Recognition Of Viral Pathogens Research Articles

Viral piracy of host RNA phosphatase DUSP11 by avipoxviruses.

Proper recognition of viral pathogens is an essential part of the innate immune response. A common viral replicative intermediate and chemical signal that cells use to identify pathogens is the presence of a triphosphorylated 5' end (5'ppp) RNA, which activates the cytosolic RNA sensor RIG-I and initiates downstream antiviral signaling. While 5'pppRNA generated by viral RNA-dependent RNA polymerases (RdRps) can be a potent activator of the immune response, endogenous RNA polymerase III (RNAPIII) transcripts can retain the 5'pppRNA generated during transcription and induce a RIG-I-mediated immune response. We have previously shown that host RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) can act on both host and viral RNAs, altering their levels and reducing their ability to induce RIG-I activation. Our previous work explored how artificially altered DUSP11 can impact immune activation, prompting further exploration into natural contexts of altered DUSP11. Here, we have identified viral DUSP11 homologs (vDUSP11s) present in some avipoxviruses. Consistent with the known functions of endogenous DUSP11, we have shown that expression of vDUSP11s: 1) reduces levels of endogenous RNAPIII transcripts, 2) reduces a cell's sensitivity to 5'pppRNA-mediated immune activation, and 3) restores virus infection defects seen in the absence of DUSP11. Our results identify a virus-relevant context where DUSP11 activity has been co-opted to alter RNA metabolism and influence the outcome of infection.

Co-expression analysis suggests lncRNA-mRNA interactions enhance antiviral immune response during acute Chikungunya fever in whole blood of pediatric patients.

Chikungunya virus is an arbovirus that causes the neglected tropical disease chikungunya fever, common in tropical areas worldwide. There is evidence that arboviruses alter host transcriptome and modulate immune response; this modulation may involve transcriptional and post-transcriptional control mechanisms mediated by long non-coding RNAs (lncRNAs). Herein, we employed bioinformatic analysis to evaluate co-expression of lncRNAs and their putative target mRNAs in whole blood during natural Chikungunya infection in adolescent boys. Sequencing data from GSE99992 was uploaded to the Galaxy web server, where data was aligned with HISAT2, gene counts were estimated with HTSeq-count, and differential expression was run with DESeq2. After gene classification with Biomart, Pearson's correlation was applied to identify potential interactions between lncRNAs and mRNAs, which were later classified into cis and trans according to genomic location (FEELnc) and binding potential (LncTar), respectively. We identified 1,975 mRNAs and 793 lncRNAs that were differentially expressed between the acute and convalescent stages of infection in the blood. Of the co-expressed lncRNAs and mRNAs, 357 potentially interact in trans and 9 in cis; their target mRNAs enriched pathways related to immune response and viral infections. Out of 52 enriched KEGG pathways, the RIG-I like receptor signaling is enriched by the highest number of target mRNAs. This pathway starts with the recognition of viral pathogens, leading to innate immune response mediated by the production of IFN-I and inflammatory cytokines. Our findings indicate that alterations in lncRNA expression in adolescent boys, induced by acute Chikungunya infection, potentially modulate mRNAs that contribute to antiviral immune responses.

PSII-1 Beef steers divergent in average daily gain have differential expressions of immunity-related genes in whole blood

