Foot-and-Mouth Disease Virus 3C Protease Antagonizes Interferon Signaling and C142T Substitution Attenuates the FMD Virus.

3C protease (3Cpro), a chymotrypsin-like cysteine protease encoded by the foot-and-mouth disease virus (FMDV), plays an essential role in processing the FMDV P1 polyprotein into individual viral capsid proteins in FMDV replication. Previously, it has been shown that 3Cpro is involved in the blockage of the host type-I interferon (IFN) responses by FMDV. However, the underlying mechanisms are poorly understood. Here, we demonstrated that the protease activity of 3Cpro contributed to the degradation of RIG-I and MDA5, key cytosolic sensors of the type-I IFN signaling cascade in proteasome, lysosome and caspase-independent manner. And also, we examined the degradation ability on RIG-I and MDA5 of wild-type FMDV 3Cpro and FMDV 3Cpro C142T mutant which is known to significantly alter the enzymatic activity of 3Cpro. The results showed that the FMDV 3Cpro C142T mutant dramatically reduce the degradation of RIG-I and MDA5 due to weakened protease activity. Thus, the protease activity of FMDV 3Cpro governs its RIG-I and MDA5 degradation ability and subsequent negative regulation of the type-I IFN signaling. Importantly, FMD viruses harboring 3Cpro C142T mutant showed the moderate attenuation of FMDV in a pig model. In conclusion, our results indicate that a novel mechanism evolved by FMDV 3Cpro to counteract host type-I IFN responses and a rational approach to virus attenuation that could be utilized for future vaccine development.

Pluripotency and immunomodulatory signatures of canine induced pluripotent stem cell-derived mesenchymal stromal cells are similar to harvested mesenchymal stromal cells.

With a view towards harnessing the therapeutic potential of canine mesenchymal stromal cells (cMSCs) as modulators of inflammation and the immune response, and to avoid the issues of the variable quality and quantity of harvested cMSCs, we examined the immunomodulatory properties of cMSCs derived from canine induced pluripotent stem cells (ciMSCs), and compared them to cMSCs harvested from adipose tissue (cAT-MSC) and bone marrow (cBM-MSC). A combination of deep sequencing and quantitative RT-PCR of the ciMSC transcriptome confirmed that ciMSCs express more genes in common with cBM-MSCs and cAT-MSCs than with the ciPSCs from which they were derived. Both ciMSCs and harvested cMSCs express a range of pluripotency factors in common with the ciPSCs including NANOG, POU5F1 (OCT-4), SOX-2, KLF-4, LIN-28A, MYC, LIF, LIFR, and TERT. However, ESRRB and PRDM-14, both factors associated with naïve, rather than primed, pluripotency were expressed only in the ciPSCs. CXCR-4, which is essential for the homing of MSCs to sites of inflammation, is also detectable in ciMSCs, cAT- and cBM-MSCs, but not ciPSCs. ciMSCs constitutively express the immunomodulatory factors iNOS, GAL-9, TGF-ß1, PTGER-2α and VEGF, and the pro-inflammatory mediators COX-2, IL-1ß and IL-8. When stimulated with the canine pro-inflammatory cytokines tumor necrosis factor-α (cTNF-α), interferon-γ (cIFN-γ), or a combination of both, ciMSCs upregulated their expression of IDO, iNOS, GAL-9, HGF, TGF-ß1, PTGER-2α, VEGF, COX-2, IL-1ß and IL-8. When co-cultured with mitogen-stimulated lymphocytes, ciMSCs downregulated their expression of iNOS, HGF, TGF-ß1 and PTGER-2α, while increasing their expression of COX-2, IDO and IL-1ß. Taken together, these findings suggest that ciMSCs possess similar immunomodulatory capabilities as harvested cMSCs and support further investigation into their potential use for the management of canine immune-mediated and inflammatory disorders.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells from the Tasmanian Devil (Sarcophilus harrisii) Express Immunomodulatory Factors and a Tropism Toward Devil Facial Tumor Cells.

Marsupials have long attracted scientific interest because of their unique biological features and their position in mammalian evolution. Mesenchymal stem cells (MSCs) are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory, and regenerative properties. MSCs have been harvested from various tissues in numerous eutherian species; however, there are no descriptions of MSCs derived from a marsupial. In this study, we have generated Tasmanian devil (Sarcophilus harrisii) MSCs from devil induced pluripotent stem cells (iPSCs), thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. Devil iPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and through inhibition of the transforming growth factor beta/activin signaling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the MSC-specific surface proteins CD73, CD90, and CD105, in addition to the pluripotency transcription factors OCT4/POU5F1, SOX2, and NANOG. Expression of the marsupial pluripotency factor POU5F3, a paralogue of OCT4/POU5F1, is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic, and chondrogenic pathways in vitro, confirming their trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Devil iMSCs show a tropism toward medium conditioned by devil facial tumor cells and express a range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against Devil Facial Tumor Disease.

Induced Pluripotent Stem Cells from a Marsupial, the Tasmanian Devil (Sarcophilus harrisii): Insight into the Evolution of Mammalian Pluripotency.

We demonstrate the generation of Tasmanian devil (Sarcophilus harrisii) induced pluripotent stem cells (DeviPSCs) from dermal fibroblasts by lentiviral delivery of human transcription factors. DeviPSCs display characteristic pluripotent stem cell colony morphology, with individual cells having a high nuclear-to-cytoplasmic ratio and alkaline phosphatase activity. DeviPSCs are leukemia inhibitory factor dependent and have reactivated endogenous octamer-binding transcription factor 4 [OCT4, POU domain, class 5, transcription factor 1 (POU5F1)], POU2 [POU domain, class 5, transcription factor 3 (POU5F3)], sex determining region Y-box 2 (SOX2), Nanog homeobox (NANOG) and dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) genes, retained a normal karyotype, and concurrently silenced exogenous human transgenes. Notably, co-expression of both OCT4 and POU2 suggests that they are representative of cells of the epiblast, the marsupial equivalent of the inner cell mass. DeviPSCs readily form embryoid bodies and in vitro teratomas containing derivatives of all three embryonic germ layers. To date, DeviPSCs have been stably maintained for more than 45 passages. Our DeviPSCs provide an invaluable resource for studies into marsupial pluripotency and development, and they may also serve as an important tool in efforts to combat the threat of devil facial tumor disease.

Dense Granule Protein-7 (GRA-7) of Toxoplasma gondii inhibits viral replication in vitro and in vivo.

Dense granule protein-7 (GRA-7) is an excretory protein of Toxoplasma gondii. It is a potential serodiagnostic marker and vaccine candidate for toxoplasmosis. Previous reports demonstrated that GRA-7 induces innate immune responses in macrophages by interacting with TRAF6 via the MyD88-dependent pathway. In the present study, we evaluated the antiviral activity and induction of an antiviral state by GRA-7 both in vitro and in vivo. It was observed that GRA-7 markedly reduced the replication of vesicular stomatitis virus (VSV-GFP), influenza A virus (PR8-GFP), coxsackievirus (H3-GFP), herpes simplex virus (HSV-GFP), and adenovirus-GFP in epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. These antiviral activities of GRA-7 were attributed to the induction of type I interferon (IFN) signaling, resulting in the secretion of IFNs and pro-inflammatory cytokines. Additionally, in BALB/c mice, intranasal administration of GRA-7 prevented lethal infection by influenza A virus (H1N1) and exhibited prophylactic effects against respiratory syncytial virus (RSV-GFP). Collectively, these results suggested that GRA-7 exhibits immunostimulatory and broad spectrum antiviral activities via type I IFN signaling. Thus, GRA-7 can be potentially used as a vaccine adjuvant or as a candidate drug with prophylactic potential against viruses.

Coptidis Rhizoma extract inhibits replication of respiratory syncytial virus in vitro and in vivo by inducing antiviral state.

Coptidis Rhizoma is derived from the dried rhizome of Ranunculaceous plants and is a commonly used traditional Chinese medicine. Although Coptidis Rhizoma is commonly used for its many therapeutic effects, antiviral activity against respiratory syncytial virus (RSV) has not been reported in detail. In this study, we evaluated the antiviral activities of Coptidis Rhizoma extract (CRE) against RSV in human respiratory tract cell line (HEp2) and BALB/c mice. An effective dose of CRE significantly reduces the replication of RSV in HEp2 cells and reduces the RSV-induced cell death. This antiviral activity against RSV was through the induction of type I interferon-related signaling and the antiviral state in HEp2 cells. More importantly, oral administration of CRE exhibited prophylactic effects in BALB/c mice against RSV. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we confirmed that palmatine was related to the antiviral properties and immunemodulation effect. Taken together, an extract of Coptidis Rhizoma and its components play roles as immunomodulators and could be a potential source as promising natural antivirals that can confer protection to RSV. These outcomes should encourage further allied studies in other natural products.

Epimedium koreanum Nakai displays broad spectrum of antiviral activity in vitro and in vivo by inducing cellular antiviral state.

Epimedium koreanum Nakai has been extensively used in traditional Korean and Chinese medicine to treat a variety of diseases. Despite the plant's known immune modulatory potential and chemical make-up, scientific information on its antiviral properties and mode of action have not been completely investigated. In this study, the broad antiviral spectrum and mode of action of an aqueous extract from Epimedium koreanum Nakai was evaluated in vitro, and moreover, the protective effect against divergent influenza A subtypes was determined in BALB/c mice. An effective dose of Epimedium koreanum Nakai markedly reduced the replication of Influenza A Virus (PR8), Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV) and Newcastle Disease Virus (NDV) in RAW264.7 and HEK293T cells. Mechanically, we found that an aqueous extract from Epimedium koreanum Nakai induced the secretion of type I IFN and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in cells. Among various components present in the extract, quercetin was confirmed to have striking antiviral properties. The oral administration of Epimedium koreanum Nakai exhibited preventive effects on BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3 and H9N2). Therefore, an extract of Epimedium koreanum Nakai and its components play roles as immunomodulators in the innate immune response, and may be potential candidates for prophylactic or therapeutic treatments against diverse viruses in animal and humans.