furthermore, the P240-based live-attenuated vaccine provided complete protection to piglets against the challenge of virulent PDCoV. In conclusion, we showed that a PDCoV strain was completely attenuated through serial passaging in vitro. These results provide insights into the potential molecular mechanisms of PDCoV attenuation and the development of a promising live-attenuated PDCoV vaccine.IMPORTANCEPorcine deltacoronavirus (PDCoV) is one of the most important enteropathogenic pathogens that cause diarrhea in pigs of various ages, especially in suckling piglets, and causes enormous economic losses in the global commercial pork industry. There are currently no effective measures to prevent and control PDCoV. As reported in previous porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus studies, inactivated vaccines usually elicit less robust protective immune responses than live-attenuated vaccines in native sows. Therefore, identifying potential attenuation mechanisms, gene evolution, pathogenicity differences during PDCoV passaging, and immunogenicity as live-attenuated vaccines is important for elucidating the mechanism of attenuation and developing safe and effective vaccines for virulent PDCoV strains. In this study, we demonstrated that the virulence of the PDCoV strain CH/XJYN/2016 was completely attenuated following serial cell passaging in vitro, and changes in the biological characteristics and protection efficacy of the strain were evaluated. Our results help elucidate the mechanism of PDCoV attenuation and support the development of appropriate designs for the study of live PDCoV vaccines.

Focal adhesion kinase (FAK) inhibition induces aquaporin 2 (AQP2) plasma membrane accumulation in renal epithelial cells by remodeling the actin cytoskeleton

The actin cytoskeleton is involved in the cellular traffcking of the AQP2 water channel in kidney collecting duct (CD) principal cells (PC): this plays an important role in water homeostasis. Focal adhesion kinase (FAK) is known to modulate the actin cytoskeleton through small GTPase-mediated pathways and we, therefore, investigated the potential role of FAK in AQP2 traffcking. We and others have previously shown that AQP2 constitutively traffcs and recycles independently of vasopressin (VP) and that this involves actin depolymerization/polymerization; therefore, FAK could regulate AQP2 traffcking through actin remodeling, bypassing the VP-V2R (cAMP/PKA) signaling pathway. To examine this, we used small molecule FAK inhibitors, VS-4718, GSK2256098 (GSK), or Defactinib-Hydrochloride (DH). We used immunofluorescence staining, a transferrin endocytosis assay ± cold block treatment, a fluorometric exocytosis assay using cells expressing secreted yellow fluorescent protein (ssYFP), a RhoA activity assay, and western blotting. Inhibiting FAK with 1mM VS-4718 for 30 min increased AQP2 membrane accumulation in LLCPK1 and IMCD kidney epithelial cells expressing AQP2. VS-4718 resulted in reduced clathrin-mediated endocytosis, as well as increased vesicle exocytosis, both mechanisms contributing to increased AQP2 membrane accumulation. This effect of FAK inhibition was independent of vasopressin/cAMP signaling and did not require phosphorylation of the Serine 256 residue of AQP2, since AQP2 membrane accumulation also occurred in cells expressing the constitutive dephosphorylation AQP2 S256A mutant. The FAK inhibitor VS-4718 promoted actin depolymerization (phalloidin staining assay) which would facilitate AQP2 vesicle exocytosis into the plasma membrane. We then examined the activity of the small GTPase, RhoA, whose activity increases actin polymerization and vesicle traffcking in the exocytic and endocytic pathways. We found that FAK inhibition by VS-4718 downregulated RhoA activity using GST-RBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Similar results were obtained with GSK and DH. In summary, our study revealed that the interaction between FAK and cytoskeletal architecture plays a crucial role in regulating AQP2 recycling and its subsequent entry into the endocytic pathway. Thus, targeting FAK could be a potential therapeutic strategy for water balance disorders involving defective AQP2 reabsorption, such as nephrogenic diabetes insipidus. This work was supported by NIDDK R01DK096015 (JHL) and DK096586 (DB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oxidative stress induced by menadione (vitamin K3) results in non-canonical AQP2 membrane traffcking in LLC-AQP2 cells

