Radical S-adenosyl Methionine Domain Containing 2 Research Articles

Protumorigenic Interferon-Stimulated Genes in Cancer: A Comprehensive Review.

Interferon-stimulated genes (ISGs), whose production is triggered by interferons, are known to defend the host from pathogenic and cancer-specific antigens, one of which is by inducing apoptosis in infected or mutated cells. It has been reported recently that specific ISGs aid cancer cells in evading immunosurveillance and inflammatory cells by inhibiting the apoptosis process. This report reviewed four apoptosis-regulating ISG proteins: interferon-stimulated gene 15 (ISG15), interferon alpha-inducible protein 27 (IFI27), interferon alpha-inducible protein 6 (IFI6), and radical S-adenosyl methionine domain containing 2 (RSAD2), demonstrating anti-apoptosis function, and considered them protumorigenic.

Induction of the antiviral factors APOBEC3A and RSAD2 upon CCL2 neutralization in primary human macrophages involves NF-kB, JAK/STAT, and gp130 signaling.

The CC chemokine ligand 2 (CCL2)/CC chemokine receptor 2 axis plays key roles in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. We previously reported that exposure of monocyte-derived macrophages (MDMs) to CCL2 neutralizing antibody (αCCL2 Ab) restricted HIV-1 replication at post-entry steps of the viral life cycle. This effect was associated with induction of transcripts coding for innate antiviral proteins, amongst which apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A) and radical S-adenosyl methionine domain containing 2 (RSAD2). This study aimed at identifying the signaling pathways involved in induction of these factors by CCL2 blocking in MDMs. Through a combination of pharmacologic inhibition, quantitative RT-PCR, western blotting, and confocal laser-scanning microscopy, we demonstrated that CCL2 neutralization activates the canonical NF-kB and JAK/STAT pathways, as assessed by time-dependent phosphorylation of IkB, STAT1, and STAT3 and p65 nuclear translocation. Furthermore, pharmacologic inhibition of I kappa B kinase and JAKs strongly reduced APOBEC3A and RSAD2 transcript accumulation elicited by αCCL2 Ab treatment. Interestingly, exposure of MDMs to αCCL2 Ab resulted in induction of IL-6 family cytokines, and interfering with glycoprotein 130, the common signal-transducing receptor subunit shared by these cytokines, inhibited APOBEC3A and RSAD2 up-regulation triggered by CCL2 neutralization. These results provide novel insights into the signal transduction pathways underlying the activation of innate responses triggered by CCL2 neutralization in macrophages. Since this response was found to be associated with protective antiviral effects, the new findings may help design innovative therapeutic approaches targeting CCL2 to strengthen host innate immunity.

Expression analysis and antiviral activity of koi carp (Cyprinus carpio) viperin against carp edema virus (CEV)

Viperin, also known as radical S-Adenosyl methionine domain containing 2 (RSAD2), is an IFN stimulated protein that plays crucial roles in innate immunity. Here, we identified a viperin gene from the koi carp (Cyprinus carpio) (kVip). The ORF of kVip is 1047 bp in length, encoding a polypeptide of 348 amino acids with neither signal peptide nor transmembrane protein. The predicted molecular weight is 40.37 kDa and the isoelectric point is 7.7. Multiple sequence alignment indicated that putative kVip contains a radical SAM superfamily domain and a conserved C-terminal region. kVip was highly expressed in the skin and spleen of healthy koi carps, and significantly stimulated in both natural and artificial CEV-infected koi carps. In vitro immune stimulation analysis showed that both extracellular and intracellular poly (I: C) or poly (dA: dT) caused a significant increase in kVip expression of spleen cells. Furthermore, intraperitoneal injection of recombinant kVip (rkVip) not only reduced the CEV load in the gills, but also improved the survival of koi carps following CEV challenge. Additionally, rkVip administration effectively regulated inflammatory and anti-inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) and interferon-related molecules (cGAS, STING, MyD88, IFN-γ, IFN-α, IRF3 and IRF9). Collectively, kVip effectively responded to CEV infection and exerted antiviral function against CEV partially by regulation of inflammatory and interferon responses.

