Porcine Cell Lines Research Articles

A novel method for isolation and culture of primary swine gastric epithelial cells

BackgroundCulturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.ResultsMechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.ConclusionsA new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

Beneficial Effects of Rosmarinic Acid on IPEC-J2 Cells Exposed to the Combination of Deoxynivalenol and T-2 Toxin.

Mycotoxin contamination in feedstuffs is a worldwide problem that causes serious health issues both in humans and animals, and it contributes to serious economic losses. Deoxynivalenol (DON) and T-2 toxin (T-2) are major trichothecene mycotoxins and are known to challenge mainly intestinal barrier functions. Polyphenolic rosmarinic acid (RA) appeared to have antioxidant and anti-inflammatory properties in vitro. The aim of this study was to investigate protective effects of RA against DON and T-2 or combined mycotoxin-induced intestinal damage in nontumorigenic porcine cell line, IPEC-J2. It was ascertained that simultaneous treatment of DON and T-2 (DT2: 1 μmol/L DON + 5 nmol/L T − 2) for 48 h and 72 h reduced transepithelial electrical resistance of cell monolayer, which was restored by 50 μmol/L RA application. It was also found that DT2 for 48 h and 72 h could induce oxidative stress and elevate interleukin-6 (IL-6) and interleukin-8 (IL-8) levels significantly, which were alleviated by the administration of RA. DT2 administration contributed to the redistribution of claudin-1; however, occludin membranous localization was not altered by combined mycotoxin treatment. In conclusion, beneficial effect of RA was exerted on DT2-deteriorated cell monolayer integrity and on the perturbated redox status of IPEC-J2 cells.

Pyridaben leads to inhibition of cell growth and induction of cell death through intracellular mechanisms in early pregnancy

Recently, infertility has become a major global issue. It is crucial to identify environmental factors that lead to infertility. The prevalent use of pesticides in agriculture results in the exposure of livestock and humans to these pesticides. Studies have reported the harmful effects of pesticides on pregnancy. Pyridaben, a pesticide that inhibits mitochondrial complex 1, has been reported to have detrimental effects on neurons, spermatogenesis, hormonal balance, and embryonic development. However, the effect of pyridaben on the female reproductive system has not yet been studied. Therefore, in this study, we evaluated the effects of pyridaben on early pregnancy in porcine reproductive cell lines, which are known to mimic the female reproductive system. Results demonstrated that pyridaben decreased cell growth in porcine endometrial luminal epithelial and porcine trophectoderm cell lines through inhibition of cell signal transduction. Further, pyridaben increased subG1 phase and late apoptosis through the induction of reactive oxygen species production, mitochondrial dysfunction, calcium unbalances, pro-apoptotic signals, and endoplasmic reticulum (ER) stress. Moreover, we found that pyridaben induced autophagy and inhibition of placentation through the regulation of ER-mitochondria axis proteins. Overall, pyridaben was found to be harmful in early pregnancy in pigs and may have similar effects in human pregnancy.

Farm Animal-derived Models of the Intestinal Epithelium: Recent Advances and Future Applications of Intestinal Organoids

Farm animals play an important role in translational research as large animal models of the gastrointestinal (GI) tract. The mechanistic investigation of zoonotic diseases of the GI tract, in which animals can act as asymptomatic carriers, could provide important information for therapeutic approaches. In veterinary medicine, farm animals are no less relevant, as they can serve as models for the development of diagnostic and therapeutic approaches of GI diseases in the target species. However, farm animal-derived cell lines of the intestinal epithelium are rarely available from standardised cell banks and, in addition, are not usually specific for certain sections of the intestine. Immortalised porcine or bovine enterocytic cell lines are more widely available, compared to goat or sheep-derived cell lines; no continuous cell lines are available from the chicken. Other epithelial cell types with intestinal section-specific distribution and function, such as goblet cells, enteroendocrine cells, Paneth cells and intestinal stem cells, are not represented in those cell line-based models. Therefore, intestinal organoid models of farm animal species, which are already widely used for mice and humans, are gaining importance. Crypt-derived or pluripotent stem cell-derived intestinal organoid models offer the possibility to investigate the mechanisms of inter-cell or host-pathogen interactions and to answer species-specific questions. This review is intended to give an overview of cell culture models of the intestinal epithelium of farm animals, discussing species-specific differences, culture techniques and some possible applications for intestinal organoid models. It also highlights the need for species-specific pluripotent stem cell-derived or crypt-derived intestinal organoid models for promotion of the Three Rs principles (replacement, reduction and refinement).

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

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

Effects of gE/gI deletions on the miRNA expression of PRV-infected PK-15 cells

Pseudorabies virus (PRV) belongs to the Alphaherpesvirinae subfamily of Herpesviridae. PRV-induced pseudorabies is a highly contagious disease that has caused huge economic losses to the global swine industry. The PRV gE/gI gene deletion vaccine strain (Fa ΔgE/gI strain) constructed from the PRV Fa wild-type strain was shown to have a protective effect against infection. However, the interaction between PRV gE/gI genes and host miRNA needs further exploration, and little is known about the regulatory mechanisms of non-coding RNAs during PRV infection. miRNAs play a key regulatory role in viral infection and immune responses, so we analyzed the differential expression of miRNAs induced by the PRV Fa ΔgE/gI strain and Fa wild-type strain in the PK15 cell line. High-throughput sequencing reads were aligned to known Sus scrofa pre-miRNAs in the miRBase database. Target genes of differentially expressed miRNAs were predicted using the miRGen 3.0 database, then filtered miRNA target genes were subjected to Gene Ontology (GO) analysis and Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis. Stem-loop quantitative real-time PCR was performed to confirm the accuracy of high-throughput sequencing data. In total, 387, 472, and 490 annotated and novel mature miRNAs were identified from PRV Fa ΔgE/gI strain-infected, Fa wild-type strain-infected, and non-infected PK-15 cells, respectively. Five PRV-encoded miRNAs were also identified. GO analysis showed that target genes of differentially expressed miRNAs in PRV Fa ΔgE/gI strain-infected and Fa wild-type strain-infected PK-15 cells were mainly involved in biological regulation and metabolic processes. STRING analysis showed that immune-related target genes of differentially expressed miRNAs in the Toll-like receptor signaling pathway, B cell receptor signaling pathway, T cell receptor signaling pathway, nuclear factor-κB signaling pathway, and transforming growth factor-β signaling pathway were interrelated. This is the first report of the small RNA transcriptome in PRV mutant wild-type strain-infected and Fa ΔgE/gI strain-infected porcine cell lines. Our findings will contribute to the prevention and treatment of PRV mutant strains.

Establishment and characterization of a porcine B cell lymphoma cell line

The lack of available, well characterized, established, domestic porcine cell lines hinders the advancement of porcine cellular immunology. A case of multicentric lymphoma was diagnosed in a market weight pig at the time of slaughter. Affected lymph nodes and spleen were collected and used for single cell isolation and analysis. Cell lines were established by 3 rounds of limiting dilution from splenic and subiliac lymph node lymphomas. Surface marker staining identified the cells as CD21+, CD79a+, CD20+, PAX5+, and CD3− and cells were grown and easily passaged in cell culture. Transcriptome analysis was carried out to further characterize these rapidly proliferating cells validating the initial cytometric findings, confirming their identity as B cell lymphomas, and suggesting that they arose from germinal center centroblasts with aberrant control of BCL6 expression. Functional analysis identified the cells as being involved in cancer, cell movement, cell survival, and apoptosis. These new porcine B cell lymphoma cell lines will be a valuable resource for more in-depth cellular investigations into the porcine immune system and cancer, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.

Susceptibility of swine cells and domestic pigs to SARS-CoV-2

ABSTRACT The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naïve sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.

Genomic characteristics of classical swine fever virus strains of bovine origin according to primary and secondary sequence-structure analysis.

Background:Classical swine fever virus (CSFV), species member of the family Flaviviridae, is generally considered restricted to domestic and wild suids. The circulation of CFSV has been detected in cattle herds in China and India. Natural infection appeared associated with clinical signs in some cases.Aim:The secondary structures of the internal ribosome entry site in the 5′ untranslated region (UTR) were used for the genomic characterization of bovine strains.Methods:Sequences have been compared to the representative CSFV strains isolated from pigs, vaccines, and contaminants from porcine cell lines and an ovine strain isolated in Spain.Results:The observed sequences from cattle showed a genetic relatedness with live-attenuated vaccine strains used in pigs. Sequence characteristics of the Chinese strain S171 are genetically distant from the previously reported CSFV genotypes, suggesting a new outgroup in the species, described for the first time, and named CSFV-d. Other Chinese strains were genetically closely related to CSFV genotype a2 (Alfort type) pig strains. Indian strains, reported from the states of Tamil Nadu and Meghalaya, were genetically closely related to CSFV genotype a1 (Brescia type) and a5 pig strains, respectively.Conclusion:These preliminary observations are new and relevant in countries, where CSFV control and eradication strategies are applied.

A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain of African swine fever virus

ABSTRACT The main target cells for African swine fever virus (ASFV) replication in pigs are of monocyte macrophage lineage and express markers typical of the intermediate to late stages of differentiation. The lack of a porcine cell line, which accurately represents these target cells, limits research on virus host interactions and the development of live-attenuated vaccine strains. We show here that the continuously growing, growth factor dependent ZMAC-4 porcine macrophage cell line is susceptible to infection with eight different field isolates of ASFV. Replication in ZMAC-4 cells occurred with similar kinetics and to similar high titres as in primary porcine bone marrow cells. In addition we showed that twelve passages of an attenuated strain of ASFV, OURT88/3, in ZMAC-4 cells did not reduce the ability of this virus to induce protection against challenge with virulent virus. Thus, the ZMAC-4 cells provide an alternative to primary cells for ASFV replication.

MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages

MYH9 has been identified as an indispensable cellular protein for porcine reproductive and respiratory syndrome virus (PRRSV) entry into permissive cells using the monoclonal anti-idiotypic antibody (Mab2-5G2) recognizing an antibody that specifically interacts with PRRSV glycoprotein 5 (GP5). More recently, we found that Mab2-5G2 interacted with the MYH9 C-terminal domain, designated PRA, which is required for PRRSV internalization. In this study, we demonstrate that blocking of MYH9 with Mab2-5G2 significantly diminished PRRSV internalization by porcine alveolar macrophage (PAM) via interruption of direct interaction between GP5 and MYH9, and thus remarkably inhibited subsequent infection of PAMs by PRRSV-2 isolates. Moreover, the three-dimensional structure of the Mab2-5G2 Fab-PRA complex determined via homology modeling predicted potential docking sites required for PRRSV internalization. Further analysis of Mab2-5G2-binding sites within PRA highlighted that the amino acids E1670, K1673, E1679, and I1683 in PRA are the key Mab2-5G2-binding residues. Notably, recombinant PRA protein blocked the interaction between PRRSV GP5 and cellular MYH9 by preventing translocation of MYH9 from the cytoplasm to the cell membrane, an essential step for PRRSV virion internalization. Meanwhile, porcine cell line permissive for PRRSV bearing point mutation of E1670A in MYH9 demonstrated reduced susceptibility for PRRSV infection. In conclusion, this work increases understanding of both PRRSV pathogenesis and the mechanistic role played by MYH9 in PRRSV infection.

Functional Characterization and Proteomic Analysis of Porcine Deltacoronavirus Accessory Protein NS7.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in neonatal piglets. Like other coronaviruses, PDCoV encodes at least three accessory or species-specific proteins; however, the biological roles of these proteins in PDCoV replication remain undetermined. As a first step toward understanding the biology of the PDCoV accessory proteins, we established a stable porcine cell line constitutively expressing the PDCoV NS7 protein in order to investigate the functional characteristics of NS7 for viral replication. Confocal microscopy and subcellular fractionation revealed that the NS7 protein was extensively distributed in the mitochondria. Proteomic analysis was then conducted to assess the expression dynamics of the host proteins in the PDCoV NS7-expressing cells. Highresolution two-dimensional gel electrophoresis initially identified 48 protein spots which were differentially expressed in the presence of NS7. Seven of these spots, including two upregulated and five down-regulated protein spots, showed statistically significant alterations, and were selected for subsequent protein identification. The affected cellular proteins identified in this study were classified into functional groups involved in various cellular processes such as cytoskeleton networks and cell communication, metabolism, and protein biosynthesis. A substantial down-regulation of α-actinin-4 was confirmed in NS7-expressing and PDCoV-infected cells. These proteomic data will provide insights into the understanding of specific cellular responses to the accessory protein during PDCoV infection.

03:27 PM Abstract No. 221 Porcine and human hepatocellular carcinoma cell lines present similar drug-metabolizing enzyme expression levels and comparable sorafenib and doxorubicin cytotoxic responses

03:27 PM Abstract No. 221 Porcine and human hepatocellular carcinoma cell lines present similar drug-metabolizing enzyme expression levels and comparable sorafenib and doxorubicin cytotoxic responses

Adhesion of Salmonella to Pancreatic Secretory Granule Membrane Major Glycoprotein GP2 of Human and Porcine Origin Depends on FimH Sequence Variation.

