Efficacy of pulmonary telerehabilitation on exercise tolerance, fatigue, perceived exertion, depression, and quality of life in COVID-19 survivors / Eficácia da telereabilitação pulmonar na tolerância ao exercício, fadiga, percepção de esforço, depressão e qualidade de vida em sobreviventes de COVID-19

INTRODUCTION: Telerehabilitation advanced significantly with the emergence of COVID-19 and the recommendation of limiting physiotherapist-patient contact time whenever practicable. The effectiveness of telerehabilitation on those who had a longer stay in hospital and on oxygen support following discharge is still under question. OBJECTIVES: To evaluate the effects after six weeks of pulmonary telerehabilitation on exercise tolerance, fatigue level, perceived exertion, symptoms of depression and quality of life in patients surviving COVID-19. MATERIALS AND METHODS: A quasi-experimental study was conducted on 25 post-COVID-19 patients following discharge in a home environment setting. The participants were advised to prepare equipment such as oxygen concentrator, B-type oxygen cylinder (backup), lengthy oxygen tubes, finger pulse oximeter, mini static pedal exerciser, incentive spirometry, weight cuffs or water bottles and sandbags. After six weeks of telerehabilitation, the patients underwent assessments including initial oxygen saturation (SPO2), heart rate, peak oxygen demand during exercise to maintain baseline SPO2, peak heart rate, maximum drop in SPO2, recovery time to baseline SPO2 measured with a pulse oximeter and stopwatch, peak perceived exertion using the Borg Dyspnea Scale, peak fatigue score using the visual analog scale (VASF), quality of life assessed with the SF-36 questionnaire, and mental health status evaluated with the Hamilton Depression Scale (HAMD). One-way repeated measure ANOVA and paired t-test were used. RESULTS: Significant improvements following the intervention on the initial SPO2 (F (2.12, 23.13) = 21.0, p< 0.05) and quality of life also showed significant improvement (mean difference =29.92, p< 0.05). CONCLUSION: Six weeks of comprehensive pulmonary telerehabilitation with simple equipment improved tolerance to exercise, fatigue, perceived exertion, symptoms of depression and quality of life for post-COVID-19 patients.

INTRODUÇÃO: A telereabilitação avançou significativamente com o surgimento da COVID-19 e a recomendação de limitar o tempo de contato entre fisioterapeutas e pacientes sempre que possível. A eficácia da telereabilitação em pacientes que permaneceram mais tempo no hospital e necessitaram de suporte de oxigênio após a alta ainda está em questão. OBJETIVO: Para avaliar os efeitos após seis semanas de telereabilitação pulmonar na tolerância ao exercício, nível de fadiga, percepção de esforço, sintomas de depressão e qualidade de vida em pacientes sobreviventes de COVID-19. MATERIAIS E MÉTODOS: Foi realizado um estudo quase-experimental com dezesseis pacientes pós-COVID-19 selecionados de um hospital multispecializado. Os participantes foram orientados a preparar equipamentos como concentrador de oxigênio, cilindro de oxigênio tipo B (reserva), tubos de oxigênio longos, oxímetro de pulso, exercitador de pedal estático, espirômetro incentivador, pesos de tornozelo ou garrafas d'água e sacos de areia. Após seis semanas de telereabilitação, os pacientes foram submetidos a avaliações, incluindo saturação inicial de oxigênio (SPO2), frequência cardíaca, demanda máxima de oxigênio durante o exercício para manter a SPO2 basal, frequência cardíaca máxima, queda máxima na SPO2, tempo de recuperação para SPO2 basal medido com um oxímetro de pulso e cronômetro, esforço percebido máximo usando a Escala de Dispneia de Borg, pontuação máxima de fadiga usando a escala analógica visual (VASF), qualidade de vida avaliada com o questionário SF-36 e estado de saúde mental avaliado com a Escala de Depressão de Hamilton (HAMD). RESULTADOS: Melhorias significativas após a intervenção foram observadas na SPO2 inicial (F (2,12, 23,13) = 21,0, p <0,05) e na frequência cardíaca (F (1,839, 20,23) = 43,73, p <0,05), demanda máxima de oxigênio durante o exercício para manter a SPO2 basal (F (1,487, 16,36) = 8,96, p <0,05), esforço percebido máximo (F (5, 55) = 112,51, p <0,05), pontuação máxima de fadiga (F (1,755, 19,30) = 67,44, p <0,05), frequência cardíaca máxima (F (1,798, 19,78) = 50,99, p <0,05), queda máxima na SPO2 (F (2,467, 27,14) = 41,46, p <0,05) e tempo máximo de recuperação para alcançar a SPO2 basal (F (5, 55) = 78,89, p <0,05). A análise de seis semanas pós-intervenção nos sintomas depressivos (diferença média = 11,25, p <0,05) e na qualidade de vida também mostrou melhoria significativa (diferença média = 29,92, p <0,05). CONCLUSÃO: Seis semanas de telereabilitação pulmonar abrangente com equipamentos simples melhoraram a tolerância ao exercício, a fadiga, a percepção de esforço, os sintomas de depressão e a qualidade de vida em pacientes pós-COVID-19.

Ebola Virus NP Binding to Host Protein Phosphatase-1 Regulates Capsid Formation.

The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. Our results point to a new role for PP1 in EBOV replication in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.

Ebolavirus Species-Specific Interferon Antagonism Mediated by VP24.

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-ß), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms.

Clinical outcomes of bronchiectasis in India: data from the EMBARC/Respiratory Research Network of India registry.

BACKGROUND: Identifying risk factors for poor outcomes can help with risk stratification and targeting of treatment. Risk factors for mortality and exacerbations have been identified in bronchiectasis but have been almost exclusively studied in European and North American populations. This study investigated the risk factors for poor outcome in a large population of bronchiectasis patients enrolled in India. METHODS: The European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India (EMBARC-India) registry is a prospective observational study of adults with computed tomography-confirmed bronchiectasis enrolled at 31 sites across India. Baseline characteristics of patients were used to investigate associations with key clinical outcomes: mortality, severe exacerbations requiring hospital admission, overall exacerbation frequency and decline in forced expiratory volume in 1 s. RESULTS: 1018 patients with at least 12-month follow-up data were enrolled in the follow-up study. Frequent exacerbations (≥3 per year) at baseline were associated with an increased risk of mortality (hazard ratio (HR) 3.23, 95% CI 1.39-7.50), severe exacerbations (HR 2.71, 95% CI 1.92-3.83), future exacerbations (incidence rate ratio (IRR) 3.08, 95% CI 2.36-4.01) and lung function decline. Coexisting COPD, dyspnoea and current cigarette smoking were similarly associated with a worse outcome across all end-points studied. Additional predictors of mortality and severe exacerbations were increasing age and cardiovascular comorbidity. Infection with Gram-negative pathogens (predominantly Klebsiella pneumoniae) was independently associated with increased mortality (HR 3.13, 95% CI 1.62-6.06), while Pseudomonas aeruginosa infection was associated with severe exacerbations (HR 1.41, 95% CI 1.01-1.97) and overall exacerbation rate (IRR 1.47, 95% CI 1.13-1.91). CONCLUSIONS: This study identifies risk factors for morbidity and mortality among bronchiectasis patients in India. Identification of these risk factors may support treatment approaches optimised to an Asian setting.

A single intranasal dose of human parainfluenza virus type 3-vectored vaccine induces effective antibody and memory T cell response in the lungs and protects hamsters against SARS-CoV-2.

Respiratory tract vaccination has an advantage of needle-free delivery and induction of mucosal immune response in the portal of SARS-CoV-2 entry. We utilized human parainfluenza virus type 3 vector to generate constructs expressing the full spike (S) protein of SARS-CoV-2, its S1 subunit, or the receptor-binding domain, and tested them in hamsters as single-dose intranasal vaccines. The construct bearing full-length S induced high titers of neutralizing antibodies specific to S protein domains critical to the protein functions. Robust memory T cell responses in the lungs were also induced, which represent an additional barrier to infection and should be less sensitive than the antibody responses to mutations present in SARS-CoV-2 variants. Following SARS-CoV-2 challenge, animals were protected from the disease and detectable viral replication. Vaccination prevented induction of gene pathways associated with inflammation. These results indicate advantages of respiratory vaccination against COVID-19 and inform the design of mucosal SARS-CoV-2 vaccines.

Attenuated activation of pulmonary immune cells in mRNA-1273-vaccinated hamsters after SARS-CoV-2 infection.