Revealing common differential mRNAs, signaling pathways, and immune cells in blood, glomeruli, and tubulointerstitium of lupus nephritis patients based on transcriptomic data

Lupus nephritis (LN) is a potentially fatal autoimmune disease. The purpose of this study was to find potential key molecular markers of LN to aid in the early diagnosis and management of the disease. Datasets GSE99967_blood, GSE32591_glomeruli, and GSE32591_tubulointerstitium were included in this study. Differentially expressed mRNAs (DEmRNAs) were identified between the normal control and LN groups using the limma package in R. Common DEmRNAs in the three datasets were taken. Subsequently, functional enrichment analysis, immune correlation analysis, receiver operating characteristic (ROC) curve analysis and real-time polymerase chain reaction (RT-PCR) verification were performed. In this study, 11 common DEmRNAs were obtained and all of them were up-regulated. In protein–protein interaction (PPI) networks, we found that MX dynamin like GTPase 1 (MX1) and radical S-adenosyl methionine domain containing 2 (RSAD2) had the highest interaction score (0.997). Functional enrichment analysis revealed that MX1 and RSAD2 were enriched in influenza A and hepatitis C signaling pathways. The area under the curve (AUC) values of interferon-induced protein 44 (IFI44) and MX1 in GSE32591_glomeruli and GSE32591_tubulointerstitium datasets are 1, which is worthy of further study on their diagnostic value and molecular mechanism. The xCell analysis showed abnormal distribution of granulocyte-macrophage progenitor (GMP) cells in blood, glomeruli, and tubulointerstitium. Pearson’s correlation analysis found that GMP cells were significantly correlated with lactotransferrin (LTF) and cell cycle. Identification of common DEmRNAs and key pathways in the blood, glomeruli, and tubulointerstitium of patients with LN provides potential research directions for exploring the molecular mechanisms of the disease.

Interferon-α regulates abnormally increased expression of RSAD2 in Th17 and Tfh cells in systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that often involves abnormal activation of regulatory IFN genes and regulation of B cells by CD4+ T cells. Radical S-adenosyl methionine domain containing 2 (RSAD2) is a viral suppressor protein regulated by type I IFN, and it has been proven to play an important regulatory role in SLE. However, the mechanism by which RSAD2 participates in the pathogenesis of SLE is unclear. In this study, we observed higher expression levels of RSAD2 in CD4+ T-cell subsets from the peripheral blood of SLE patients than in those from healthy controls by bioinformatics analysis and validation experiments. We analyzed the expression of RSAD2 in CD4+ T cells of patients with SLE and other autoimmune diseases. In addition, we found that the expression of RSAD2 in CD4+ T cells might be regulated by IFN-α, and RSAD2 significantly affected the differentiation of Th17 cells and T follicular helper (Tfh) cells. Our findings underlined that RSAD2 may promote B-cell activation by promoting the differentiation of Th17 and Tfh cells in SLE patients, a process that is regulated by IFN-α.

Interferon-λ3 alleviates intestinal epithelium injury induced by porcine rotavirus in mice

Interferons are a group of glycoproteins that are expressed in various cell types in their inflammatory responses to infections. In this study, we explored the protective effects of porcine interferon-λ3 (PIFN-λ3) on intestinal inflammation and injury in mice induced by porcine rotavirus (PRV). BALB/c mice were administrated by PIFN-λ3 or phosphate buffer solution (PBS) for three days prior to PRV infection. We show that PRV infection caused acute inflammatory responses in mice, as indicated by increases in serum concentrations of inflammatory cytokines such as the interlukin-1β (IL-1β), interlukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (P < 0.05). However, PIFN-λ3 administration not only decreased their concentrations but also elevated the concentrations of immunoglobulin (Ig) M and IgG in the PRV challenged mice (P < 0.05). PRV infection significantly decreased the jejunal villus height and the ratio of villus height to crypt depth (V/C); however, PIFN-λ3 treatment significantly elevated the villus height and the abundance of tight junction protein ZO-1 in the jejunum (P < 0.05). Moreover, PIFN-λ3 decreased the replication of PRV in the jejunal epithelium, but significantly increased the abundance of sIgA and the activities of maltase and sucrase in the PRV-challenged mice (P < 0.05). Interestingly, PIFN-λ3 elevated the expression levels of sodium/glucose cotransporter 1 (SGLT1) and mucin 2 (MUC2) in the PRV-challenged mice (P < 0.05). Moreover, PIFN-λ3 significantly increased the expression levels of IL-10, signal transducer and activator of transcription 1 (STAT1), and critical interferon-stimulated genes such as the 2′-5′ oligoadenylate synthetase-like 1 (OASL1), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and radical S-adenosyl methionine domain containing 2 (RSAD2) in the jejunum upon PRV infection (P < 0.05). The anti-virus and anti-inflammatory effect of PIFN-λ3 should make it an attractive candidate to prevent various pathogen-induced bowel diseases.