Abstract We evaluated the whole-blood immune-related gene expression profiles in beef steers divergent in average daily gain (ADG). Forty-eight healthy Angus crossbred beef steers (21 d post-weaning; 210 ± 12 kg of BW) from a single source were housed in individual slatted floor pens and were fed similar corn silage-based total mixed ration ad libitum. After 42 days of feeding, the steers were assigned into two groups with lowest ADG (LF: n = 8) and highest (HF: n = 8) ADG. The average daily DM intake of the steers in LF and HF were 6.08 kg ± 0.57 and 6.04 kg ± 0.42, respectively, and was similar between the two groups (P = 0.72). Whole blood samples were taken from both LF and HF steers and were immediately processed for RNA extraction. Messenger RNA expressions of 84 genes related to innate and adaptive immune responses were analyzed using the RT2 Profiler cow innate and adaptive immune responses PCR Array (PABT-052ZA; Qiagen). Immune genes with FC ≥ 1.2 or ≤ 0.83 having P-value ≤ 0.05 were considered to be differentially expressed. A total number of 11 immune genes were differentially expressed between HF and LF steers. The mRNA expressions of 10 immune genes (IRF3, TLR3, CCR4, MAPK3, TYK2, STAT3, STAT4, STAT6, CCR8, and GATA3) were upregulated in HF steers; these genes were involved in viral pathogen recognition and eradication, defense against intracellular and extracellular pathogens and parasites, and immune response homeostasis. A pro-inflammatory cytokine, IL-2, was upregulated in LF steers. These findings demonstrate that beef steers with divergent ADG had differential expressions of immune-related genes in the blood and future studies are needed to evaluate the mechanisms that cause differences in the expression of these immune genes and how these mechanisms can be employed to drive improved animal performance.

Human PSC-Derived Hepatocytes Express Low Levels of Viral Pathogen Recognition Receptors, but Are Capable of Mounting an Effective Innate Immune Response.

Hepatocytes are key players in the innate immune response to liver pathogens but are challenging to study because of inaccessibility and a short half-life. Recent advances in in vitro differentiation of hepatocyte-like cells (HLCs) facilitated studies of hepatocyte–pathogen interactions. Here, we aimed to define the anti-viral innate immune potential of human HLCs with a focus on pattern recognition receptor (PRR)-expression and the presence of a metabolic switch. We analysed cytoplasmic PRR and endosomal toll-like receptor (TLR)-expression, as well as activity and adaptation of HLCs to an inflammatory environment. We found that transcript levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA5), and TLR3 became downregulated during differentiation, indicating the acquisition of a more tolerogenic phenotype, as expected in healthy hepatocytes. HLCs responded to activation of RIG-I by producing interferons (IFNs) and IFN-stimulated genes. Despite low-level levels of TLR3, receptor expression was upregulated in an inflammatory environment. TLR3 signalling induced expression of proinflammatory cytokines at the gene level, indicating that several PRRs need to interact for successful innate immune activation. The inflammatory responsiveness of HLCs was accompanied by the downregulation of cytochrome P450 3A and 1A2 activity and decreased serum protein production, showing that the metabolic switch seen in primary hepatocytes during anti-viral responses is also present in HLCs.

Average daily gain divergence in beef steers is associated with altered plasma metabolome and whole blood immune-related gene expression.

We evaluated the plasma amine/phenol- and carbonyl-metabolome and whole-blood immune gene expression profiles in beef steers with divergent average daily gain (ADG). Forty-eight Angus crossbred beef steers (21 days postweaning; 210 ± 8.5 kg of body weight) were fed the same total mixed ration ad libitum for 42 days with free access to water. After 42 days of feeding, the steers were divided into two groups of lowest (LF: n = 8) and highest ADG (HF: n = 8). Blood samples were taken from all steers. The blood samples from LF and HF steers were used for further analysis. A subsample of the whole blood was immediately transferred into RNA-protect tubes for RNA extraction and messenger RNA expressions of 84 genes involved in innate and adaptive immune responses. Another subsample of the whole blood was immediately centrifuged to harvest the plasma for subsequent metabolome analysis. The average daily dry matter intake of the steers in LF and HF was 6.08 kg ± 0.57 and 6.04 kg ± 0.42, respectively, and was similar between the two groups (P = 0.72). The ADG (1.09 kg ± 0.13) of LF was lower (P = 0.01) than that of HF (1.63 kg ± 0.20). The expressions of 10 immune-related genes were upregulated (FC ≥ 1.2; P ≤ 0.05) in HF steers; these genes were involved in viral pathogen recognition and eradication, defense against intracellular and extracellular pathogens and parasites, and immune response homeostasis. A total number of 42 carbonyl-containing metabolites and 229 amine/phenol-containing metabolites were identified in the plasma samples of both groups. No alteration in carbonyl-metabolome was detected. Ten metabolites with immunomodulatory, anti-inflammatory, and reactive oxygen-scavenging properties were greater (FDR ≤ 0.05) in HF steers, whereas eight metabolites including arginine, phenylalanine, guanidoacetic acid, and aspartyl-threonine were greater in LF steers. This study demonstrated that beef steers with divergent ADG had altered plasma amine/phenol metabolome and immune-related gene expressions in the blood. Notably, plasma metabolites and immune-related genes of great health benefits were greater in steers with high ADG.

TLR9 Polymorphisms Might Contribute to the Ethnicity Bias for EBV-Infected Nasopharyngeal Carcinoma.

SummaryNasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world, but is endemic in some ethnic groups. The association of NPC with the Epstein-Barr virus (EBV) is firmly established; however, the mechanism is still unclear. TLR9 is well known for its essential role in viral pathogen recognition and activation of innate immunity. Here, we report a set of TLR9 polymorphisms in the TIR-2 domain of the TLR9 protein collected from the EBV-infected NPC samples from northeast Indian populations sharing the aforesaid ethnicity. The occurrence of mutations is significantly high in these samples as we found a p value of <0.0001 at a significance level of 0.05. These might play an important role for the lack of function of TLR9 and thus for the higher occurrence of EBV-mediated NPC in such ethnic groups.

An Undergraduate Research Project Utilizing CRISPR-Cas9 Gene Editing Technology to Study Gene Function in Arabidopsis thaliana.

The CRISPR-Cas9 system functions in microbial viral pathogen recognition pathways by identifying and targeting foreign DNA for degradation. Recently, biotechnological advances have allowed scientists to use CRISPR-Cas9-based elements as a molecular tool to selectively modify DNA in a wide variety of other living systems. Given the emerging need to bring engaging CRISPR-Cas9 laboratory experiences to an undergraduate audience, we incorporated a CRISPR-based research project into our Genetics class laboratories, emphasizing its use in plants. Our genetic manipulations were designed for Arabidopsis thaliana, which despite serving as a plant research model, has traditionally been difficult to use in a classroom setting. For this project, students transformed plasmid DNA containing the essential CRISPR-Cas9 gene editing elements into A. thaliana. Expression of these elements in the plant genome was expected to create a deletion at one of six targeted genes. The genes we chose had a known seedling and/or juvenile loss-of-function phenotype, which made genetic analysis by students with a limited background possible. It also allowed the project to reach completion in a typical undergraduate semester timeframe. Assessment efforts demonstrated several learning gains, including students’ understanding of CRISPR-Cas9 content, their ability to apply CRISPR-Cas9 gene editing tools using bioinformatics and genetics, their ability to employ elements of experimental design, and improved science communication skills. They also felt a stronger connection to their scientific education and were more likely to continue on a STEM career path. Overall, this project can be used to introduce CRISPR-Cas9 technology to undergraduates using plants in a single-semester laboratory course.

IFN-λ and microRNAs are important modulators of the pulmonary innate immune response against influenza A (H1N2) infection in pigs.

The innate immune system is paramount in the response to and clearance of influenza A virus (IAV) infection in non-immune individuals. Known factors include type I and III interferons and antiviral pathogen recognition receptors, and the cascades of antiviral and pro- and anti-inflammatory gene expression they induce. MicroRNAs (miRNAs) are increasingly recognized to participate in post-transcriptional modulation of these responses, but the temporal dynamics of how these players of the antiviral innate immune response collaborate to combat infection remain poorly characterized. We quantified the expression of miRNAs and protein coding genes in the lungs of pigs 1, 3, and 14 days after challenge with swine IAV (H1N2). Through RT-qPCR we observed a 400-fold relative increase in IFN-λ3 gene expression on day 1 after challenge, and a strong interferon-mediated antiviral response was observed on days 1 and 3 accompanied by up-regulation of genes related to the pro-inflammatory response and apoptosis. Using small RNA sequencing and qPCR validation we found 27 miRNAs that were differentially expressed after challenge, with the highest number of regulated miRNAs observed on day 3. In contrast, the number of protein coding genes found to be regulated due to IAV infection peaked on day 1. Pulmonary miRNAs may thus be aimed at fine-tuning the initial rapid inflammatory response after IAV infection. Specifically, we found five miRNAs (ssc-miR-15a, ssc-miR-18a, ssc-miR-21, ssc-miR-29b, and hsa-miR-590-3p)–four known porcine miRNAs and one novel porcine miRNA candidate–to be potential modulators of viral pathogen recognition and apoptosis. A total of 11 miRNAs remained differentially expressed 14 days after challenge, at which point the infection had cleared. In conclusion, the results suggested a role for miRNAs both during acute infection as well as later, with the potential to influence lung homeostasis and susceptibility to secondary infections in the lungs of pigs after IAV infection.

A comparative study of three akirin genes from big belly seahorse Hippocampus abdominalis: Molecular, transcriptional and functional characterization

Akirins, members of the NF-κB signaling pathway, are highly conserved nuclear proteins, which regulate gene expression in many physiological processes, including immunity, myogenesis, carcinogenesis, and embryogenesis. The akirin family in teleost fish consists of two to three genes. In the present study, three akirin genes from Hippocampus abdominalis were identified from a transcriptome database and designated as HaAkirin1, HaAkirin2(1), and HaAkirin2(2). The nuclear localization of HaAkirin1 and HaAkirin2(1) was confirmed by subcellular localization analysis. In contrast, diffused localization of HaAkirin2(2) was identified in the nucleus and cytoplasm that confirmed the aberrant nature of the nuclear localization signal. Phylogenetic analysis revealed a closer relationship of HaAkirins with other known teleost akirins. All three HaAkirin transcripts were ubiquitously expressed in all examined tissues with higher expression in ovary tissue. Immune challenge with LPS, poly I:C, and Streptococcus iniae exhibited a significant increase in the expression of all three HaAkirins in kidney and liver tissues. NF-κB luciferase assays revealed that relative luciferase activity was significantly higher for all three HaAkirin genes than mock controls. These results suggest that HaAkirin genes might play a role in regulating NF-κB dependent immune gene expression and their expression could be induced by bacterial and viral pathogen recognition molecular patterns.

PATTERN-RECOGNIZING RECEPTORS AND THE INNATE IMMUNE RESPONSE TO VIRAL INFECTION

The innate immune response to viral pathogens is crucial in mobilizing defensive reactions of an organism during the development of an acute viral infection. Cells of the innate immunity system detect viral antigens due to genetically programmed pattern-recognition receptors (PRRs), which are located either on the cell surface or inside the certain intracellular components. These image-recognizing receptors include Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RIG-I-like receptors), nucleotide oligomerization domain-like receptors (NOD-like receptors), also known as NACHT, LRR and PYD domains of the protein, and cytosolic DNA sensors. The trigger mechanisms for these receptors are viral proteins, and nucleic acids serve as activators. The presence of PRRs that are responsible for the determination of viral antigens in cellular components allows the cells of innate immunity to recognize a wide range of viral agents that replicate in various cellular structures, and develop an immune response to them. This article summarizes the disparate data presented in modern English literature on the role of PRRs and the associated signaling pathways. Understanding the recognition of viral pathogens required triggering a cascade of cytokine and interferon production provides insights into how viruses activate the signal paths of PRRs and the effect of the interaction of viral antigens and these receptors on the formation of the antiviral immune response.

The cytosolic sensors RIG-I and DAI can mediate glial inflammatory responses to neurotropic viruses (162.17)

Abstract The rapid onset of severe neuroinflammation is a defining feature of viral encephalitis. Glial cells are ideally positioned to respond to CNS pathogens and produce potent inflammatory mediators following exposure to neurotropic viruses. However, the mechanisms by which glia perceive viruses remain unclear. Recently the cytosolic proteins RIG-I and DAI have been reported to function as intracellular sensors of RNA and DNA viruses, respectively, but their role in virally-induced neuroinflammation has not been defined. We have shown that primary glial cells express such receptors constitutively and that their expression can be further induced following viral challenge. In the present study we demonstrate that resident CNS cells express these cytosolic pattern recognition receptors in situ and we show that their expression is elevated in murine models of viral encephalitis. Importantly, we have utilized specific ligands for RIG-I and DAI to demonstrate that these viral sensors can mediate the production of inflammatory/neurotoxic mediators by microglia and astrocytes. Furthermore, we have employed siRNA approaches to demonstrate the importance of RIG-I and DAI in such responses to the neurotropic viruses, VSV and HSV-1, respectively. Together, our studies indicate that these novel cytosolic viral sensors may play a critical role in the recognition of viral pathogens and the initiation of the potentially lethal neuroinflammation associated with acute viral CNS infection.

Critical Role of IRF-8 in Negative Regulation of TLR3 Expression by Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase-2 Activity in Human Myeloid Dendritic Cells

Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1- and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8-induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase-dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation.

Recognition of single-stranded RNA viruses by Toll-like receptor 7.

Viral infection of mammalian host results in the activation of innate immune responses. Toll-like receptors (TLRs) have been shown to mediate the recognition of many types of pathogens, including viruses. The genomes of viruses possess unique characteristics that are not found in mammalian genomes, such as high CpG content and double-stranded RNA. These genomic nucleic acids serve as molecular signatures associated with viral infections. Here we show that TLR7 recognizes the single-stranded RNA viruses, vesicular stomatitis virus and influenza virus. The recognition of these viruses by plasmacytoid dendritic cells and B cells through TLR7 results in their activation of costimulatory molecules and production of cytokines. Moreover, this recognition required intact endocytic pathways. Mice deficient in either the TLR7 or the TLR adaptor protein MyD88 demonstrated reduced responses to in vivo infection with vesicular stomatitis virus. These results demonstrate microbial ligand recognition by TLR7 and provide insights into the pathways used by the innate immune cells in the recognition of viral pathogens.

Novel Regulation of an MHC Class I Gene Response to Interferon-γ

The ability of IFN-γ, to increase the expression of MHC class I gene products is likely to enhance cytolytic T lymphocyte recognition of viral pathogens and tumor cells. The murine lymphoma AKR SL3-cl.F AZ R (SL3-cl.F) responds aberrantly to treatment with interferon-γ such that H-2D k surface expression is augmented, but H-2K k expression remains at constitutive levels. Somatic cell fusions have been used to demonstrate that the lesion responsible for this phenotype is cis -dominant, implicating a primary lesion within the SL3-cl.F H-2K k gene. In this communication, we have used PCR to analyze the nucleotide sequence in regions of the SL3-cl.F H-2K k promoter known to contain interferon-responsive enhancer elements. Comparison of the SL3-cl.F H-2K k sequences to known consensus elements revealed complete identity. In order to identify the lesion responsible for the SL3-cl.F phenotype, two H-2K k genomic clones were independently isolated from SL3-cl.F. Each clone exists as a 10.5-kbp EcoRI fragment containing the entire structural gene. The site of transcription initiation is at the center of this fragment; therefore, all regulatory elements within 5 kbp of the transcript start site which could alter steady-slate message levels are included. Interestingly, IFN-γ-augmented expression of the H-2K k gene was restored following DNA-mediated transfection of either of these clones into fibroblast cell lines and the parental cell line SL3-cl.F. Because isolation of these clones required passage of the DNA through a prokaryotic host, which alters the pattern of DNA methylation, there was the possibility that demethylation was responsible for the newly acquired IFN-γ-responsive phenotype. Treatment of SL3-cl.F with 5-azacytidine, which inhibits de novo methylation, did not restore IFN-γ-augmented expression, however, thus excluding H-2K k specific methylation as a potential mechanism. Collectively, these data demonstrate that the alteration responsible for the phenotype observed in SL3-cl.F does not involve known transcriptional regulatory elements. Potential mechanisms which might account for the mutant phenotype are discussed.