Bacterial host tropism is a primary determinant of the range of host organisms they can infect. Salmonella serotypes are differentiated into host-restricted and host-adapted specialists, and host-unrestricted generalists. In order to elucidate the underlying molecular mechanisms of host specificity in Salmonella infection, we investigated the role of the intestinal host cell receptor zymogen granule membrane glycoprotein 2 (GP2), which is recognized by FimH adhesin of type 1 fimbriae found in Enterobacteriaceae. We compared four human and two porcine GP2 isoforms. Isoforms were expressed in Sf9 cells as well as in one human (HEp-2) and one porcine (IPEC-J2) cell line. FimH genes of 128 Salmonella isolates were sequenced and the 10 identified FimH variants were compared regarding adhesion (static adhesion assay) and infection (cell line assay) using an isogenic model. We expressed and characterized two functional porcine GP2 isoforms differing in their amino acid sequence to human isoforms by approximately 25%. By comparing all isoforms in the static adhesion assay, FimH variants were assigned to high, low or no-binding phenotypes. This FimH variant-dependent binding was neither specific for one GP2 isoform nor for GP2 in general. However, cell line infection assays revealed fundamental differences: using HEp-2 cells, infection was also FimH variant-specific but mainly independent of human GP2. In contrast, this FimH variant dependency was not obvious using IPEC-J2 cells. Here, we propose an alternative GP2 adhesion/infection mechanism whereby porcine GP2 is not a receptor that determined host-specificity of Salmonella. Salmonella specialists as well as generalists demonstrated similar binding to GP2. Future studies should focus on spatial distribution of GP2 isoforms in the human and porcine intestine, especially comparing health and disease.

Assessing abundance and specificity of different types of sgRNA targeting miRNA precursors

CRISPR/Cas technology enables efficient and specific editing the genome. Since different bacterial sources or artificially modified Cas9, as well as Cpf1 and other nucleases, recognize different PAMs (protospacer adjacent motifs), different gene editing nucleases may use different types of sgRNAs (small guide RNA). MicroRNAs (miRNAs) are a class of regulatory small non-coding RNAs. To determine whether specific targets for sgRNAs in miRNA precursor exit, the abundance and specificity of 11 different types of sgRNA targeting 28 645 miRNA precursors were analyzed in the present study using the CRISPR-offinder, a bioinformatics software developed in our own laboratory. The CRISPR/Cas9 lentivirus technology was used to target the miR-302/367 cluster in a porcine cell line, and its knockout efficiency for the miRNA target was evaluated. The results show that there are about 8 different types of sgRNAs that can target individual miRNA precursors. By assessing the off-target effect, only 18.2% of the sgRNAs showed high specificity for targeting the porcine miRNA precursors. Lastly, using the miR-302/367 cluster target as an example, we showed that the CRISPR/Cas9 lentivirus technology was 40% efficient in successfully establishing correct knockout of the target miRNA in the porcine cell line. This present study provides an important resource for the use of CRISPR/Cas technology to target miRNAs for knockout studies.

Iberian pig mesenchymal stem/stromal cells from dermal skin, abdominal and subcutaneous adipose tissues, and peripheral blood: in vitro characterization and migratory properties in inflammation

BackgroundRecently, the capacity of mesenchymal stem/stromal cells (MSCs) to migrate into damaged tissues has been reported. For MSCs to be a promising tool for tissue engineering and cell and gene therapy, it is essential to know their migration ability according to their tissue of origin. However, little is known about the molecular mechanisms regulating porcine MSC chemotaxis. The aim of this study was to examine the migratory properties in an inflammatory environment of porcine MSC lines from different tissue origins: subcutaneous adipose tissue (SCA-MSCs), abdominal adipose tissue (AA-MSCs), dermal skin tissue (DS-MSCs) and peripheral blood (PB-MSCs).MethodsSCA-MSCs, AA-MSCs, DS-MSCs and PB-MSCs were isolated and analyzed in terms of morphological features, alkaline phosphatase activity, expression of cell surface and intracellular markers of pluripotency, proliferation, in vitro chondrogenic, osteogenic and adipogenic differentiation capacities, as well as their ability to migrate in response to inflammatory cytokines.ResultsSCA-MSCs, AA-MSCs, DS-MSCs and PB-MSCs were isolated and showed plastic adhesion with a fibroblast-like morphology. All MSC lines were positive for CD44, CD105, CD90 and vimentin, characteristic markers of MSCs. The cytokeratin marker was also detected in DS-MSCs. No expression of MHCII or CD34 was detected in any of the four types of MSC. In terms of pluripotency features, all MSC lines expressed POU5F1 and showed alkaline phosphatase activity. SCA-MSCs had a higher growth rate compared to the rest of the cell lines, while the AA-MSC cell line had a longer population doubling time. All MSC lines cultured under adipogenic, chondrogenic and osteogenic conditions showed differentiation capacity to the previously mentioned mesodermal lineages. All MSC lines showed migration ability in an agarose drop assay. DS-MSCs migrated greater distances than the rest of the cell lines both in nonstimulated conditions and in the presence of the inflammatory cytokines TNF-α and IL-1β. SCA-MSCs and DS-MSCs increased their migration capacity in the presence of IL-1β as compared to PBS control.ConclusionsThis study describes the isolation and characterization of porcine cell lines from different tissue origin, with clear MSC properties. We show for the first time a comparative study of the migration capacity induced by inflammatory mediators of porcine MSCs of different tissue origin.