5-Methoxyflavone-induced AMPKα activation inhibits NF-κB and P38 MAPK signaling to attenuate influenza A virus-mediated inflammation and lung injury in vitro and in vivo

Influenza-related acute lung injury (ALI) is a life-threatening condition that results mostly from uncontrolled replication of influenza virus (IV) and severe proinflammatory responses. The methoxy flavonoid compound 5-methoxyflavone (5-MF) is believed to have superior biological activity in the treatment of cancer. However, the effects and underlying mechanism of 5-MF on IV-mediated ALI are still unclear. Here, we showed that 5-MF significantly improved the survival of mice with lethal IV infection and ameliorated IV-mediated lung edema, lung histological changes, and inflammatory cell lung recruitment. We found that 5-MF has antiviral activity against influenza A virus (IAV), which was probably associated with increased expression of radical S-adenosyl methionine domain containing 2 (RSAD2) and suppression of endosomal acidification. Moreover, IV-infected A549 cells with 5-MF treatment markedly reduced proinflammatory mediator expression (IL-6, CXCL8, TNF-α, CXCL10, CCL2, CCL3, CCL4, GM-CSF, COX-2, and PGE2) and prevented P-IKBα, P-P65, and P-P38 activation. Interestingly, we demonstrated that 5-MF treatment could trigger activation of AMP-activated protein kinase (AMPK)α in IV-infected A549 cells, as evidenced by activation of the AMPKα downstream molecule P53. Importantly, the addition of AMPKα blocker compound C dramatically abolished 5-MF-mediated increased levels of RSAD2, the inhibitory effects on H1N1 virus-elicited endosomal acidification, and the suppression expression of proinflammatory mediators (IL-6, TNF-α, CXCL10, COX-2 and PGE2), as well as the inactivation of P-IKBα, P-P65, and P-P38 MAPK signaling pathways. Furthermore, inhibition of AMPKα abrogated the protective effects of 5-MF on H1N1 virus-mediated lung injury and excessive inflammation in vivo. Taken together, these results indicate that 5-MF alleviated IV-mediated ALI and suppressed excessive inflammatory responses through activation of AMPKα signaling.

Intratracheal instillation of graphene oxide decreases anti-virus responses and lipid contents via suppressing Toll-like receptor 3 in mouse livers.

Recent studies revealed a causal relationship between Toll-like receptors (TLRs) and lipid droplet biogenesis. Interestingly, it has been reported before that nanomaterials (NMs) were capable to modulate TLRs, but it remains unclear if NMs could affect lipid levels via TLR signaling pathways. In this study, we investigated the influences of airway exposure to graphene oxide (GO) on TLR3 signaling pathways and lipid levels in mouse livers. Intratracheal instillation of GO (0.1, 1, and 5mg/kg, once a day, totally 5days) induced inflammatory cell infiltrations as indicated by hematoxylin-eosin (H&amp;E) staining and fibrosis as indicated by Masson staining in lungs, accompanying with decreased TLR3 proteins. Consistently, a TLR3-regulated anti-virus protein, namely interferon induced protein with tetratricopeptide repeats 1 (IFIT1), as well as two TLR3-regulated lipid proteins, namely radical S-adenosyl methionine domain containing 2 (RSAD2) and perilipin 2 (PLIN2), were decreased in lungs. The protein levels of interferon-β in serum were also decreased. In livers, GO exposure induced disorganization of liver cells but not fibrosis. In agreement with the trends observed in lungs, TLR3, IFIT1, RSAD2, and PLIN2 proteins were decreased in livers. As a possible consequence, GO exposure dose-dependently decreased lipid levels in livers as indicated by oil red O and BODIPY 493/503 staining. We concluded that airway exposure to GO decreased anti-virus responses and lipid levels in mouse livers via the suppression of TLR3.