Oxidative stress affects a wide variety of cellular processes including morphology, protein traffcking, and cell death. Several studies show that reactive oxygen species (ROS) are able to pass through some water channels, and that ROS may modulate aquaporin expression and traffcking. However, a relationship between ROS status and the traffcking of aquaporin-2 (AQP2) has yet to be well-defined. To address this question, we first treated LLC-PK1 cells expressing cmyc-tagged AQP2 (LLC-AQP2 cells) for 30 min with menadione (MK3, 20 μM), an agent widely used to generate ROS in cell culture. Similar to vasopressin (VP), MK3 induced significant AQP2 accumulation in the plasma membrane of these cells. Using LLC-AQP2 cells expressing secreted soluble green fluorescent protein (ss-GFP), we revealed significantly increased vesicle exocytosis upon treatment with MK3. Surprisingly, MK3 did not affect rhodamine-labeled dextran endocytosis, suggesting that the MK3 effect is mainly due an increase in AQP2 exocytosis into the plasma membrane. Next, we studied the role of AQP2 c-terminal serine 256 phosphorylation in this process. MK3 treatment resulted in significant plasma membrane accumulation of the non-phosphorylatable AQP2 S256A mutant, whereas VP had no effect, as previously reported. To understand the contribution of protein kinase A (PKA) to MK3-mediated AQP2 traffcking, we used CRISPR-generated LLC-AQP2,koPKA cells that lack PKA isoforms. Interestingly, an increase in AQP2 membrane accumulation was still observed, suggesting that the MK3 effect is PKA-independent. By western blotting with anti-phospho-AQP2 antibodies, we found that MK3 did not affect phosphorylation of the critical S256 residue, but in contrast to VP, MK3 actually increased the phosphorylation level of S261 instead of reducing it. Finally, we showed that AQP2 accumulation in both LLC-AQP2 and LLC-AQP2 S256A cells was inhibited in the presence of glutathione (5 mM), an anti-oxidant or reducing agent. Interestingly, western blot analysis showed that glutathione abolished the MK3-induced increase of S261 AQP2 phosphorylation. In summary, our study shows that menadione (MK3) induces AQP2 membrane accumulation in LLC-AQP2 cells, suggesting a role of ROS in this process. This effect is largely due a change in AQP2 vesicle exocytosis. It does not require phosphorylation of the c-terminal S256 residue of AQP2, but MK3 does cause increased S261 phosphorylation. While incompletely understood at present, these data reveal that alternative, probably ROS-related, regulatory mechanisms can be involved in AQP2 traffcking and recycling. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Identification of two flavonoids antiviral inhibitors targeting 3C-like protease of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) has caused severe damage to the global pig industry in the past 20 years, creating an urgent demand for the development of associated medications. Flavonoids have emerged as promising candidates for combating coronaviruses. It is believed that certain flavonoids can directly inhibit the 3C-like protease (3CLpro), thus displaying antiviral activity against coronaviruses. In this investigation, we applied a flavonoid library to screen for natural compounds against PEDV 3CLpro. Baicalein and baicalin were found to efficiently inhibit PEDV 3CLproin vitro, with the IC50 value of 9.50 ± 1.02 μM and 65.80 ± 6.57 μM, respectively. A docking analysis supported that baicalein and baicalin might bind to the active site and binding pocket of PEDV 3CLpro. Moreover, both baicalein and baicalin successfully suppressed PEDV replication in Vero and LLC-PK1 cells, as indicated by reductions in viral RNA, protein, and titer. Further investigation revealed that baicalein and baicalin mainly inhibited the early viral replication of the post-entry stage. Furthermore, baicalein showed potential effects on the attachment or invasion step of PEDV. Collectively, our findings provide experimental proof for the inhibitory effects of baicalein and baicalin on PEDV 3CLpro activity and PEDV infection. These discoveries may introduce novel therapeutic strategies for controlling porcine epidemic diarrhea (PED).

