Search Results (10)

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes’ transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4. Full article

The proper supplementation of boron, an essential trace element, can enhance animal immune function. We utilized the method of TMT peptide labeling in conjunction with LC-MS/MS quantitative proteomics for the purpose of examining the effects of boric acid on a rat model and analyzing proteins from the duodenum. In total, 5594 proteins were obtained from the 0, 10, and 320 mg/L boron treatment groups. Two hundred eighty-four proteins that exhibit differential expression were detected. Among the comparison, groups of 0 vs. 10 mg/L, 0 vs. 320 mg/L, and 10 vs. 320 mg/L of boron, 110, 32, and 179 proteins, respectively, demonstrated differential expression. The results revealed that these differential expression proteins (DEPs) mainly clustered into two profiles. GO annotations suggested that most of the DEPs played a role in the immune system process, in which 2′-5′-oligoadenylate synthetase-like, myxovirus resistance 1, myxovirus resistance 2, dynein cytoplasmic 1 intermediate chain 1, and coiled-coil domain containing 88B showed differential expression. The DEPs had demonstrated an augmentation in the signaling pathways, which primarily include phagosome, antigen processing, and presentation, as well as cell adhesion molecules (CAMs). Our study found that immune responses in the duodenum were enhanced by lower doses of boron and that this effect is likely mediated by changes in protein expression patterns in related signaling pathways. It offers an in-depth understanding of the underlying molecular mechanisms that lead to immune modulation in rats subjected to dietary boron treatment. Full article

Influenza A virus (IAV) is a leading cause of human respiratory infections and poses a major public health concern. IAV replication can affect the expression of DNA methyltransferases (DNMTs), and the subsequent changes in DNA methylation regulate gene expression and may lead to abnormal gene transcription and translation, yet the underlying mechanisms of virus-induced epigenetic changes from DNA methylation and its role in virus–host interactions remain elusive. Here in this paper, we showed that DNMT1 expression could be suppressed following the inhibition of miR-142-5p or the PI3K/AKT signaling pathway during IAV infection, resulting in demethylation of the promotor region of the 2′-5′-oligoadenylate synthetase-like (OASL) protein and promotion of its expression in A549 cells. OASL expression enhanced RIG-I-mediated interferon induction and then suppressed replication of IAV. Our study elucidated an innate immunity mechanism by which up-regulation of OASL contributes to host antiviral responses via epigenetic modifications in IAV infection, which could provide important insights into the understanding of viral pathogenesis and host antiviral defense. Full article

The innate immune pathway serves as the first line of defense against viral infections and plays a crucial role in the host’s immune response in clearing viruses. Prior research has indicated that the influenza A virus has developed various strategies to avoid host immune responses. Nevertheless, the role of the NS1 protein of the canine influenza virus (CIV) in the innate immune pathway remains unclear. In this study, eukaryotic plasmids of NS1, NP, PA, PB1, and PB2 were constructed, and it was found that these proteins interact with melanoma differentiation-associated gene 5 (MDA5) and antagonize the activation of IFN-β promoters by MDA5. We selected the NS1 protein for further study and found that NS1 does not affect the interaction between the viral ribonucleoprotein (RNP) subunit and MDA5, but that it downregulates the expression of the laboratory of genetics and physiology 2 (LGP2) and retinoic acid-inducible gene-I (RIG-I) receptors in the RIG-I pathway. Additionally, NS1 was found to inhibit the expression of several antiviral proteins and cytokines, including MX dynamin like GTPase 1 (MX1), 2′-5′oligoadenylate synthetase (OAS), Signal Transducers and Activators of Transcription (STAT1), tripartite motif 25 (TRIM25), interleukin-2 (IL-2), IFN, IL-8, and IL-1β. To further investigate the role of NS1, a recombinant H3N2 virus strain (rH3N2) and an NS1-null virus (rH3N2ΔNS1) were rescued using reverse-genetic technology. The rH3N2ΔNS1 virus exhibited lower viral titers compared to rH3N2, but had a stronger activation effect on the receptors LGP2 and RIG-I. Furthermore, when compared to rH3N2, rH3N2ΔNS1 exhibited a more pronounced activation of antiviral proteins such as MX1, OAS, STAT1, and TRIM25, as well as antiviral cytokines such as IL-6, IFN-β, and IL-1β. These findings suggest a new mechanism by which NS1, a nonstructural protein of CIV, facilitates innate immune signaling and provides new avenues for the development of antiviral strategies. Full article

Mono-lactate glyceride (LG) is a short-chain fatty acid ester. It has been shown that short-chain fatty acid esters play an important role in maintaining intestinal structure and function. The aim of this study is to investigate the effects of mono-lactate glyceride on growth performance and intestinal morphology and function in weaned piglets. Sixteen 21-day-old weaned piglets of similar weight were distributed arbitrarily to two treatments: The control group (basal diet) and the LG group (basal diet + 0.6% mono-lactate glyceride). The experiment lasted for 21 days. On day 21 of the trial, piglets were weighed, and blood and intestinal samples were collected for further analysis. Results showed that dietary supplementation with 0.6% mono-lactate glyceride decreased (p < 0.05) the diarrhea rate and the contents of malondialdehyde and hydrogen peroxide in the ileum and jejunum and increased (p < 0.05) the expression of intestinal tight junction protein (Occludin) and the activities of superoxide dismutase and catalase in the ileum and colon. In addition, mono-lactate glyceride supplementation could enhance intestinal mucosal growth by increasing (p < 0.05) the mRNA levels of extracellular regulated protein kinases, promote intestinal mucosal water and nutrient transport and lipid metabolism by increasing (p < 0.05) the mRNA levels of b^0,+ amino acid transporter, aquaporin 3, aquaporin 10, gap junction protein alpha 1, intestinal fatty acid-binding protein, and lipoprotein lipase, enhance antiviral and immune function by increasing (p < 0.05) the mRNA levels of nuclear factor kappa-B, interferon-β, mucovirus resistance protein II, 2’-5’-oligoadenylate synthetase-like, interferon-γ, C-C motif chemokine ligand 2, and toll-like receptor 4, and enhance antioxidant capacity by increasing (p < 0.05) the mRNA levels of NF-E2-related factor 2 and glutathione S-transferase omega 2 and decreasing (p < 0.05) the mRNA level of NADPH oxidase 2. These results suggested that dietary supplementation with mono-lactate glyceride could decrease the diarrhea rate by improving intestinal antioxidant capacity, intestinal mucosal barrier, intestinal immune defense function, and intestinal mucosal water and nutrient transport. Collectively, dietary supplementation with 0.6% mono-lactate glyceride improved the intestinal function of weaned piglets. Full article

The importance of the IFN-induced oligoadenylate synthetase (OAS) proteins and the OAS/RNase L pathway in the innate response against viral pathogens is well-established, however the observed differences in anti-viral activity between the human OAS1 p46 and p42 isoforms are not fully understood. The protein expression of these isoforms is determined by the SNP rs10774671, either being an A or a G allele resulting in expression of either the p42 or the p46 isoform. Using fluorescence microscopy and immunoblot analysis of fractionated cell samples, we show here that the CaaX motif is of key importance to the cellular localization. The OAS1 p42 isoform is mainly located in the cytosol, whereas the p46 isoform with a C-terminal CaaX motif is translocated to membranous organelles, like the mitochondria. We furthermore observed differences between p42 and p46 in their effect on mitochondrial physiology using high resolution respirometry and fluorometry. Overexpression of OAS1 p42 and IFN-β treatment of HeLa cells (AA genotype) resulted in significantly increased respiration, which was not seen with p46 overexpression. The difference in subcellular localization and mitochondrial effect of these two OAS1 isoforms might help to explain the anti-viral mechanisms that differentiate these proteins. Full article

Porcine epidemic diarrhea virus (PEDV) is currently detected as the main pathogen causing severe diarrhea in pig farms. The phenotypic alterations induced by pathogenic infections are usually tightly linked with marked changes in epigenetic modification and gene expression. We performed global mapping of H3K4 trimethylation (H3K4me3) and transcriptomic analyses in the jejunum of PEDV-infected and healthy piglets using chromatin immunoprecipitation sequencing and RNA-seq techniques. A total of 1885 H3K4me3 peaks that are associated with 1723 genes were characterized. Moreover, 290 differentially expressed genes were identified, including 104 up-regulated and 186 down-regulated genes. Several antiviral genes including 2’-5’-oligoadenylate synthetase 1 (OAS1), 2’-5’-oligoadenylate synthetase 2 (OAS2), ephrin B2 (EFNB2), and CDC28 protein kinase regulatory subunit 1B (CKS1B) with higher H3K4me3 enrichment and expression levels in PEDV-infected samples suggested the potential roles of H3K4me3 deposition in promoting their expressions. Transcription factor annotation analysis highlighted the potential roles of two transcription factors interferon regulatory factor 8 (IRF8) and Kruppel like factor 4 (KLF4) in modulating the differential expression of genes involved in PEDV infection. The results provided novel insights into PEDV infection from the transcriptomic and epigenetic layers and revealed previously unknown and intriguing elements potentially involved in the host responses. Full article

Interferon (IFN)-induced 2′-5′-oligoadenylate synthetase (OAS) proteins exhibit an extensive and efficient antiviral effect against flavivirus infection in mammals and birds. Only the 2′-5′-oligoadenylate synthetase-like (OASL) gene has been identified thus far in birds, except for ostrich, which has both OAS1 and OASL genes. In this study, we first investigated the antiviral activity of goose OASL (goOASL) protein against a duck-origin Tembusu virus (DTMUV) in duck embryo fibroblast cells (DEFs). To investigate the relationship of conserved amino acids that are related to OAS enzyme activity and ubiquitin-like (UBL) domains with the antiviral activity of goOASL, a series of mutant goOASL plasmids was constructed, including goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T. Interestingly, all these mutant proteins significantly inhibited the replication of DTMUV in DEFs in a dose-dependent manner. Immunofluorescence analysis showed that the goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T proteins were located not only in the cytoplasm where DTMUV replicates but also in the nucleus of DEFs. However, the goOASL and goOASL mutant proteins were mainly colocalized with DTMUV in the cytoplasm of infected cells. Our data indicated that goOASL could significantly inhibit DTMUV replication in vitro, while the active-site residues S64, D76, D78 and D144, which were associated with OAS enzyme activity, the UBL domains were not required for the antiviral activity of goOASL protein. Full article

Interferons are a group of antiviral cytokines acting as the first line of defense in the antiviral immunity. Here, we describe the antiviral activity of goose type I interferon (IFNα) and type II interferon (IFNγ) against duck plague virus (DPV). Recombinant goose IFNα and IFNγ proteins of approximately 20 kDa and 18 kDa, respectively, were expressed. Following DPV-enhanced green fluorescent protein (EGFP) infection of duck embryo fibroblast cells (DEFs) with IFNα and IFNγ pre-treatment, the number of viral gene copies decreased more than 100-fold, with viral titers dropping approximately 100-fold. Compared to the control, DPV-EGFP cell positivity was decreased by goose IFNα and IFNγ at 36 hpi (3.89%; 0.79%) and 48 hpi (17.05%; 5.58%). In accordance with interferon-stimulated genes being the “workhorse” of IFN activity, the expression of duck myxovirus resistance (Mx) and oligoadenylate synthetases-like (OASL) was significantly upregulated (p < 0.001) by IFN treatment for 24 h. Interestingly, duck cells and goose cells showed a similar trend of increased ISG expression after goose IFNα and IFNγ pretreatment. Another interesting observation is that the positive feedback regulation of type I IFN and type II IFN by goose IFNα and IFNγ was confirmed in waterfowl for the first time. These results suggest that the antiviral activities of goose IFNα and IFNγ can likely be attributed to the potency with which downstream genes are induced by interferon. These findings will contribute to our understanding of the functional significance of the interferon antiviral system in aquatic birds and to the development of interferon-based prophylactic and therapeutic approaches against viral disease. Full article

The interferon (IFN)-regulated endoribonuclease RNase-L is involved in multiple aspects of the antimicrobial innate immune response. It is the terminal component of an RNA cleavage pathway in which dsRNA induces the production of RNase-L-activating 2-5A by the 2′-5′-oligoadenylate synthetase. The active nuclease then cleaves ssRNAs, both cellular and viral, leading to downregulation of their expression and the generation of small RNAs capable of activating retinoic acid-inducible gene-I (RIG-I)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome. This leads to IFNβ expression and IL-1β activation respectively, in addition to broader effects on immune cell function. RNase-L is also one of a growing number of innate immune components that interact with the cell cytoskeleton. It can bind to several cytoskeletal proteins, including filamin A, an actin-binding protein that collaborates with RNase-L to maintain the cellular barrier to viral entry. This antiviral activity is independent of catalytic function, a unique mechanism for RNase-L. We also describe here the interaction of RNase-L with the E3 ubiquitin ligase and scaffolding protein, ligand of nump protein X (LNX), a regulator of tight junction proteins. In order to better understand the significance and context of these novel binding partners in the antimicrobial response, other innate immune protein interactions with the cytoskeleton are also discussed. Full article