The long non-coding RNA LNC_000397 negatively regulates PRRSV replication through induction of interferon-stimulated genes

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most significant threats to the global swine industry. It is of great importance to understand viral-host interactions to develop novel antiviral strategies. Long non-coding RNAs (lncRNAs) have emerged as critical factors regulating host antiviral immune responses. However, lncRNAs participating in virus-host interactions during PRRSV infection remain largely unexplored.MethodRNA transcripts of porcine alveolar macrophages (PAMs) infected with two different PRRSV strains, GSWW/2015 and VR2332, at 24 h post-infection were sequenced by high-throughput sequencing. Four programs namely, CNCI, CPC, PFAM, and phyloCSF, were utilized to predict the coding potential of transcripts. mRNAs co-localized or co-expressed with differentially expressed lncRNAs were considered as their targets. Fuction of lncRNAs was predicted by GO and KEGG analysis of their target mRNAs. The effect of LNC_000397 on PRRSV replication was validated by knockdown its expression using siRNA. Target genes of LNC_000397 were identified by RNA-Sequencing and validated by RT-qPCR.ResultIn this study, we analyzed lncRNA and mRNA expression profiles of PRRSV GSWW/2015 and VR2332 infected porcine alveolar macrophages. A total of 1,147 novel lncRNAs were characterized, and 293 lncRNAs were differentially expressed. mRNAs co-localized and co-expressed with lncRNAs were enriched in pathogen-infection-related biological processes such as Influenza A and Herpes simplex infection. Functional analysis revealed the lncRNA, LNC_000397, which was up-regulated by PRRSV infection, negatively regulated PRRSV replication. Knockdown of LNC_000397 significantly impaired expression of antiviral ISGs such as MX dynamin-like GTPase 1 (MX1), ISG15 Ubiquitin-like modifier (ISG15), and radical S-adenosyl methionine domain containing 2 (RSAD2).ConclusionsLNC_000397 negatively regulated PRRSV replication by inducing interferon-stimulated genes (ISGs) expression. Our study is the first report unveiling the role of host lncRNA in regulating PRRSV replication, which might be beneficial for the development of novel antiviral therapeutics.

Graphene oxide links alterations of anti-viral signaling pathways with lipid metabolism via suppressing TLR3 in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs), the main cells constructing blood vessels, are important in the regulation of the pathophysiology of vascular systems; however, relatively few studies have investigated the influence of nanomaterials (NMs) on VSMCs. In this study, we found that the interaction between graphene oxide and human VSMCs led to the cytotoxicity and morphological changes of cells. Because transcriptomic data suggested that graphene oxide decreased anti-viral signaling pathways via decreasing Toll-like receptor 3 (TLR3), we further verified that graphene oxide decreased interferon induced protein with tetratricopeptide repeats 1 (IFIT1) and the radical S-adenosyl methionine domain containing 2 (RSAD2), and TLR3-downstream genes involved in anti-viral responses. Due to the involvement of RSAD2 in lipid dysfunction, we also verified that graphene oxide disrupted lipid homeostasis and increased adipose triglyceride lipase (ATGL). Adding TLR3 agonist polyinosinic:polycytidylic acid (Poly IC) partially increased TLR3-downstream protein interleukin-8 (IL-8) and some lipid classes, particularly lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), in graphene oxide-exposed VSMCs. In mice receiving repeated intravenous injection of graphene oxide, significantly decreased TLR3, IFIT1 and RSAD2 but increased ATGL proteins were observed in aortas. We conclude that graphene oxide altered anti-viral signaling pathways and lipid metabolism via decreasing TLR3 in VSMCs.

Weighted gene co-expression network analysis reveals that CXCL10, IRF7, MX1, RSAD2, and STAT1 are related to the chronic stage of spinal cord injury.

BackgroundThe process of spinal cord injury involves acute, subacute, and chronic stages; however, the specific pathological mechanism remains unclear. In this study, weighted gene co-expression network analysis (WGCNA) was used to clarify specific modules and hub genes that associated with SCI.MethodsThe gene expression profiles GEO Series (GSE)45006 and GEO Series (GSE)2599 were downloaded, and the co-expression network modules were identified by the WGCNA package. The protein-protein interaction (PPI) network and Venn diagram were constructed to identify hub genes. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to quantify the degree of the top five candidate genes. Correlation analysis was also carried out between hub genes and immune infiltration.ResultsIn total, 14,402 genes and seven modules were identified. The brown module was considered to be the most critical module for the chronic stage of SCI, which contained 775 genes that were primarily associated with various biological processes, including extracellular structure organization, lysosome, isoprenoid biosynthesis, response to nutrients, response to wounding, sulfur compound metabolic process, cofactor metabolic process, and ossification. Furthermore, C-X-C motif chemokine ligand 10 (CXCL10), myxovirus (influenza virus) resistance 1 (MX1), signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 7 (IRF7) and radical S-adenosyl methionine domain containing 2 (RSAD2) were identified as the hub genes in the PPI and Venn diagram network, and verified by qRT-PCR. Immune infiltration analysis revealed that CD8+ T cells, macrophages, neutrophils, plasmacytoid dendritic cells, helper T cells, Th2 cells, and tumor-infiltrating lymphocytes may be involved in the SCI process.ConclusionsThere were significant differences among the five hub genes (CXCL10, IRF7, MX1, RSAD2, and STAT1) of the brown module, which may be potential diagnostic and prognostic markers of SCI, and immune cell infiltration may play an important role in the chronic stage of SCI.

Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis

Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV) is an arterivirus newly discovered in Chinese softshell turtles. Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles. In this study, a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system, and its polyclonal antibody was generated. The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay (dot-ELISA). The distribution of TSHSV in different organs of T. sinensis was examined by immunohistochemistry (IHC) and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed, and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles. The IHC assay indicated that the virus was highly localized in various cells, including intestinal lymphocytes, enterocytes, kidney epithelial cells, spleen cells, lung macrophages, and cardiomyocytes. The qRT-PCR analysis revealed that TSHSV was detected in all organs tested, including the lungs, liver, kidneys, spleen, and heart. The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group. The interferon-stimulated genes (ISGs), myxovirus resistance protein 2 (MX2) and radical S-adenosyl methionine domain containing 2 (RSAD2) were highly upregulated in all groups of infected turtles. Antibody-dependent enhancement (ADE) seemed to occur after stimulation by the polyclonal antibody, because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group. Overall, these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

Knockdown of RSAD2 attenuates B cell hyperactivity in patients with primary Sjögren's syndrome (pSS) via suppressing NF-κb signaling pathway.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that is mainly characterized as abnormal activation of B cells. It is reported that radical s-adenosyl methionine domain-containing 2 (RSAD2) is overexpressed in CD19+ B cells of pSS patients, but its role in pSS B cells remains unknown. Herein, RSAD2 expression was upregulated in CD19+ B cells of pSS patients and positively correlated with the expression of interleukin-10 (IL-10) in serum. After CD40L stimulation, knockdown of RSAD2 significantly attenuated cell viability, the production levels of immunoglobins and the expression of IL-10, while promoted cell apoptosis of pSS CD19+ B cells. Mechanistically, knockdown of RSAD2 negatively regulated nuclear factor kappa-b (NF-κb) signaling pathway. In addition, overexpression of p65 prominently alleviated the inhibitory effect of RSAD2 knockdown on proliferation, immunoglobin production and IL-10 expression in CD40L-induced CD19+ B cells. Our study indicated that silencing RSAD2 attenuated pSS B cell hyperactivity via suppressing NF-κb signaling pathway, which might provide a potential therapeutic target for pSS treatment.

Expression of USP18 and IL2RA Is Increased in Individuals Receiving Latent Tuberculosis Treatment with Isoniazid.