A new workflow for the effective curation of membrane permeability data from open ADME information.

Membrane permeability is an in vitro parameter that represents the apparent permeability (Papp) of a compound, and is a key absorption, distribution, metabolism, and excretion parameter in drug development. Although the Caco-2 cell lines are the most used cell lines to measure Papp, other cell lines, such as the Madin-Darby Canine Kidney (MDCK), LLC-Pig Kidney 1 (LLC-PK1), and Ralph Russ Canine Kidney (RRCK) cell lines, can also be used to estimate Papp. Therefore, constructing in silico models for Papp estimation using the MDCK, LLC-PK1, and RRCK cell lines requires collecting extensive amounts of in vitro Papp data. An open database offers extensive measurements of various compounds covering a vast chemical space; however, concerns were reported on the use of data published in open databases without the appropriate accuracy and quality checks. Ensuring the quality of datasets for training in silico models is critical because artificial intelligence (AI, including deep learning) was used to develop models to predict various pharmacokinetic properties, and data quality affects the performance of these models. Hence, careful curation of the collected data is imperative. Herein, we developed a new workflow that supports automatic curation of Papp data measured in the MDCK, LLC-PK1, and RRCK cell lines collected from ChEMBL using KNIME. The workflow consisted of four main phases. Data were extracted from ChEMBL and filtered to identify the target protocols. A total of 1661 high-quality entries were retained after checking 436 articles. The workflow is freely available, can be updated, and has high reusability. Our study provides a novel approach for data quality analysis and accelerates the development of helpful in silico models for effective drug discovery. Scientific Contribution: The cost of building highly accurate predictive models can be significantly reduced by automating the collection of reliable measurement data. Our tool reduces the time and effort required for data collection and will enable researchers to focus on constructing high-performance in silico models for other types of analysis. To the best of our knowledge, no such tool is available in the literature.

Prediction of drug-drug interaction risk of P-glycoprotein substrate in drug discovery

We aimed at predicting the drug-drug interaction (DDI) risk of P-glycoprotein (P-gp) substrates by using P-gp expressing LLC-PK1 cells and its knockout mice (KO). The area under the curve (AUC) of 16 marketed drugs and plasma concentration (Cplasma) of 207 screening compounds, with corrected efflux ratio (CER) ≥ 2, were compared between P-gp KO mice and wild type mice (WT). At permeability (Papp) ≥ 10 × 10−6 cm/s in parent LLC-PK1 cells, AUC ratios (KO/WT) and Cplasma ratios (KO/WT) of these compounds were within 3-fold. AUC ratios (KO/WT) of clinical P-gp substrates, with human AUC ratios with and without P-gp inhibitor administration ≥2, were higher than 8.7. These observations led us to establish a work-flow of P-gp substrate assessment with the threshold AUC ratio (KO/WT) ≥ 9 leading to a DDI risk of AUC ratio (human) ≥ 2. A screening compound showing high CER (=57.6) was found, but its AUC ratio (KO/WT) was 3.7, had been presumed to be a weak risk and its AUC ratio (human) was 1.2 in a later clinical DDI study. Our proposed workflow should be useful for predicting the DDI risk of P-gp substrates in drug discovery.

