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Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp’s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation. IMPORTANCE The RNA replication of positive stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles (ROs). Double-membrane vesicles are a prominent type of viral ROs. They are induced by coronaviruses, such as SARS-CoV and MERS-CoV, as well as by a number of other important pathogens, yet little is known about their biogenesis. In this study, we explored the viral protein requirements for the formation of MERS-CoV- and SARS-CoV-induced DMVs and established that coexpression of two of the three transmembrane subunits of the coronavirus replicase polyprotein, nonstructural proteins (nsp’s) 3 and 4, is required and sufficient to induce DMV formation. Moreover, release of nsp3 and nsp4 from the polyprotein by proteolytic maturation is essential for this process. These findings provide a strong basis for further research on the biogenesis and functionality of coronavirus ROs and may point to more general principles of viral DMV formation. The RNA replication of positive stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles (ROs). Double-membrane vesicles are a prominent type of viral ROs. They are induced by coronaviruses, such as SARS-CoV and MERS-CoV, as well as by a number of other important pathogens, yet little is known about their biogenesis. In this study, we explored the viral protein requirements for the formation of MERS-CoV- and SARS-CoV-induced DMVs and established that coexpression of two of the three transmembrane subunits of the coronavirus replicase polyprotein, nonstructural proteins (nsp’s) 3 and 4, is required and sufficient to induce DMV formation. Moreover, release of nsp3 and nsp4 from the polyprotein by proteolytic maturation is essential for this process. These findings provide a strong basis for further research on the biogenesis and functionality of coronavirus ROs and may point to more general principles of viral DMV formation.

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons. How HIV reservoirs are shaped over time on antiviral therapy is poorly understood. Here, the authors analyze the dynamics of the HIV reservoir by longitudinal proviral sequencing revealing that HIV reservoir expression can contribute to its clearance and paradoxically even to its persistence.

Human metapneumovirus (HMPV) is one of the major causative agents of human respiratory tract infections. Monitoring of virus evolution could aid toward the development of new antiviral treatments or vaccine designs. Human metapneumovirus (HMPV) is one of the leading causes of respiratory illness (RI), primarily in infants. Worldwide, two genetic lineages (A and B) of HMPV are circulating that are antigenically distinct and can each be further divided into genetic sublineages. Surveillance combined with large-scale whole-genome sequencing studies of HMPV are scarce but would help to identify viral evolutionary dynamics. Here, we analyzed 130 whole HMPV genome sequences obtained from samples collected from individuals hospitalized with RI and partial fusion ( n  = 144) and attachment ( n  = 123) protein gene sequences obtained from samples collected from patients with RI visiting general practitioners between 2005 and 2021 in the Netherlands. Phylogenetic analyses demonstrated that HMPV continued to group in the four sublineages described in 2004 (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected in the Netherlands after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between samples obtained from patients with RI being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. In the past, different names for the newly energing lineages have been proposed, demonstrating the need for a consistent naming convention. Here, criteria are proposed for the designation of new genetic lineages to aid in moving toward a systematic HMPV classification. IMPORTANCE Human metapneumovirus (HMPV) is one of the major causative agents of human respiratory tract infections. Monitoring of virus evolution could aid toward the development of new antiviral treatments or vaccine designs. Here, we studied HMPV evolution between 2005 and 2021, with viruses obtained from samples collected from hospitalized individuals and patients with respiratory infections consulting general practitioners. Phylogenetic analyses demonstrated that HMPV continued to group in the four previously described sublineages (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between patients being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. These data were used to propose criteria for the designation of new genetic lineages to aid toward a systematic HMPV classification.

ObjectivesSystemic lupus erythematosus (SLE) is characterised by an activated interferon (IFN) system as well as by the formation of multiple autoantibodies (autoAbs). Interestingly, even autoAbs against IFN type I have been described to occur in SLE and are known to increase susceptibility to viral infections. Epstein-Barr Virus (EBV) causes chronic infection with strong evidence for an association with autoimmune diseases, and is known for increased reactivation rates in SLE. We hypothesize that EBV reactivation is linked to autoAbs against type I IFN.Methods378 patient plasma samples collected in the context of the Swiss SLE Cohort study (SSCS) were analysed for autoAbs against type I IFN subtypes alpha2, beta and omega using a bead-based Luminex assay, and their neutralisation activity as well as the level of type I-III IFN activation in a cell-based assay. EBV reactivation was assessed in anti-IFN autoAbs positive samples and matched controls using VCA-IgM antibodies ELISA and viral plasma DNA PCR.ResultsIFN activation levels were high in 105 (28%) of the SLE samples and correlated closely with disease activity as measured by SELENA-SLEDAI and physician’s global assessment (PGA) score. SLE patients showed a high rate of autoAbs against type I IFNs (anti-alpha2 n=40 (11%), anti-beta n=27 (7%), anti-omega n=31 (8%)). Only a minority of autoAbs was neutralising in the cell-based assay used. Subtypes of the type I IFN autoAbs related differentially to clinical manifestation.EBV reactivation was rare (n=7, 4%). While the majority of samples with EBV reactivation had anti-IFN autoAbs (5 out of 7) this did not reach statistical significance. Anti-IFN beta was the most prevalent autoAb (n=4).ConclusionsWe confirm high anti-type I IFN autoAbs rates in SLE patients and can observe subtype specific relations to disease activity. EBV reactivation was more common in samples with anti-IFN autoAbs, predominantly anti-IFN beta. However, the incidence of EBV reactivation was low resulting in insufficient statistical power. Future studies should be performed on larger cohorts. Furthermore, longitudinal studies will be important to explore the stability of anti-IFN autoAbs and the temporal relation of EBV reactivation, IFN activation and development of anti-IFN autoAbs.

Crassignatha danaugirangensis sp. n. (Araneae: Symphytognathidae) was discovered during a tropical ecology field course held at the Danau Girang Field Centre in Sabah, Malaysia. A taxonomic description and accompanying ecological study were completed as course activities. To assess the ecology of this species, which belongs to the ground-web-building spider community, three habitat types were surveyed: riparian forest, recently inundated riverine forest, and oil palm plantation. Crassignatha danaugirangensis sp. n. is the most abundant ground-web-building spider species in riparian forest; it is rare or absent from the recently inundated forest and was not found in a nearby oil palm plantation. The availability of this taxonomic description may help facilitate the accumulation of data about this species and the role of inundated riverine forest in shaping invertebrate communities.

Knowledge of food web interactions is essential for understanding the role of carnivores in an ecosystem and designing appropriate conservation and management strategies to preserve them. These interactions can only be understood by studying carnivores' diets and obtaining comprehensive and unbiased diet data. For large carnivores—which typically rely on large herbivores as prey—the role of smaller prey species has not received attention. This study aims to quantify the contribution of small (5–50 kg) and very small (<5 kg) prey taxa in the diet of lions (Panthera leo melanochaita) in four Kenyan National Parks (NPs). We use DNA metabarcoding to achieve higher‐resolution insights into prey composition, which is less biased toward large prey species compared to traditional methods, such as carcass counts. Our study identified 24 prey taxa in a total of 171 lion fecal samples. Small and very small prey taxa together contributed 18.7% out of 278 prey occurrences in all fecal samples, with comparable small prey presence (ranging from 8% to 15%) in the diet for each NP studied. This approach proved to be useful in detecting small and very small prey species in the diet of lions and can therefore be used in future research to uncover the diverse diet composition of these large carnivores. The consistent presence of smaller prey species in the diet indicates that lions generally supplement their large prey diet with smaller prey. Proportions of occurrences (%TX) of the four prey categories in the diet of the lion in four National Parks (NPs) in Kenya. Prey taxa identified and their contribution in percentages are given, and the diet contribution of medium‐to‐large, small, and very small taxa are depicted, as well as the presence of mesopredator (Mesop) taxa. Diet composition by prey taxon and prey category is presented as a proportion of occurrence (%TX). Total prey occurrences found were 278 in a total of 171 scat samples. The “Other” in the “Medium to Large” prey category consists of Giraffa (3%), Suidae (2%), Hippopotamus (0.4%), and Crocodylus (0.4%). In the “Medium to Large” category, the Equidae consisted of only one genus, Equus, whereas the Bovidae consisted of seven taxa, Syncerus (20%), Connochaetes (14%), Alcelaphus (6%), Bos (1%), Bovidae sp. (0.7%), Kobus (0.4%), and Tragelaphus (0.4%). Taxa abbreviations used: Aepyceros (Aepyc), Charadrius (Chara), Gallus (Gallu), Hystrix (Hystr), Ichneumia (Ichne), Lemniscomys (Lemni), Leptailurus (Lepta), Madoqua (Madoq), Nanger (Nange), and Phoenicopterus roseus (Phoen).

Interferons (IFNs) are a critical component of innate immune defenses and limit viral disease severity. To advance studies on IFNs and their neutralization by pathogenic autoantibodies, we generated a Renilla luciferase-based reporter cell line capable of detecting the activities of IFN-Is, IFN-II, and IFN-IIIs. The reporter cell line exhibits a 125-to 2000-fold higher sensitivity to IFNs than a commonly-used alternative biological reporter system, and allows for a rapid and simple live-cell workflow for detecting low titer amounts of neutralizing anti-IFN antibodies.

Spinal muscular atrophy (SMA) is a devastating motor neuron disease that predominantly affects children and represents the most common cause of hereditary infant mortality. The condition results from deleterious variants in SMN1, which lead to depletion of the survival motor neuron protein (SMN). Now, 20 years after the discovery of this genetic defect, a major milestone in SMA and motor neuron disease research has been reached with the approval of the first disease-modifying therapy for SMA by US and European authorities -- the antisense oligonucleotide nusinersen. At the same time, promising data from early-stage clinical trials of SMN1 gene therapy have indicated that additional therapeutic options are likely to emerge for patients with SMA in the near future. However, the approval of nusinersen has generated a number of immediate and substantial medical, ethical and financial implications that have the potential to resonate beyond the specific treatment of SMA. Here, we provide an overview of the rapidly evolving therapeutic landscape for SMA, highlighting current achievements and future opportunities. We also discuss how these developments are providing important lessons for the emerging second generation of combinatorial ('SMN-plus') therapies that are likely to be required to generate robust treatments that are effective across a patient's lifespan.

Clinical trials demonstrate that screening can reduce lung cancer mortality by over 20%. However, lung cancer screening effectiveness (reduction in lung cancer specific mortality) may vary by personal risk-factors. Here we evaluate heterogeneity in lung cancer screening effectiveness through traditional sub-group analyses, predictive modelling approaches and machine-learning in individual-level data from the Dutch-Belgian lung cancer screening trial (NELSON; 14,808 participants, 12,429 men, 2377 women, 2 persons with an unknown sex) and the National Lung Screening Trial (NLST; 53,405 participants, 31,501 men, 21,904 women). We find that screening effectiveness varies by pack-years (screening effectiveness ranges across trials: lowest groups = 26.8-50.9%, highest groups = 5.5-9.5%), smoking status (screening effectiveness ranges across trials: former smokers = 37.8-39.1%, current smokers = 16.1-22.7%) and sex (screening effectiveness ranges across trials: women = 24.6-25.3%; men = 8.3-24.9%). Furthermore, screening effectiveness varies by histology (screening effectiveness ranges across trials: adenocarcinoma = 17.8-23.0%, other lung cancers = 24.5-35.5%, small-cell carcinoma = 9.7%-11.3%). Screening is ineffective for squamous-cell carcinoma in NLST (screening effectiveness = 27.9% (95% confidence interval: 69.8% increase to 4.5% decrease) mortality increase) but effective in NELSON (screening effectiveness = 52.2% (95% confidence interval: 25.7-69.1% decrease) mortality reduction). We find that variations in screening effectiveness across pack-years, smoking status, and sex are primarily explained by a greater prevalence of histologies with favourable screening effectiveness in these groups. Our study shows that heterogeneity in lung screening effectiveness is primarily driven by histology and that relaxing smoking-related screening eligibility criteria may enhance screening effectiveness. Lung cancer screening effectiveness can be subject to variation based on personal risk factors. Here the authors report that heterogeneity in lung cancer screening effectiveness is primarily driven by histology.

US guidelines now recommend lung cancer screening with low-dose CT for high-risk individuals. Reports of new nodules after baseline screening have been scarce and are inconsistent because of differences in definitions used. We aimed to identify the occurrence of new solid nodules and their probability of being lung cancer at incidence screening rounds in the Dutch-Belgian Randomized Lung Cancer Screening Trial (NELSON). In the ongoing, multicentre, randomised controlled NELSON trial, between Dec 23, 2003, and July 6, 2006, 15 822 participants who had smoked at least 15 cigarettes a day for more than 25 years or ten cigarettes a day for more than 30 years and were current smokers, or had quit smoking less than 10 years ago, were enrolled and randomly assigned to receive either screening with low-dose CT (n=7915) or no screening (n=7907). From Jan 28, 2004, to Dec 18, 2006, 7557 individuals underwent baseline screening with low-dose CT; 7295 participants underwent second and third screening rounds. We included all participants with solid non-calcified nodules, registered by the NELSON radiologists as new or smaller than 15 mm3 (study detection limit) at previous screens. Nodule volume was generated semiautomatically by software. We calculated the maximum volume doubling time for nodules with an estimated percentage volume change of 25% or more, representing the minimum growth rate for the time since the previous scan. Lung cancer diagnosis was based on histology, and benignity was based on histology or stable size for at least 2 years. The NELSON trial is registered at trialregister.nl, number ISRCTN63545820. We analysed data for participants with at least one solid non-calcified nodule at the second or third screening round. In the two incidence screening rounds, the NELSON radiologists registered 1222 new solid nodules in 787 (11%) participants. A new solid nodule was lung cancer in 49 (6%) participants with new solid nodules and, in total, 50 lung cancers were found, representing 4% of all new solid nodules. 34 (68%) lung cancers were diagnosed at stage I. Nodule volume had a high discriminatory power (area under the receiver operating curve 0·795 [95% CI 0·728–0·862]; p<0·0001). Nodules smaller than 27 mm3 had a low probability of lung cancer (two [0·5%] of 417 nodules; lung cancer probability 0·5% [95% CI 0·0–1·9]), nodules with a volume of 27 mm3 up to 206 mm3 had an intermediate probability (17 [3·1%] of 542 nodules; lung cancer probability 3·1% [1·9–5·0]), and nodules of 206 mm3 or greater had a high probability (29 [16·9%] of 172 nodules; lung cancer probability 16·9% [12·0–23·2]). A volume cutoff value of 27 mm3 or greater had more than 95% sensitivity for lung cancer. Our study shows that new solid nodules are detected at each screening round in 5–7% of individuals who undergo screening for lung cancer with low-dose CT. These new nodules have a high probability of malignancy even at a small size. These findings should be considered in future screening guidelines, and new solid nodules should be followed up more aggressively than nodules detected at baseline screening. Zorgonderzoek Nederland Medische Wetenschappen and Koningin Wilhelmina Fonds Kankerbestrijding.