Background The treatment of latent tuberculosis infection (LTBI) in individuals at risk of reactivation is essential for tuberculosis control. However, blood biomarkers associated with LTBI treatment have not been identified. Methods Blood samples from tuberculin skin test (TST) reactive individuals were collected before and after one and six months of isoniazid (INH) therapy. Peripheral mononuclear cells (PBMC) were isolated, and an in-house interferon-γ release assay (IGRA) was performed. Expression of chemokine ligand 4 (CCL4), chemokine ligand 10 (CXCL10), chemokine ligand 11 (CXCL11), interferon alpha (IFNA), radical S-adenosyl methionine domain-containing 2 (RSAD2), ubiquitin-specific peptidase 18 (USP18), interferon-induced protein 44 (IFI44), interferon-induced protein 44 like (IFI44L), interferon-induced protein tetratricopeptide repeats 1(IFIT1), and interleukin 2 receptor subunit alpha (IL2RA) mRNA levels were assessed by qPCR before, during, and after INH treatment. Results We observed significantly lower relative abundances of USP18, IFI44L, IFNA, and IL2RA transcripts in PBMC from IGRA-positive individuals compared to levels in IGRA-negative individuals before INH therapy. Also, relative abundance of CXCL11 was significantly lower in IGRA-positive than in IGRA-negative individuals before and after one month of INH therapy. However, the relative abundance of CCL4, CXCL10, and CXCL11 mRNA was significantly decreased and that of IL2RA and USP18 significantly increased after INH therapy, regardless of the IGRA result. Our results show that USP18, IFI44L, IFIT1, and IL2RA relative abundances increased significantly, meanwhile the relative abundance of CCL4, CXCL11, and IFNA decreased significantly after six months of INH therapy in TST-positive individuals. Conclusions Changes in the profiles of USP18, IL2RA, IFNA, CCL4, and CXCL11 expressions during INH treatment in TST-positive individuals, regardless of IGRA status, are potential tools for monitoring latent tuberculosis treatment.

Molecular characterization and expression analysis of rockfish (Sebastes schlegelii) viperin, and its ability to enervate RNA virus transcription and replication in vitro

Viperin, also known as RSAD2 (Radical S-adenosyl methionine domain containing 2), is an interferon-induced endoplasmic reticulum-associated antiviral protein. Previous studies have shown that viperin levels are elevated in the presence of viral RNA, but it has rarely been characterized in marine organisms. This study was designed to functionally characterize rockfish viperin (SsVip), to examine the effects of different immune stimulants on its expression, and to determine its subcellular localization. SsVip is a 349 amino acid protein with a predicted molecular mass of 40.24 kDa. It contains an S-adenosyl l-methionine binding conserved domain with a CNYKCGFC sequence. Unchallenged tissue expression analysis using quantitative real time PCR (qPCR) revealed SsVip expression to be the highest in the blood, followed by the spleen. When challenged with poly I:C, SsVip was upregulated by approximately 60-fold in the blood after 24 h, and approximately 50-fold in the spleen after 12 h. Notable upregulation was detected throughout the poly I:C challenge experiment in both tissues. Significant expression of SsVip was detected in the blood following Streptococcus iniae and lipopolysaccharide challenge, and viral hemorrhagic septicemia virus (VHSV) gene transcription was significantly downregulated during SsVip overexpression. Furthermore, cell viability assay and virus titer quantification with the presence of SsVip revealed a significant reduction in virus replication. As with previously identified viperin counterparts, SsVip was localized in the endoplasmic reticulum. Our findings show that SsVip is an antiviral protein crucial to innate immune defense.

Duplex Quantitative Polymerase Chain Reaction of ISG15 and RSAD2 Increases Accuracy of Early Pregnancy Diagnosis in Dairy Cows

Abstract Early diagnosis of pregnancy is important in livestock production, but there is no reliable technology used for pregnancy diagnosis within the first three weeks after insemination. During early pregnancy, the expression of interferon-stimulating genes (ISGs) in peripheral blood leukocytes (PBL) is significantly increased. However, due to different strains, detection sample types, detection methods, threshold value, etc. the specific effectiveness of early pregnancy diagnosis using ISGs is worth further study. The purpose of this study was to test interferon-stimulated protein 15 (ISG15), 2'–5'-oligoadenylate synthetase 1 (OAS1) and radical S-adenosyl methionine domain containing 2 (RSAD2) for early pregnancy diagnosis in dairy cows. The expression of ISG15, OAS1, and RSAD2 in PBL of pregnant and non-pregnant heifers on days 0, 14, 18, 21 and 28 after artificial insemination (AI) was detected by fluorescence quantitative polymerase chain reaction (PCR). The sensitivity and specificity of the pregnancy diagnosis was analyzed using expression of these three genes separately or in combination by receiver operating characteristic curve. The combination with the highest accuracy used probe primers and duplex fluorescence quantitative PCR. The single quantitative PCR results showed that expression of ISG15, OAS1 and RSAD2 on day 18 after AI was significantly higher in pregnant than in non-pregnant cows. When these three genes were used separately, or in combination, for early pregnancy diagnosis, the sensitivity for the RSAD2 gene was 100%, and the combination of ISG15 with RSAD2 was 94.7%. The duplex quantitative PCR showed that, although the sensitivity of ISG15 alone was 100%, its specificity was only 88.2% (cut-off value 1.402). The sensitivity of RSAD2 alone was 89.5%, and the specificity was 88.2%; however, when the two genes were used in combination, the sensitivity, specificity and diagnostic cut-off value were consistent with the results of single quantitative PCR. These results indicated that a duplex quantitative PCR assay system for early pregnancy diagnosis in cows using ISG15 and RSAD2 was established. Simultaneous detection of expression of ISG15 and RSAD2 by duplex quantitative PCR can effectively improve the diagnostic accuracy for dairy cows.

A Western-type dietary pattern and atorvastatin induce epicardial adipose tissue interferon signaling in the Ossabaw pig

Epicardial adipose tissue (EAT) inflammation is thought to potentiate the development of coronary artery disease (CAD). Overall diet quality and statin therapy are important modulators of inflammation and CAD progression. Our objective was to examine the effects and interaction of dietary patterns and statin therapy on EAT gene expression in the Ossabaw pig. Pigs were randomized to 1 of 4 groups; Heart Healthy diet (high in unsaturated fat, unrefined grain, fruits/vegetables [HHD]) or Western diet (high in saturated fat, cholesterol, refined grain [WD]), with or without atorvastatin. Diets were fed in isocaloric amounts for 6 months. A two-factor edge R analysis identified the differential expression of 21 genes. Relative to the HHD, the WD resulted in a significant 12-fold increase of radical s-adenosyl methionine domain containing 2 (RSAD2), a gene induced by interferon signaling. Atorvastatin led to the significant differential expression of 17 genes predominately involved in interferon signaling. Results were similar using the Porcine Translational Research Database. Pathway analysis confirmed the up-regulation of interferon signaling in response to the WD and atorvastatin independently. An expression signature of the largely interferon related differentially expressed genes had no predictive capability on a histological assessment of atherosclerosis in the underlying coronary artery. These results suggest that a WD and atorvastatin evoke an interferon mediated immune response in EAT of the Ossabaw pig, which is not associated with the presence of atherosclerosis.

A key anti-viral protein, RSAD2/VIPERIN, restricts the release of measles virus from infected cells

Measles virus (MV), a paramyxovirus, is one of the most contagious human pathogens and is responsible for thousands of deaths annually. Wild-type MV evolved to counter the innate immune system by avoiding both type I interferon (IFN) induction and inhibiting IFN signaling through the JAK/STAT pathway. However, virus replication is significantly inhibited in IFN-pretreated cells. Similarly, MV vaccine derived strains are inhibited by IFN pretreatment, but vaccine strains also induce IFN. Despite the significant progress in understanding the interactions between MV and the IFN pathway, the IFN stimulated genes (ISGs) that inhibit MV replication remain largely unknown. The aim of this study is to identify specific ISGs that mediate restriction of MV. In this study, we report that Radical S-adenosyl methionine domain containing 2 (RSAD2) restricts MV infection at the stage of virus release in infected 293T cells. Furthermore, attenuated MV strains are currently being developed as a novel treatment for solid and hematological malignancies. Therefore, we tested the impact of RSAD2 expression in an oncolytic virotherapy context using a MV permissive ovarian cancer line (SR-B2). As measured in 293T cells, MV release was also impaired in SR-B2 cells transduced to express RSAD2 in vitro. Additionally, oncolytic MV therapeutic efficacy was impaired in SR-B2 cells transduced to express RSAD2 in vivo. Overall, we identify RSAD2 as a novel restriction factor for MV by inhibiting the release of virus. These results provide important information regarding the interaction between MV and the innate immune system, as well as implications for the design of oncolytic MV platforms.

RSAD2 and AIM2 Modulate Coxsackievirus A16 and Enterovirus A71 Replication in Neuronal Cells in Different Ways That May Be Associated with Their 5' Nontranslated Regions.

Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) are closely related enteroviruses that cause the same hand, foot, and mouth disease (HFMD), but neurological complications occur only very rarely in CV-A16 compared to EV-A71 infections. To elucidate host responses that may be able to explain these differences, we performed transcriptomic analysis and real-time quantitative PCR (RT-qPCR) in CV-A16-infected neuroblastoma cells (SK-N-SH), and the results showed that the radical S-adenosylmethionine domain containing 2 (RSAD2) was the highest upregulated gene in the antimicrobial pathway. Increased RSAD2 expression was correlated with reduced viral replication, while RSAD2 knockdown cells were correlated with increased replication. EV-A71 replication showed no apparent correlation to RSAD2 expressions. Absent in melanoma 2 (AIM2), which is associated with pyroptotic cell death, was upregulated in EV-A71-infected neurons but not in CV-A16 infection, suggesting that the AIM2 inflammasome played a significant role in suppressing EV-A71 replication. Chimeric viruses derived from CV-A16 and EV-A71 but containing swapped 5' nontranslated regions (5' NTRs) showed that RSAD2 expression/viral replication and AIM2 expression/viral replication patterns may be linked to the 5' NTRs of parental viruses. Differences in secondary structure of internal ribosomal entry sites within the 5' NTR may be responsible for these findings. Overall, our results suggest that CV-A16 and EV-A71 elicit different host responses to infection, which may help explain the apparent lower incidence of CV-A16-associated neurovirulence in HFMD outbreaks compared to EV-A71 infection.IMPORTANCE Although coxsackievirus A16 (CV-A16) and enterovirus A17 (EV-A71) both cause hand, foot, and mouth disease, EV-A71 has emerged as a leading cause of nonpolio, enteroviral fatal encephalomyelitis among young children. The significance of our research is in the identification of the possible differing and novel mechanisms of CV-A16 and EV-A71 inhibition in neuronal cells that may impact viral neuropathogenesis. We further showed that viral 5' NTRs may play significant roles in eliciting different host response mechanisms.

Effect of exogenous progesterone on embryo size and ewe uterine gene expression in an ovine 'dam size' model of maternal constraint.

Progesterone (P4), acting via its receptor, regulates uterine function and histotroph production, which are crucial to embryo growth. This study aimed to examine exogenous P4 effects on embryo size and differential endometrial gene expression at Day 19 of gestation using a 'dam size' sheep model of maternal constraint. Purebred Suffolk (S, genotypically large) embryos were transferred into recipient groups of Cheviot (C, genotypically small) or Suffolk ewes that had, or had not, been pre-treated with P4 from Days 0 to 6 of pregnancy. At Day 19S embryos were collected from four experimental groups: P4 pretreated S ewes (SP4; n=5), untreated S ewes (SnP4; n=15), P4 pretreated C ewes (CP4; n=7) and untreated C ewes (CnP4; n=21). Day-19 embryos from CP4 ewes were larger (P<0.05) than those from CnP4 ewes and similar in size (P>0.05) to embryos from SnP4 and SP4 ewes. Expression of mucin 1 (MUC1) and prostaglandin-endoperoxide synthase 2 (PTGS2) was upregulated in uterine horns ipsilateral to the corpus luteum from CP4 ewes. Prostaglandin receptor (PGR), MUC1 and PTGS2 expression was upregulated, whilst cathepsin L (CTSL) and radical S-adenosyl methionine domain-containing 2 (RSAD2) expression was downregulated in the ipsilateral horn of SP4 ewes. This suggests that pretreating ewes with exogenous P4 may alleviate early pregnancy maternal constraint via mechanisms that alter uterine function. However, further research is required to investigate the timing of P4 administration and its impact on conception rates.