Transcriptome Analysis of LLC-PK Cells Single or Coinfected with Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two most prevalent swine enteric coronaviruses worldwide. They commonly cause natural coinfections, which worsen as the disease progresses and cause increased mortality in piglets. To better understand the transcriptomic changes after PEDV and PDCoV coinfection, we compared LLC porcine kidney (LLC-PK) cells infected with PEDV and/or PDCoV and evaluated the differential expression of genes by transcriptomic analysis and real-time qPCR. The antiviral efficacy of interferon-stimulated gene 20 (ISG20) against PDCoV and PEDV infections was also assessed. Differentially expressed genes (DEGs) were detected in PEDV-, PDCoV-, and PEDV + PDCoV-infected cells at 6, 12, and 24 h post-infection (hpi), and at 24 hpi, the number of DEGs was the highest. Furthermore, changes in the expression of interferons, which are mainly related to apoptosis and activation of the host innate immune pathway, were found in the PEDV and PDCoV infection and coinfection groups. Additionally, 43 ISGs, including GBP2, IRF1, ISG20, and IFIT2, were upregulated during PEDV or PDCoV infection. Furthermore, we found that ISG20 significantly inhibited PEDV and PDCoV infection in LLC-PK cells. The transcriptomic profiles of cells coinfected with PEDV and PDCoV were reported, providing reference data for understanding the host response to PEDV and PDCoV coinfection.

Inhibition of porcine deltacoronavirus entry and replication by Cepharanthine

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that mainly causes acute diarrhea/vomiting, dehydration, and mortality in piglets, possessing economic losses and public health concerns. However, there are currently no proven effective antiviral agents against PDCoV. Cepharanthine (CEP) is a naturally occurring alkaloid used as a traditional remedy for radiation-induced symptoms, but its underlying mechanism of CEP against PDCoV has remained elusive. The aim of this study was to investigate the anti-PDCoV effects and mechanisms of CEP in LLC-PK1 cells. The results showed that the antiviral activity of CEP was based on direct action on cells, preventing the virus from attaching to host cells and virus replication. Importantly, Surface Plasmon Resonance (SPR) results showed that CEP has a moderate affinity to PDCoV receptor, porcine aminopeptidase N (pAPN) protein. AutoDock predicted that CEP can form hydrogen bonds with amino acid residues (R740, N783, and R790) in the binding regions of PDCoV and pAPN. In addition, RT-PCR results showed that CEP treatment could significantly reduce the transcription of ZBP1, cytokine (IL-1β and IFN-α) and chemokine genes (CCL-2, CCL-4, CCL-5, CXCL-2, CXCL-8, and CXCL-10) induced by PDCoV. Western blot analysis revealed that CEP could inhibit viral replication by inducing autophagy. In conclusion, our results suggest that the anti-PDCoV activity of CEP is not only relies on competing the virus binding with pAPN, but also affects the proliferation of the virus in vitro by downregulating the excessive immune response caused by the virus and inducing autophagy. CEP emerges as a promising candidate for potential anti-PDCoV therapeutic development.

P Glycoprotein Mediated Drug Interaction between Digoxin and Orange Juice- Exploratory Study by In-vitro Approach

P.gp is an efflux transporter that plays a vital role in drug transportation. Aim: This exploratory study is aimed to find the P-glycoprotein (P-gp) mediated drug interaction between digoxin and orange juice, conducted for a period of six months. The 3D structure of the human P-gp was predicted from the peptide by molecular threading using default settings. The structural analysis and verification server and the PDB were used to assess the stereo-chemical quality of these structures. The molecular structure of P-gp in a transport cycle has been investigated using a variety of methods. Molecular docking has been performed to predict the highest binding affinity to P gp with ligands of orange juice and ligands of ketoconazole. In 50% ethyl acetate extract of orange juice was prepared and a transcellular transport study was done using MDR1 transfectants and MRP2 transfectants LLC-PK1, LLC-GAS-COL150, LLC-pCI and LLC-MRP cells grown in M199 medium supplemented with 10% fetal calf serum at 37℃ in a humidified atmosphere of 5% CO2/95% air. The minimum inhibitory concentration is found to be 125 mcg/mL. Hence, the loading concentration along with orange juice is found to be 24.562 mcg/mL and the concentration less than that is found with poor sensitivity. Whereas the concentration above 125 mcg/mL loaded with orange juice is found to inhibit P-gp. The absorbance of orange juice and digoxin was measured at 470 nm. Since digoxin is not sensitive to orange juice beyond the concentration of 125 mcg/mL, it is considered as the MIC. Hence orange juice is a moderate p-gp Inhibitor and it escalates the serum digoxin toxicity. Orange juice is inhibiting the P-gp which increases the serum toxicity levels of digoxin.

Chlorogenic acid inhibits porcine deltacoronavirus release by targeting apoptosis

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae, genus Deltacoronavirus, can cause acute diarrhea in piglets, and also possesses cross-species transmission potential, leading to severe economic losses and threatening public health. However, no approved drug against PDCoV infection is available. Here, we investigated the antiviral effect of chlorogenic acid (CGA), the main active component of Lonicerae Japonicae Flos, against PDCoV infection. The results showed that CGA inhibited the replication of PDCoV significantly both in LLC-PK1 and ST cells, with a selectivity index greater than 80. CGA decreased the synthesis of PDCoV viral RNA and protein, and viral titers in a dose-dependent manner. The results of the time-of-addition assay indicated that CGA mainly affected the early stage of virus replication and viral release. Moreover, CGA significantly reduced apoptosis caused by PDCoV infection, and the application of apoptosis agonist and inhibitor revealed that apoptosis could promote progeny virus release. Further study demonstrated that CGA can inhibit virus release by directly targeting apoptosis caused by PDCoV infection. In conclusion, CGA is an effective agent against PDCoV, which provides a foundation for drug development for the treatment of PDCoV and other coronavirus infections.

HSP90AB1 is a host factor that promotes porcine deltacoronavirus replication

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus. It causes mortality in neonatal piglets and is of growing concern because of its broad host range, including humans. To date, the mechanism of PDCoV infection remains poorly understood. Here, based on a genome-wide CRISPR screen of PDCoV-infected cells, we found that HSP90AB1 (heat shock protein 90 alpha family class B1) promotes PDCoV infection. Knockdown or KO of HSP90AB1 in LLC-PK cells resulted in a significantly suppressed PDCoV infection. Infected cells treated with HSP90 inhibitors 17-AAG and VER-82576 also showed a significantly suppressed PDCoV infection, although KW-2478, which does not affect the ATPase activity of HSP90AB1, had no effect on PDCoV infection. We found that HSP90AB1 interacts with the N, NS7, and NSP10 proteins of PDCoV. We further evaluated the interaction between N and HSP90AB1 and found that the C-tail domain of the N protein is the HSP90AB1-interacting domain. Further studies showed that HSP90AB1 protects N protein from degradation via the proteasome pathway. In summary, our results reveal a key role for HSP90AB1 in the mechanism of PDCoV infection and contribute to provide new host targets for PDCoV antiviral research.

Evaluation of the immunoprotective effects of porcine deltacoronavirus subunit vaccines

Porcine deltacoronavirus (PDCoV), a new porcine enteric coronavirus, has seriously endangered the pig breeding industry and caused great economic losses. However, a PDCoV vaccine is not commercially available. Therefore, new and efficient PDCoV vaccines must be developed without delay. In this study, we used the ExpiCHO eukaryotic expression system to express and purify the following 3 structural proteins of PDCoV: S, N and M. Subsequently, the level of humoral and cellular immunity induced by the S protein (immunization with the S protein alone) and a protein mixture (immunization with a mixture of S, N and M proteins) were evaluated in mice and piglets, respectively, and the performances of the 2 immunizations in a challenge protection test were assessed in piglets. The results showed that both the S protein and the protein mixture induced the production of high levels of specific IgG antibodies and neutralizing antibodies and effectively neutralized PDCoV-infected LLC-PK cells in vitro. Furthermore, compared with the S protein, the N and M proteins in the protein mixture promoted the expression of CD8+ T cells and IFN-γ, induced a stronger cellular immune response, and effectively protected 4/5 of the piglets from PDCoV infection. In conclusion, the results of this study showed that the N and M proteins play important roles in inducing an immunoprotective response. Using N and M antigens as effective antigenic components in the development of PDCoV vaccines in the future will effectively increase the immune efficacy of the vaccines.