Abstract 5914: The porcine model for onocolytic adenovirus-based therapy

Abstract Oncolytic adenoviruses (Ad) are promising tools in the development of cancer therapeutics. A majority of Ad-based therapies utilize serotypes of species C, with Adenovirus type 5 (Ad5) being the most commonly employed. Previously, clinical trials have demonstrated the low efficiency of Ad5 vectors, mainly due to absence of the Ad5 primary receptor (Coxsackie Adenovirus Receptor, CAR) in cancer cells. Engineering Ad vectors utilizing the species B (Ad3, Ad35, Ad11) receptors have greatly improved the oncolytic potential of Ad-based therapies. However, the lack of a viable animal model has impeded clinical translation of these tropism-modified vectors. Mouse models are insufficient because Ad does not replicate in murine tissue. Non-human apes are not feasible due to availability and cost. Cotton rats and Syrian hamsters, although permissive of Ad5 replication, are not suitable for Ad3-, Ad35-, and Ad11-retargeted vectors due to the lack of species B primary receptors (CD46 and desmoglein 2) in rodent systems. In this study, we explored pigs as a model to study performance of the group B oncolytic adenoviruses by employing the fiber-modified Ad5/Ad3 chimeric vector. As a control, the Ad5 fiber-unmodified virus was used. First, we demonstrated the ability of swine cell lines to support replication of both Ad5 and Ad5/Ad3. Second, we analyzed binding, gene transfer, cytolytic, and replication ability of Ad5 and Ad5/Ad3 in various non-human cell lines (swine, hamster, murine, rat, bovine, canine). Our data confirmed that while hamster cell lines (HP1 and HapT1) were able to support binding and replication of Ad5-based vectors, they failed to support that with the Ad3-retargeted vectors. Additionally, we showed that among all tested cell lines, only porcine cells (PK15 and PTK75) were supportive of both binding and replication of the Ad3-retargeted virus. Of note, Ad5/Ad3 outperformed Ad5 in its cytolytic effect in porcine cell lines. These in vitro results prompted evaluation of the vectors in vivo. Immunocompetent Yorkshire pigs were systemically injected with a single dose of Ad5- and Ad5/3- expressing luciferase (Luc) from the Ad E3 region. Quantitative PCR analyses of the primary organs collected 7 days post-infection revealed Ad5 and Ad5/3 viral DNA in the lungs and spleen. Replication-dependent Luc expression was also observed in these tissue samples suggesting active viral replication. The quantity of viral DNA in other tissue such as the kidneys, liver, and pancreas was negligible. The results of these in vitro and in vivo studies indicate that pigs are a promising model to assess unmodified and tropism-modified adenoviral vectors. Citation Format: Malavika Chandrashekar, Lisa Koodie, Michele Dunning, George Ruth, Richard Bianco, Masato Yamamoto, Julia Davydova. The porcine model for onocolytic adenovirus-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5914.

Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry.

Identification of cellular receptors used by coronavirus (CoV) entry into the host cells is critical to an understanding of pathogenesis and to development of intervention strategies. The fourth CoV genus, Deltacoronavirus, evolutionarily related to the Gammacoronavirus, has just been defined recently. In the current study, we demonstrate that porcine aminopeptidase N (pAPN) acts as a cross-genus CoV functional receptor for both enteropathogenic porcine deltacoronovirus (PDCoV) and alphacoronovirus (AlphaCoV) (transmissible gastroenteritis virus [TGEV]) based upon three lines of evidence. First, the soluble S1 protein of PDCoV bound to the surface of target porcine cell lines known to express pAPN as efficiently as TGEV-S1, which could be blocked by soluble pAPN pretreatment. Second, both PDCoV-S1 and TGEV-S1 physically recognized and interacted with pAPN by coimmunoprecipitation in pAPN cDNA-transfected cells and by dot blot hybridization assay. Finally, exogenous expression of pAPN in refractory cells conferred susceptibility to PDCoV-S1 binding and to PDCoV entry and productive infection. PDCoV-S1 appeared to have a lower pAPN-binding affinity and likely consequent lower infection efficiency in pAPN-expressing refractory cells than TGEV-S1, suggesting that there may be differences between these two viruses in the virus-binding regions in pAPN. This study paves the way for dissecting the molecular mechanisms of PDCoV-host interactions and pathogenesis as well as facilitates future vaccine development and intervention strategies against PDCoV infection.IMPORTANCE The emergence of new human and animal coronaviruses is believed to have occurred through interspecies transmission that is mainly mediated by a species-specific receptor of the host. Among the four genera of the Coronavirinae, a couple of functional receptors for the representative members in the genera Alphacoronavirus and Betacoronavirus have been identified, whereas receptors for Gammacoronavirus and Deltacoronavirus, which are believed to originate from birds, are still unknown. Porcine coronaviruses, including the newly discovered porcine deltacoronavirus (PDCoV) associated with diarrhea in newborn piglets, have posed a serious threat to the pork industry in Asia and North America. Here, we report that PDCoV employs the alphacoronavirus TGEV functional receptor porcine aminopeptidase N (pAPN) for cellular entry, demonstrating the usage of pAPN as a cross-genus CoV functional receptor. The identification of the PDCoV receptor provides another example of the expanded host range of CoV and paves the way for further investigation of PDCoV-host interaction and pathogenesis.

Crosstalk Between Nuclear Glucose-Regulated Protein 78 and Tumor Protein 53 Contributes to the Lipopolysaccharide Aggravated Apoptosis of Endoplasmic Reticulum Stress-Responsive Porcine Intestinal Epithelial Cells

Background/Aims: Lipopolysaccharides (LPSs) act as virulence factors that trigger intestinal inflammation and thereby compromise the production of pigs worldwide. Intestinal diseases and dysfunction have been attributed to endoplasmic reticulum stress (ERS) and the subsequent apoptosis of intestinal epithelial cells. Therefore It is important to explore whether LPSs aggravate ERS-mediated apoptosis of intestinal epithelial cells. Methods: ERS and inflammation models were established in porcine cell line J2 (IPEC-J2) and the cells were treated with tunicamycin or LPS at specific times. The expression of marker proteins was determined by western blot and immunofluorescence. Possible crosstalk between proteins was analyzed by co-immunoprecipitation. Small interfering RNA transfection was employed to verify the mechanisms. Results: We found that Escherichia coli-derived LPS aggravated ERS and ERS-mediated apoptosis in ERS-responsive IPEC-J2 cells. The crosstalk between nuclear glucose-regulated protein 78 (GRP78) and tumor protein 53 (p53) was verified to trigger this LPS-aggravated apoptosis of ERS-responsive intestinal cells. Conclusion: This novel finding implies that intestinal malfunctions might solely originate from the effects of Gram-negative bacteria on ERS-responsive intestinal cells. The regulation of ERS signaling (especially the crosstalk between nuclear GRP78 and p53) in ERS-responsive/rapidly growing intestines may help intestinal cells survive from Gram-negative bacterial infections.

Phenotyping and susceptibility of established porcine cells lines to African Swine Fever Virus infection and viral production

African swine fever virus (ASFV) is a highly pathogenic, double-stranded DNA virus with a marked tropism for cells of the monocyte-macrophage lineage, affecting swine species and provoking severe economic losses and health threats. In the present study, four established porcine cell lines, IPAM-WT, IPAM-CD163, C∆2+ and WSL, were compared to porcine alveolar macrophage (PAM) in terms of surface marker phenotype, susceptibility to ASFV infection and virus production. The virulent ASFV Armenia/07, E70 or the naturally attenuated NHV/P68 strains were used as viral models. Cells expressed only low levels of specific receptors linked to the monocyte/macrophage lineage, with low levels of infection overall, with the exception of WSL, which showed more efficient production of strain NHV/P68 but not of strains E70 and Armenia/07.