Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

Summary Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases, RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded (ds) RNAs with a triphosphorylated 5′ (5′ppp) terminus. However, identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5′ppp dsRNA “panhandle” structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and super-resolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection, and establish viral nucleocapsids with a 5′ppp dsRNA panhandle as a RIG-I activator.

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Summary The cytoplasmic RNA helicase RIG-I mediates innate sensing of RNA viruses. The genomes of influenza A virus (FLUAV) are encapsidated by the nucleoprotein and associated with RNA polymerase, posing potential barriers to RIG-I sensing. We show that RIG-I recognizes the 5’-triphosphorylated dsRNA on FLUAV nucleocapsids but that polymorphisms at position 627 of the viral polymerase subunit PB2 modulate RIG-I sensing. Compared to mammalian-adapted PB2-627K, avian FLUAV nucleocapsids possessing PB2-627E are prone to increased RIG-I recognition, and RIG-I-deficiency partially restores PB2-627E virus infection of mammalian cells. Heightened RIG-I sensing of PB2-627E nucleocapsids correlates with previously established lower affinity of 627E-containing PB2 for nucleoprotein and is increased by further nucleocapsid instability. The effect of RIG-I on PB2-627E nucleocapsids is independent of antiviral signaling, suggesting that RIG-I-nucleocapsid binding alone can inhibit infection. These results indicate that RIG-I is a direct avian FLUAV restriction factor and highlight nucleocapsid disruption as an antiviral strategy.

The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation.

Background: Traditionally, exercise programmes for improving functional performance and reducing falls are organised as structured sessions. An alternative approach of integrating functional exercises into everyday tasks has emerged in recent years. Objectives: Summarising the current evidence for the feasibility and effectiveness of interventions integrating functional exercise into daily life. Methods: A systematic literature search was conducted including articles based on the following criteria: (1) individuals ≥60 years; (2) intervention studies of randomised controlled trials (RCTs) and non-randomised studies (NRS); (3) using a lifestyle-integrated approach; (4) using functional exercises to improve strength, balance, or physical functioning; and (5) reporting outcomes on feasibility and/or effectiveness. Methodological quality of RCTs was evaluated using the PEDro scale. Results: Of 4,415 articles identified from 6 databases, 14 (6 RCTs) met the inclusion criteria. RCT quality was moderate to good. Intervention concepts included (1) the Lifestyle-integrated Functional Exercise (LiFE) programme integrating exercises into everyday activities and (2) combined programmes using integrated and structured training. Three RCTs evaluated LiFE in community dwellers and reported significantly improved balance, strength, and functional performance compared with controls receiving either no intervention, or low-intensity exercise, or structured exercise. Two of these RCTs reported a significant reduction in fall rate compared with controls receiving either no intervention or low-intensity exercise. Three RCTs compared combined programmes with usual care in institutionalised settings and reported improvements for some (balance, functional performance), but not all (strength, falls) outcomes. NRS showed behavioural change related to LiFE and feasibility in more impaired populations. One NRS comparing a combined home-based programme to a gym-based programme reported greater sustainability of effects in the combined programme. Conclusions: This review provides evidence for the effectiveness of integrated training for improving motor performances in older adults. Single studies suggest advantages of integrated compared with structured training. Combined programmes are positively evaluated in institutionalised settings, while little evidence exists in other populations. In summary, the approach of integrating functional exercise into daily life represents a promising alternative or complement to structured exercise programmes. However, more RCTs are needed to evaluate this concept in different target populations and the potential for inducing behavioural change.

<b><i>Background:</i></b> The Lifestyle-integrated Functional Exercise (LiFE) program is an intervention integrating balance and strength activities into daily life, effective at reducing falls in at-risk people ≥70 years. There is potential for LiFE to be adapted to young seniors in order to prevent age-related functional decline. <b><i>Objective:</i></b> We aimed to (1) develop an intervention by adapting Lifestyle-integrated Functional Exercise (aLiFE) to be more challenging and suitable for preventing functional decline in young seniors in their 60s and (2) perform an initial feasibility evaluation of the program. Pre-post changes in balance, mobility, and physical activity (PA) were also explored. <b><i>Methods:</i></b> Based on a conceptual framework, a multidisciplinary expert group developed an initial aLiFE version, including activities for improving strength, neuromotor performances, and PA. Proof-of-concept was evaluated in a 4-week pre-post intervention study measuring (1) feasibility including adherence, frequency of practice, adverse events, acceptability (i.e., perceived helpfulness, adaptability, level of difficulty of single activities), and safety, and (2) changes in balance/mobility (Community Balance and Mobility Scale) and PA (1 week activity monitoring). The program was refined based on the study results. <b><i>Results:</i></b> To test the initial aLiFE version, 31 young seniors were enrolled and 30 completed the study (mean age 66.4 ± 2.7 years, 60% women). Of a maximum possible 16 activities, participants implemented on average 12.1 ± 1.8 activities during the intervention, corresponding to mean adherence of 76%. Implemented activities were practiced 3.6–6.1 days/week and 1.8–7.8 times/day, depending on the activity type. One noninjurious fall occurred during practice, although the participant continued the intervention. The majority found the activities helpful, adaptable to individual lifestyle, appropriately difficult, and safe. CMBS score increased with medium effect size (d = 0.72, <i>p</i> = 0.001). Increase in daily walking time (d = 0.36) and decrease in sedentary time (d = –0.10) were nonsignificant. Refinements included further increasing the task challenge of some strength activities and defining the most preferred activities in the trainer’s manual to facilitate uptake of the program. <b><i>Conclusion:</i></b> aLiFE has the potential to engage young seniors in regular lifestyle-integrated activities. Effectiveness needs to be evaluated in a randomized controlled trial.

Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor– and IFN regulatory factor (Irf)9–dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow–derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.