HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells

Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear. One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6, can mediate ISGylation. Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15 conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over expression enhanced the IFNβ promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells, suggesting that HerC6-/-mice may provide a feasible model to study the role of human HerC5 in antiviral responses

The impact of emotional well-being on long-term recovery and survival in physical illness: a meta-analysis

This meta-analysis synthesized studies on emotional well-being as predictor of the prognosis of physical illness, while in addition evaluating the impact of putative moderators, namely constructs of well-being, health-related outcome, year of publication, follow-up time and methodological quality of the included studies. The search in reference lists and electronic databases (Medline and PsycInfo) identified 17 eligible studies examining the impact of general well-being, positive affect and life satisfaction on recovery and survival in physically ill patients. Meta-analytically combining these studies revealed a Likelihood Ratio of 1.14, indicating a small but significant effect. Higher levels of emotional well-being are beneficial for recovery and survival in physically ill patients. The findings show that emotional well-being predicts long-term prognosis of physical illness. This suggests that enhancement of emotional well-being may improve the prognosis of physical illness, which should be investigated by future research

Antithrombotic therapy and survival in patients with malignant disease

A broad range of studies suggest a two-way relationship between cancer and venous thromboembolism (VTE). Patients with cancer have consistently been shown to be at elevated risk for VTE; this risk is partly driven by an intrinsic hypercoagulable state elicited by the tumour itself. Conversely, thromboembolic events in patients without obvious risk factors are often the first clinical manifestation of an undiagnosed malignancy. The relationship between VTE and cancer is further supported by a number of trials and meta-analyses which, when taken together, strongly suggest that antithrombotic therapy can extend survival in patients with cancer by a mechanism that extends beyond its effect in preventing VTE. Moreover, accumulating evidence from in vitro and in vivo studies has shown that tumour growth, invasion, and metastasis are governed, in part, by elements of the coagulation system. On 22 May 2009, a group of health-care providers based in the United Kingdom met in London, England, to examine recent advances in cancer-associated thrombosis and its implications for UK clinical practice. As part of the discussion, attendees evaluated evidence for and against an effect of antithrombotic therapy on survival in cancer. This paper includes a summary of the data presented at the meeting and explores potential mechanisms by which antithrombotic agents might exert antitumour effects. The summary is followed by a consensus statement developed by the group

External quality assessment on the use of malaria rapid diagnostic tests in a non-endemic setting

<p>Abstract</p> <p>Background</p> <p>Malaria rapid diagnostic tests (RDTs) are increasingly used as a tool for the diagnosis of malaria, both in endemic and in non-endemic settings. The present study reports the results of an external quality assessment (EQA) session on RDTs in a non-endemic setting.</p> <p>Methods</p> <p>After validation of antigen stability during shipment at room temperature, three clinical samples and a questionnaire were sent to clinical laboratories in Belgium and the Grand Duchy of Luxembourg using malaria RDTs. Participants were asked to report the results of the RDTs as observations (visibility of the RDT control and test lines) and interpretations (report as formulated to the clinician). In addition, participants were invited to fill in a questionnaire on the place of RDTs in the diagnostic strategy of malaria.</p> <p>Results</p> <p>A total of 128/133 (96.2%) of clinical laboratories using RDTs participated. Six three-band and one four-band RDT brands were used. Analytical errors were rare and included (i) not recognizing invalid RDT results (1.6%) and (ii) missing the diagnosis of <it>Plasmodium falciparum </it>(0.8%). Minor errors were related to RDT test result interpretation and included (i) reporting "RDT positive" without species identification in the case of <it>P. falciparum </it>and non-<it>falciparum </it>species (16.9% and 6.5% respectively) and (ii) adding incorrect comments to the report (3.2%). Some of these errors were related to incorrect RDT package insert instructions such as (i) not reporting the possibility of mixed species infection in the case of <it>P. falciparum </it>and <it>Plasmodium vivax </it>(35.5% and 18.5% respectively) and (ii) the interpretation of <it>P. vivax </it>instead of non-falciparum species at the presence of a pan-species antigen line (4.0%). According to the questionnaire, 48.8% of participants processed ≤20 requests for malaria diagnosis in 2009. During opening hours, 93.6% of 125 participants used RDTs as an adjunct to microscopy but outside opening hours, nearly one third of 113 participants relied on RDTs as the primary (4.4%) or the single tool (25.7%) for malaria diagnosis.</p> <p>Conclusion</p> <p>In this non-endemic setting, errors in RDT performance were mainly related to RDT test line interpretations, partly due to incorrect package insert instructions. The reliance on RDTs as the primary or the single tool for the diagnosis of malaria outside opening hours is of concern and should be avoided.</p

ISG15 Modulates Development of the Erythroid Lineage

Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15-/- bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation

Inhibition of Interferon Induction and Action by the Nairovirus Nairobi Sheep Disease Virus/Ganjam Virus

The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU

Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle

MERS-CoV 4b protein interferes with the NF-κB-dependent innate immune response during infection

This work is licensed under a Creative Commons Attribution 4.0 International License.Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that emerged in 2012, causing severe pneumonia and acute respiratory distress syndrome (ARDS), with a case fatality rate of ~36%. When expressed in isolation, CoV accessory proteins have been shown to interfere with innate antiviral signaling pathways. However, there is limited information on the specific contribution of MERS-CoV accessory protein 4b to the repression of the innate antiviral response in the context of infection. We found that MERS-CoV 4b was required to prevent a robust NF-κB dependent response during infection. In wild-type virus infected cells, 4b localized to the nucleus, while NF-κB was retained in the cytoplasm. In contrast, in the absence of 4b or in the presence of cytoplasmic 4b mutants lacking a nuclear localization signal (NLS), NF-κB was translocated to the nucleus leading to the expression of pro-inflammatory cytokines. This indicates that NF-κB repression required the nuclear import of 4b mediated by a specific NLS. Interestingly, we also found that both in isolation and during infection, 4b interacted with α-karyopherin proteins in an NLS-dependent manner. In particular, 4b had a strong preference for binding karyopherin-α4 (KPNA4), which is known to translocate the NF-κB protein complex into the nucleus. Binding of 4b to KPNA4 during infection inhibited its interaction with NF-κB-p65 subunit. Thereby we propose a model where 4b outcompetes NF-κB for KPNA4 binding and translocation into the nucleus as a mechanism of interference with the NF-κB-mediated innate immune response

The Nitric Oxide Pathway Provides Innate Antiviral Protection in Conjunction with the Type I Interferon Pathway in Fibroblasts

The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection