The nucleocapsid protein facilitates p53 ubiquitination-dependent proteasomal degradation via recruiting host ubiquitin ligase COP1 in PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV.

Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

Porcine epidemic diarrhea virus activates PERK-ROS axis to benefit its replication in Vero E6 cells.

Of the three branches of unfolded protein response (UPR) that were reportedly activated by porcine epidemic diarrhea virus (PEDV), PERK is recently shown to act as an upstream regulator of oxidative response of the cells. However, it remains unknown if and how PERK activation during PEDV infection would result in oxidative stress, and whether activation of PERK and its downstream molecules affect PEDV replication. Here, we demonstrate that infection with the PEDV strain YJH/2015 triggered UPR in Vero E6 cells by activating the PERK/eIF2α pathway and led to significant increase in the expression of proapoptotic protein C/EBP homologous protein (CHOP) and ER oxidoreductase 1 alpha (ERO1α). Inhibition of PERK by short hairpin RNA (shRNA) or GSK2606414 and knockdown of CHOP by small interfering RNA reduced expression of ERO1α and generation of ROS in PEDV-infected cells. Inhibition of ERO1α by shRNA or EN460 decreased PEDV-induced ROS generation. Genetic or pharmacological inhibition of each component of PERK, CHOP, ERO1α, and ROS led to significant suppression of PEDV replication. Collectively, our study provides the first evidence that PEDV manipulates endoplasmic reticulum to perturb its redox homeostasis via the PERK-CHOP-ERO1α-ROS axis in favor of its replication.

[Alkaloids from Macleaya cordata and their cytotoxicity assay].

OBJECTIVE: To study the alkaloids of Macleaya cordata and their anti-tumor activities. METHOD: Alcohol and liquid-liquid extraction were used methods were used to extract the alkaloids constituents, and silica gel, reverse-phase octadecylsilyl (ODS), sephadex LH-20 chromatographic methods and HPLC were applied to isolate and purify compounds. MS, NMR spectroscopic methods were used to determine their structures. Furthermore, the cytotoxicity of these chemical components for MCF-7 and SF-268 cell lines was measured by MTT method. RESULT: Twelve alkaloids were isolated from the fruits of M. cordata, and their structures were identified as: maclekarpine E (1), 6-acetonyldihyrochelerythrine (2), cavidilinine (3), 6-acetonyldihyrosanguinnarine (4), O-methylzanthoxyline (5), 6-methoxy-dihydrosanguinarine (6), spallidamine (7), 6-hydroxyldihydrochelerythrine (8), arnotianamida (9), dihydrosanguinarine (10), protopine (11), and cryptopine (12). CONCLUSION: Compounds 1, 3, 7-9 were isolated from M. cordata for the first time, and compound 5 is a new natural product. The results of cytotoxic assay indicated that compound 6 showed strong cytotoxicity against MCF-7 and SF-268 cell lines with IC50 values of 0.61 µmol · L(-1) and 0.54 µmol · L(-1), respectively.

[Chemical constituents of flavonoids and their glycosides in Melastoma dodecandrum].

The chemical constituents of 95% ethanol extract of Melastoma dodecandrum were isolated and purified by chromatography on silica gel, Sephadex LH-20, and HPLC, to obtain thirteen compounds eventually. On the basis of their physico-chemical properties and spectroscopic data, these compounds were identified as quercetin (1), quercetin-3-O-ß-D-glucopyranoside (2), quercetin-3-O-(6"-O-p-coumaroyl) -ß-D-glucopyranoside (3), kaempferol (4), kaempferol-3-O-ß-D-glucopyranoside (5), kaempferol-3-O- [2",6"-di-O-(E)-coumaroyl]-ß-D-glucopyra-noside (6), luteolin (7), luteolin-7-O-(6"-p-coumaroyl) -ß-D-glucopyranoside (8), apigenin (9), apigenin-7-(6"-acetyl-glucopyranoside) (10) , naringenin (11), isovitexin (12), and epicatechin-[8,7-e] -4ß-(4-hydroxyphenyl)-3,4-dyhydroxyl-2(3H)-pyranone (13). Eight compounds(3,5,6,8-11 and 13) were obtained from M. dodecandrum for the first time.

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

[Chemical constituents from Commelina communis].

To investigate the chemical constituents from Commelina communis, fifteen compounds were separated and purified by silica gel, Sephadex LH-20, and ODS column chromatography, and semi-preparative HPLC. By analyses of NMR and MS data as well as their physical and chemical properties, the structures of these compounds were identified as chrysoeriol-7-O-beta-D-glucoside( 1), methyl gallate(2), p-coumaric acid(3), protocatechuic acid(4), caffeic acid(5), p-hydroxybenzoic acid(6), 2-phenethyl-beta-D-gly-cosidase(7) , rhaponticin(8) , (7S, 8R) -dihydrodehydrodiconiferyl alcohol-9-O-beta-D-glucoside (9), isovitexin (10) , isofurcatain (11), isorhamnetin-3-O-beta-D-glucoside(12) , quercetin-3-O-alpha-L-rhamnoside (13) , isoquercitrin (14) , and 1, 2-dihydro-6, 8-dime-thoxy-7-1-(3, 5-dimethoxy-4-hydroxyphenyl) -N1, N2-bis-[2-( 4-hydroxyphenyl) ethyl] -2, 3-naphthalene dicarboxamide (15). Compounds 2, 5-9, 11, 13 were obtained from the genus Commelina for the first time.

Explanation and verification of the rules of attack in table tennis tactics.

BACKGROUND: The rules of attack in table tennis tactics have been discovered by the coaches and researchers of the Chinese table tennis team (CTTT) through long-term practice. However, they are only empirical judgements and have not been objectively verified. METHODS: The software "Table Tennis Master" has been used to analyse 200 matches of top players of CTTT against various opponents in recent years to obtain detailed statistics by analysing the effect of attack in the end line (AIEL) and attack out of the end line (AOEL). RESULTS: (1) The scoring rate of the players was high after AIEL but very low after AOEL (p < 0.05); (2) the round of service (serve/receive) and level of skills had little influence on the effect of AIEL and AOEL; and (3) the timing of attack had a great influence on the effect of AIEL and AOEL (r > 0.9). CONCLUSIONS: In the high-level table tennis match, the rules of AIEL and AOEL are scientific. In accordance with the rules, the complex tactics can be simplified to the two concepts, AIEL and AOEL.

Machine learning based personalized promotion strategy of piglets weaned per sow per year in large-scale pig farms.

BACKGROUND: The purpose of this study was to analyze the relationship between different productive factors and piglets weaned per sow per year (PSY) in 291 large-scale pig farms and analyze the impact of the changes in different factors on PSY. We chose nine different algorithm models based on machine learning to calculate the influence of each variable on every farm according to its current situation, leading to personalize the improvement of the impact in the specific circumstances of each farm, proposing a production guidance plan of PSY improvement for every farm. According to the comparison of mean absolute error (MAE), 95% confidence interval (CI) and R2, the optimal solution was conducted to calculate the influence of 17 production factors of each pig farm on PSY improvement, finding out the bottleneck corresponding to each pig farm. The level of PSY was further analyzed when the bottleneck factor of each pig farm changed by 0.5 standard deviation (SD). RESULTS: 17 production factors were non-linearly related to PSY. The top five production factors with the highest correlation with PSY were the number of weaned piglets per litter (WPL) (0.6694), mating rate within 7 days after weaning (MR7DW) (0.6606), number of piglets born alive per litter (PBAL) (0.6517), the total number of piglets per litter (TPL) (0.5706) and non-productive days (NPD) (- 0.5308). Among nine algorithm models, the gradient boosting regressor model had the highest R2, smallest MAE and 95% CI, applied for personalized analysis. When one of 17 production factors of 291 large-scale pig farms changed by 0.5 SD, 101 pig farms (34.7%) can increase 1.41 PSY (compared to its original value) on average by adding the production days, and 60 pig farms (20.6%) can increase 1.14 PSY on average by improving WPL, 45 pig farms (15.5%) can increase 1.63 PSY by lifting MR7DW. CONCLUSIONS: The main productive factors related to PSY included WPL, MR7DW, PBAL, TPL and NPD. The gradient boosting regressor model was the optimal method to individually analyze productive factors that are non-linearly related to PSY.

Study on the influence of different production factors on PSY and its correlation.