The mRNA-1273 vaccine is effective against SARS-CoV-2 and was granted emergency use authorization by the FDA. Clinical studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibits severe SARS-CoV-2 disease similar to that in hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and nonhuman primates, low-level virus replication in mRNA-1273-vaccinated hamsters coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high-resolution analysis that is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a 2-dose schedule and provide insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

Cutting Edge: Distinct B Cell Repertoires Characterize Patients with Mild and Severe COVID-19.

Protective immunity against COVID-19 likely depends on the production of SARS-CoV-2-specific plasma cells and memory B cells postinfection or postvaccination. Previous work has found that germinal center reactions are disrupted in severe COVID-19. This may adversely affect long-term immunity against reinfection. Consistent with an extrafollicular B cell response, patients with severe COVID-19 have elevated frequencies of clonally expanded, class-switched, unmutated plasmablasts. However, it is unclear whether B cell populations in individuals with mild COVID-19 are similarly skewed. In this study, we use single-cell RNA sequencing of B cells to show that in contrast to patients with severe COVID-19, subjects with mildly symptomatic COVID-19 have B cell repertoires enriched for clonally diverse, somatically hypermutated memory B cells ∼30 d after the onset of symptoms. This provides evidence that B cell responses are less disrupted in mild COVID-19 and result in the production of memory B cells.

mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge.

The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

Effects of Overexpression of the Egyptian Fruit Bat Innate Immune Genes on Filovirus Infections in the Host Cells.

Bats constitute a large and diverse group of mammals with unique characteristics. One of these is the ability of bats to maintain various pathogens, particularly viruses, without evidence of disease. The innate immune system has been implicated as one of the important components involved in this process. However, in contrast to the human innate immune system, little data is available for bats. In the present study we generated 23 fusion constructs of innate immune genes of Egyptian fruit bat (Rousettus aegyptiacus) with mCherry as a fluorescent reporter. We evaluated the effects of overexpressing these genes on the replication of Marburg and Ebola viruses in the Egyptian fruit bat cell line R06EJ. Both viruses were substantially inhibited by overexpression of type I, II and III interferons, as well as by DDX58 (RIG-I), IFIH1, and IRF1. Our observations suggest that the broad antiviral activity of these genes reported previously in human cells is conserved in Egyptian fruit bats and these possess anti-filovirus activities that may contribute to the efficient virus clearance.

Miliary Sarcoidosis: does it exist? A case series and systematic review of literature.

BACKGROUND AND OBJECTIVES: Sarcoidosis typically presents with peribronchovascular and perilymphatic nodules on high-resolution computed tomography (HRCT); a miliary pattern is reported but not well described. DESIGN SETTING: We describe four patients with miliary sarcoidosis and results of a systematic review of all previously reported cases from 1985 onwards. RESULTS: We identified only 27 cases of "miliary" sarcoidosis in the HRCT era. These patients were older (85.2% older than 40 years), had more co-morbidities (72.7%) and were symptomatic compared to "typical" sarcoidosis. Respiratory symptoms were present in 61.9% at diagnosis. Hypercalcemia was seen in 28.5%. On review of HRCT images, only 34.6% (9/26) had a "true miliary" pattern without fissural nodules. In our series, prominent perivascular granulomas were seen on histopathology in all. 44.4% (12/27) had tuberculosis preceding or concurrent to miliary sarcoidosis. Of the eight true associations, tuberculosis preceded sarcoidosis by 52 (median, IQR 36) weeks in six and occurred concurrently in another two. The diagnosis of tuberculosis was clinical in all with concurrent diagnosis of tuberculosis and sarcoidosis. Treatment with steroids had 100% response and 14.2% relapse. CONCLUSIONS: A true miliary pattern in the HRCT era is very rare in sarcoidosis and subtle perilymphatic pattern is nearly always seen; this should be labeled "pseudo-miliary". Prominent perivascular granulomas are associated with true miliary pattern. Miliary sarcoidosis patients are older and symptomatic, needing treatment at diagnosis. "Miliary" sarcoidosis may follow treatment for tuberculosis; concurrent cases possibly indicate the difficulty in differentiating both or a "tuberculo-sarcoid" presentation. (Sarcoidosis Vasc Diffuse Lung Dis 2020; 37 (1): 53-65).

Global phosphoproteomic analysis of Ebola virions reveals a novel role for VP35 phosphorylation-dependent regulation of genome transcription.

Ebola virus (EBOV) causes severe human disease with a high case fatality rate. The balance of evidence implies that the virus circulates in bats. The molecular basis for host-viral interactions, including the role for phosphorylation during infections, is largely undescribed. To address this, and to better understand the biology of EBOV, the phosphorylation of EBOV proteins was analyzed in virions purified from infected monkey Vero-E6 cells and bat EpoNi/22.1 cells using high-resolution mass spectrometry. All EBOV structural proteins were detected with high coverage, along with phosphopeptides. Phosphorylation sites were identified in all viral structural proteins. Comparison of EBOV protein phosphorylation in monkey and bat cells showed only partial overlap of phosphorylation sites, with shared sites found in NP, VP35, and VP24 proteins, and no common sites in the other proteins. Three-dimensional structural models were built for NP, VP35, VP40, GP, VP30 and VP24 proteins using available crystal structures or by de novo structure prediction to elucidate the potential role of the phosphorylation sites. Phosphorylation of one of the identified sites in VP35, Thr-210, was demonstrated to govern the transcriptional activity of the EBOV polymerase complex. Thr-210 phosphorylation was also shown to be important for VP35 interaction with NP. This is the first study to compare phosphorylation of all EBOV virion proteins produced in primate versus bat cells, and to demonstrate the role of VP35 phosphorylation in the viral life cycle. The results uncover a novel mechanism of EBOV transcription and identify novel targets for antiviral drug development.

Pulmonary Nocardiosis Presenting as Exacerbation of Chronic Pulmonary Disease.

BACKGROUND: Pulmonary nocardiosis (PN) occurs in chronic pulmonary disease (CPD) in the absence of traditional risk factors. Clinical features that differentiate bacterial exacerbations (AE-CPDb) from PN-related exacerbations (AE-CPDPN) are not well described. OBJECTIVES: To describe a series of AE-CPDPN without traditional risk factors and compare clinical features, radiology and outcomes with age, gender and CLD-type matched AE-CPDb. MATERIALS AND METHODS: Single-center retrospective review and case-control study. RESULTS: AE-CPDPN had longer duration of symptoms and more leukocytosis at hospitalization. AE-CPDb patients were sicker with more chronic respiratory failure (OR 33.3, p = 0.01), cardiac disease and pulmonary hypertension (OR 6.2, p = 0.008) at diagnosis. More patients with AE-CPDb were discharged on domiciliary oxygen (OR 5.27, p = 0.01). On logistic regression, AE-CPDPN was independently associated with mechanical ventilation (OR 22.3, p = 0.01), length of hospital stay (median difference, 4 days, p = 0.016) but not to hospital mortality. 22.7% of AE-CPDPN died. Respiratory failure requiring oxygen, NIPPV or mechanical ventilation was associated with mortality in AE-CPDPN. CONCLUSION: PN is a rare cause of AE-CPD and can be suspected by longer symptom duration, more leukocytosis, consolidation and cavitation. AE-CPDPN is associated with longer hospital stay and mechanical ventilation. Respiratory failure is associated with mortality in AE-CPDPN. KEY MESSAGES: Pulmonary nocardiosis can present in advanced chronic lung disease as an exacerbation in the absence of traditional risk factors like immunosuppression.Bronchiectasis, followed by chronic obstructive pulmonary disease are the most common chronic lung disease risk factors.Pulmonary nocardiosis is a rare cause of acute exacerbation of chronic pulmonary disease (CPD).Compared to exacerbations of CPD due to bacterial infections, nocardiosis-related exacerbations (CPDPN) were independently related to need for mechanical ventilation and length of hospital stay.Respiratory failure requiring oxygen, noninvasive ventilation and mechanical ventilation are associated with mortality in AE-CPDPN. HOW TO CITE THIS ARTICLE: Kancherla R, Ramanathan RM PL, Appalaraju B, Srinivas R. Pulmonary Nocardiosis Presenting as Exacerbation of Chronic Pulmonary Disease. Indian J Crit Care Med 2019;23(10):467-474.

Mechanisms of African swine fever virus pathogenesis and immune evasion inferred from gene expression changes in infected swine macrophages.

African swine fever (ASF) is a swine disease caused by a large, structurally complex, double-stranded DNA virus, African swine fever virus (ASFV). In domestic pigs, acute infection by highly virulent ASF viruses causes hemorrhagic fever and death. Previous work has suggested that ASFV pathogenesis is primarily mediated by host cytokines produced by infected monocytes and macrophages. To better understand molecular mechanisms mediating virus pathogenesis and immune evasion, we used transcriptome analysis to identify gene expression changes after ASFV infection in ex vivo swine macrophages. Our results suggest that the cytokines of TNF family including FASLG, LTA, LTB, TNF, TNFSF4, TNFSF10, TNFSF13B and TNFSF18 are the major causative cytokine factors in ASF pathogenesis via inducing apoptosis. Other up-regulated proinflammatory cytokines (IL17F and interferons) and down-regulated anti-inflammatory cytokine (IL10) may also significantly contribute to ASF pathogenesis and cause excessive tissue inflammatory responses. The differential expression of genes also indicates that ASFV could evade both the innate and adaptive immune responses by (i) inhibiting MHC Class II antigen processing and presentation, (ii) avoiding CD8+ T effector cells and neutrophil extracellular traps via decreasing expression of neutrophil/CD8+ T effector cell-recruiting chemokines, (iii) suppressing M1 activation of macrophages, (iv) inducing immune suppressive cytokines, and (v) inhibiting the processes of macrophage autophagy and apoptosis. These results provide novel information to further investigate and better understand the mechanism of pathogenesis and immune evasion of this devastating swine disease.

Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection.

Ebola virus (EBOV) infections are characterized by a pronounced lymphopenia that is highly correlative with fatalities. However, the mechanisms leading to T-cell depletion remain largely unknown. Here, we demonstrate that both viral mRNAs and antigens are detectable in CD4+ T cells despite the absence of productive infection. A protein phosphatase 1 inhibitor, 1E7-03, and siRNA-mediated suppression of viral antigens were used to demonstrate de novo synthesis of viral RNAs and antigens in CD4+ T cells, respectively. Cell-to-cell fusion of permissive Huh7 cells with non-permissive Jurkat T cells impaired productive EBOV infection suggesting the presence of a cellular restriction factor. We determined that viral transcription is partially impaired in the fusion T cells. Lastly, we demonstrate that exposure of T cells to EBOV resulted in autophagy through activation of ER-stress related pathways. These data indicate that exposure of T cells to EBOV results in an abortive infection, which likely contributes to the lymphopenia observed during EBOV infections.

Bronchiectasis in India: results from the European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India Registry.

BACKGROUND: Bronchiectasis is a common but neglected chronic lung disease. Most epidemiological data are limited to cohorts from Europe and the USA, with few data from low-income and middle-income countries. We therefore aimed to describe the characteristics, severity of disease, microbiology, and treatment of patients with bronchiectasis in India. METHODS: The Indian bronchiectasis registry is a multicentre, prospective, observational cohort study. Adult patients (≥18 years) with CT-confirmed bronchiectasis were enrolled from 31 centres across India. Patients with bronchiectasis due to cystic fibrosis or traction bronchiectasis associated with another respiratory disorder were excluded. Data were collected at baseline (recruitment) with follow-up visits taking place once per year. Comprehensive clinical data were collected through the European Multicentre Bronchiectasis Audit and Research Collaboration registry platform. Underlying aetiology of bronchiectasis, as well as treatment and risk factors for bronchiectasis were analysed in the Indian bronchiectasis registry. Comparisons of demographics were made with published European and US registries, and quality of care was benchmarked against the 2017 European Respiratory Society guidelines. FINDINGS: From June 1, 2015, to Sept 1, 2017, 2195 patients were enrolled. Marked differences were observed between India, Europe, and the USA. Patients in India were younger (median age 56 years [IQR 41-66] vs the European and US registries; p<0·0001]) and more likely to be men (1249 [56·9%] of 2195). Previous tuberculosis (780 [35·5%] of 2195) was the most frequent underlying cause of bronchiectasis and Pseudomonas aeruginosa was the most common organism in sputum culture (301 [13·7%]) in India. Risk factors for exacerbations included being of the male sex (adjusted incidence rate ratio 1·17, 95% CI 1·03-1·32; p=0·015), P aeruginosa infection (1·29, 1·10-1·50; p=0·001), a history of pulmonary tuberculosis (1·20, 1·07-1·34; p=0·002), modified Medical Research Council Dyspnoea score (1·32, 1·25-1·39; p<0·0001), daily sputum production (1·16, 1·03-1·30; p=0·013), and radiological severity of disease (1·03, 1·01-1·04; p<0·0001). Low adherence to guideline-recommended care was observed; only 388 patients were tested for allergic bronchopulmonary aspergillosis and 82 patients had been tested for immunoglobulins. INTERPRETATION: Patients with bronchiectasis in India have more severe disease and have distinct characteristics from those reported in other countries. This study provides a benchmark to improve quality of care for patients with bronchiectasis in India. FUNDING: EU/European Federation of Pharmaceutical Industries and Associations Innovative Medicines Initiative inhaled Antibiotics in Bronchiectasis and Cystic Fibrosis Consortium, European Respiratory Society, and the British Lung Foundation.

Disruption of Phosphatidylserine Synthesis or Trafficking Reduces Infectivity of Ebola Virus.

The outer leaflet of the viral membrane of Ebola virus (EBOV) virions is enriched with phosphatidylserine (PtdSer), which is thought to play a central role in viral tropism, entry, and virus-associated immune evasion. We investigated the effects of inhibiting synthesis and/or export of PtdSer to the cell surface of infected cells on viral infectivity. Knockdown of both PtdSer synthase enzymes, PTDSS1 and PTDSS2, effectively decreased viral production. Decreased PtdSer expression resulted in an accumulation of virions at the plasma membrane and adjacent of intracellular organelles, suggesting that virion budding is impaired. The addition of inhibitors that block normal cellular trafficking of PtdSer to the plasma membrane resulted in a similar accumulation of virions and reduced viral replication. These findings demonstrate that plasma membrane-associated PtdSer is required for efficient EBOV budding, increasing EBOV infectivity, and could constitute a potential therapeutic target for the development of future countermeasures against EBOV.

Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions.

Viral apoptotic mimicry, which is defined by exposure of phosphatidylserine (PtdSer) into the outer leaflet of budding enveloped viruses, increases viral tropism, infectivity and promotes immune evasion. Here, we report that the calcium (Ca2+)-dependent scramblase, transmembrane protein 16F (TMEM16F), is responsible for the incorporation of PtdSer into virion membranes during Ebola virus infection. Infection of Huh7 cells with Ebola virus resulted in a pronounced increase in plasma membrane-associated PtdSer, which was demonstrated to be dependent on TMEM16F function. Analysis of virions using imaging flow cytometry revealed that short hairpin RNA-mediated down-regulation of TMEM16F function directly reduced virion-associated PtdSer. Taken together, these studies demonstrate that TMEM16F is a central cellular factor in the exposure of PtdSer in the outer leaflet of viral membranes.

Staufen1 Interacts with Multiple Components of the Ebola Virus Ribonucleoprotein and Enhances Viral RNA Synthesis.

Ebola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3' and 5' extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.IMPORTANCE Ebola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.

An unusual cause of breathlessness and profuse micronodules.

We describe a 21-year-old male with a history of smoking and subacute onset of breathlessness with normal cardiorespiratory examination. The presence of "track marks" and digital infarcts prompted evaluation for infective endocarditis and confrontational history taking revealed anorexia, weight loss over 3 months along with intravenous drug abuse of reconstituted tablets of tapentadol. Echocardiography was normal and blood cultures were sterile; computed tomography showed bilateral, diffuse, small centrilobular nodules with "tree-in-bud" appearance. In this clinicopathologic conference, we discuss the clinical and radiological differential diagnosis of centrilobular nodules, lung biopsy findings, and management options for patients with such a presentation.

Antibody-Dependent Enhancement of Ebola Virus Infection by Human Antibodies Isolated from Survivors.

Some monoclonal antibodies (mAbs) recovered from survivors of filovirus infections can protect against infection. It is currently unknown whether natural infection also induces some antibodies with the capacity for antibody-dependent enhancement (ADE). A panel of mAbs obtained from human survivors of filovirus infection caused by Ebola, Bundibugyo, or Marburg viruses was evaluated for their ability to facilitate ADE. ADE was observed readily with all mAbs examined at sub-neutralizing concentrations, and this effect was not restricted to mAbs with a particular epitope specificity, neutralizing capacity, or subclass. Blocking of specific Fcγ receptors reduced but did not abolish ADE that was associated with high-affinity binding antibodies, suggesting that lower-affinity interactions still cause ADE. Mutations of Fc fragments of an mAb that altered its interaction with Fc receptors rendered the antibody partially protective in vivo at a low dose, suggesting that ADE counteracts antibody-mediated protection and facilitates dissemination of filovirus infections.