BACKGROUND: Finding out the key reproductive performance factors, affecting piglets weaned per sow per year (PSY) can improve the production efficiency and profitability of pig farms. The objective was to understand the actual distribution of different production factors and PSY of breeding pig farms, analyze the correlation to find the main production factors affecting PSY, and formulating a Production Efficiency Improvement Plan in practice. Data included 603 breeding pig farms from September 28, 2020 to September 26, 2021. Regression analysis was used to evaluate the relationship between PSY and key production factors, and the characteristics of total pig farms versus high performance (HP) pig farms (the production performance was in the top 10%) or top 5% pig farms were compared. Spearman's rank correlation coefficient was used to analyze the correlation between production factors and find the factors related to PSY. Non-linear support vector regression (NL-SVR) was used to analyze the personalized PSY improvement through a various change of the four key factors. RESULTS: The median distribution of 15 production factors and PSY in total pig farms were different from those of HP farms. All of data were distributed nonlinearly. Mating rate within 7 days after weaning (MR7DW), farrowing rate (FR), number of piglets born alive per litter (PBAL) and number of weaned piglets per litter (WPL) were moderately correlated with PSY, and the correlation coefficients were 0.5058, 0.4427, 0.3929 and 0.3839, respectively. When the four factors in NL-SVR changed in medium (0.5 piglet or 5%) or high level (1.0 piglet or 10%), PSY can be increased by more than 0.5. CONCLUSION: NL-SVR model can be used to analyze the impact of changes in key production factors on PSY. By taking measures to improve MR7DW, FR, PBAL and WPL, it may effectively improve the current PSY and fully develop the reproductive potential of sows.

Porcine circovirus type 2 induces CHOP-ERO1α-ROS-mediated apoptosis in PK-15 cells.

Porcine circovirus type 2 (PCV2) infection induces endoplasmic reticulum (ER) stress and oxidative stress. These cellular responses could be connected with apoptosis. However, the mechanisms that link ER stress and oxidative stress in PCV2-induced apoptosis are poorly characterized. Here, we demonstrate that PCV2 infection increased expression of proapoptotic protein C/EBP homologous protein (CHOP) and ER oxidoreductase 1 alpha (ERO1α). Inhibition of CHOP by RNA silencing or inhibition of ERO1α by short hairpin RNA or EN460 repressed PCV2-induced reactive oxygen species (ROS) generation, cytosolic calcium level, and apoptotic rate in PK-15 cells. Overexpression of ERO1α enhanced PCV2-induced oxidative stress, caspase-3 cleavage, and apoptosis rate. Treatment of PCV2-infected cells with ROS scavenger N-acetyl-L-cysteine downregulated PCV2-induced ROS production, cytosolic calcium level, and apoptosis rate, but intriguingly decreased expression of CHOP and ERO1α. Thus, we propose that PCV2 induces apoptosis through ER Stress via CHOP-ERO1α-ROS signaling in host cells.

Chitosan-Coated Selenium Nanoparticles Attenuate PRRSV Replication and ROS/JNK-Mediated Apoptosis in vitro.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly prevalent and endemic swine pathogen that causes significant economic losses to the global swine industry. Selenium nanoparticles (SeNPs) have attracted increasing attention in the biomedical field, given their antiviral effects. This study aimed to investigate the inhibitory effect of chitosan-coated SeNPs (CS-SeNPs) on PRRSV replication. Methods: In this study, CS-SeNPs were synthesized by chemical reduction and characterized by assessing the morphology, size distribution, zeta potential, and element composition. Marc-145 cells were infected with r-PRRSV-EGFP (0.1 MOI) and inoculated with CS-SeNPs (10 µM). Subsequently, the concentrations of hydrogen peroxide (H2O2) and glutathione (GSH), and glutathione peroxidase (GSH-Px) activity were measured using specific commercial assay kits. ORF5 RNA expression, viral titer, and nucleocapsid (N) protein expression were assessed using qRT-PCR, TCID50, and Western blot. ROS generation, apoptosis rates, and JNK /caspase-3/PARP protein expression were evaluated using dihydroethidium staining, flow cytometry, and Western blot. Results: The results showed that CS-SeNPs treatment significantly suppressed oxidative stress induced by r-PRRSV-EGFP infection by increasing GSH-Px activity, promoting GSH production, and inhibiting H2O2 synthesis. CS-SeNPs treatment significantly inhibited ORF5 gene expression, viral titers, and N protein of r-PRRSV-EGFP at 24 and 48 hours post-infection (hpi) in Marc-145 cells. The increase in apoptosis rates induced by r-PRRSV-EGFP infection was significantly decreased by CS-SeNPs inoculation through inhibiting ROS generation, JNK phosphorylation levels, and cleavage of caspase-3 and PARP mainly at 48 hpi. Conclusion: These results demonstrated that CS-SeNPs suppress PRRSV-induced apoptosis in Marc-145 cells via the ROS/JNK signaling pathway, thereby inhibiting PRRSV replication, which suggested the potential antiviral activity of CS-SeNPs that deserves further investigation for clinical applications.

A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes.

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.

High-throughput reformatting of phage-displayed antibody fragments to IgGs by one-step emulsion PCR.

Single-chain variable fragment (scFv) is the most common format for phage display antibody library. The isolated scFvs need to be reformatted to full-length IgGs for further characterization. High throughput reformatting of scFv to IgG without disrupting VH-VL pairing is of great demanding for exhaustive screening of all antibodies in IgG format. Herein, we developed a strategy based on the overlap extension PCR in emulsion to reformat scFv to IgG while maintain the accuracy and complexity of variable region pairing. Using CD40 as an example target, we reformatted phage display derived CD40 binding scFv library to IgG mammalian display library and isolated high affinity CD40 binding IgGs. This robust and reliable antibody reformatting approach could be integrated into any phage display based antibody drug discovery.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion.

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.

Ultrastructural Characterization of Membrane Rearrangements Induced by Porcine Epidemic Diarrhea Virus Infection.

The porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) belonging to the α-CoV genus and it causes high mortality in infected sucking piglets, resulting in substantial losses in the farming industry. CoV trigger a drastic reorganization of host cell membranes to promote their replication and egression, but a detailed description of the intracellular remodeling induced by PEDV is still missing. In this study, we examined qualitatively and quantitatively, using electron microscopy, the intracellular membrane reorganization induced by PEDV over the course of an infection. With our ultrastructural approach, we reveal that, as most of CoV, PEDV initially forms double-membrane vesicles (DMVs) and convoluted membranes (CMs), which probably serve as replication/transcription platforms. Interestingly, we also found that viral particles start to form almost simultaneously in both the endoplasmic reticulum and the large virion-containing vacuoles (LVCVs), which are compartments originating from the Golgi, confirming that α-CoV assemble indistinguishably in two different organelles of the secretory pathway. Moreover, PEDV virons appear to have an immature and a mature form, similar to another α-CoV the transmissible gastroenteritis coronavirus (TGEV). Altogether, our study underlies the similarities and differences between the lifecycle of α-CoV and that of viruses belonging to other CoV subfamilies.

Progressive Motor Deficit is Mediated by the Denervation of Neuromuscular Junctions and Axonal Degeneration in Transgenic Mice Expressing Mutant (P301S) Tau Protein.

Tauopathies include a variety of neurodegenerative diseases associated with the pathological aggregation of hyperphosphorylated tau, resulting in progressive cognitive decline and motor impairment. The underlying mechanism for motor deficits related to tauopathy is not yet fully understood. Here, we use a novel transgenic tau mouse line, Tau 58/4, with enhanced neuron-specific expression of P301S mutant tau to investigate the motor abnormalities in association with the peripheral nervous system. Using stationary beam, gait, and rotarod tests, motor deficits were found in Tau 58/4 mice already 3 months after birth, which deteriorated during aging. Hyperphosphorylated tau was detected in the cell bodies and axons of motor neurons. At the age of 9 and 12 months, significant denervation of the neuromuscular junction in the extensor digitorum longus muscle was observed in Tau 58/4 mice, compared to wild-type mice. Muscle hypotrophy was observed in Tau 58/4 mice at 9 and 12 months. Using electron microscopy, we observed ultrastructural changes in the sciatic nerve of 12-month-old Tau 58/4 mice indicative of the loss of large axonal fibers and hypomyelination (assessed by g-ratio). We conclude that the accumulated hyperphosphorylated tau in the axon terminals may induce dying-back axonal degeneration, myelin abnormalities, neuromuscular junction denervation, and muscular atrophy, which may be the mechanisms responsible for the deterioration of the motor function in Tau 58/4 mice. Tau 58/4 mice represent an interesting neuromuscular degeneration model, and the pathological mechanisms might be responsible for motor signs observed in some human tauopathies.

An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication.

Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions of ATG proteins. We determined the effects of the depletion of each ATG proteome component on the replication of six different viruses. Our screen reveals that up to 36% of the ATG proteins significantly alter the replication of at least one virus in an unconventional fashion. Detailed analysis of two candidates revealed an undocumented role for ATG13 and FIP200 in picornavirus replication that is independent of their function in autophagy as part of the ULK complex. The high numbers of unveiled ATG gene-specific and pathogen-specific functions of the ATG proteins calls for caution in the interpretation of data, which rely solely on the depletion of a single ATG protein to specifically ablate autophagy.

Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells.

Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells.