SARS-CoV-2 infection induces hyaluronan production in vitro and hyaluronan levels in COVID-19 patients relate to morbidity and long-term lung impairment: a prospective cohort study.

We previously demonstrated that the lungs of deceased COVID-19 patients were filled with a clear hydrogel consisting of hyaluronan (HA). In this translational study, we investigated the role of HA at all stages of COVID-19 disease to map the consequences of elevated HA on morbidity and identify the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced HA production. A reduced alveolar surface area was observed in the lungs of deceased COVID-19 patients compared to healthy controls, as visualized by a 3D rendering of lung morphology using light-sheet fluorescence microscopy. We confirmed the presence of HA in lung biopsies and found large quantities of proinflammatory fragmented HA. The association of systemic HA in blood plasma and disease severity was assessed in patients with mild (WHO Clinical Progression Scale, WHO-CPS, 1-5) and severe COVID-19 (WHO-CPS, 6-9) during the acute and convalescent phases and related to lung function. We found that systemic levels of HA were high during acute COVID-19 disease, remained elevated during convalescence, and were associated with a reduced diffusion capacity. In vitro 3D-lung models, differentiated from primary human bronchial epithelial cells, were used to study the effects of SARS-CoV-2 infection on HA metabolism, and transcriptomic analyses revealed a dysregulation of HA synthases and hyaluronidases, both contributing to increased HA in apical secretions. Furthermore, corticosteroid treatment reduced the inflammation and downregulated HA synthases. Our findings demonstrate that HA plays a role in COVID-19 morbidity and that sustained elevated HA concentrations may contribute to long-term respiratory impairment.IMPORTANCEThis study provides insights into the role of hyaluronan (HA) in the severity and long-term impact of COVID-19 on lung function. Through extensive morphological examination of lung tissues and a multicenter study, we identified that HA levels are significantly elevated in COVID-19 patients, correlating with a reduced lung diffusion capacity during convalescence. Using a 3D-lung model, we further uncovered how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 infection causes a dysregulated HA metabolism, leading to increased HA production. Our findings provide valuable insights into the pathogenesis of SARS-CoV-2 and suggest that targeting HA metabolism could offer new therapeutic avenues for managing COVID-19, particularly to prevent long-term lung impairment. Additionally, HA holds potential as a biomarker for predicting disease severity, which could guide personalized treatment strategies.

Transcriptional Response to Tick-Borne Flavivirus Infection in Neurons, Astrocytes and Microglia In Vivo and In Vitro.

Tick-borne encephalitis virus (TBEV) is a neurotropic member of the genus Orthoflavivirus (former Flavivirus) and is of significant health concern in Europe and Asia. TBEV pathogenesis may occur directly via virus-induced damage to neurons or through immunopathology due to excessive inflammation. While primary cells isolated from the host can be used to study the immune response to TBEV, it is still unclear how well these reflect the immune response elicited in vivo. Here, we compared the transcriptional response to TBEV and the less pathogenic tick-borne flavivirus, Langat virus (LGTV), in primary monocultures of neurons, astrocytes and microglia in vitro, with the transcriptional response in vivo captured by single-nuclei RNA sequencing (snRNA-seq) of a whole mouse cortex. We detected similar transcriptional changes induced by both LGTV and TBEV infection in vitro, with the lower response to LGTV likely resulting from slower viral kinetics. Gene set enrichment analysis showed a stronger transcriptional response in vivo than in vitro for astrocytes and microglia, with a limited overlap mainly dominated by interferon signaling. Together, this adds to our understanding of neurotropic flavivirus pathogenesis and the strengths and limitations of available model systems.

An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy in neuroscience.

Introduction: Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited. Methods: T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images. Results: Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR. Discussion: The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.

The NSP3 protein of SARS-CoV-2 binds fragile X mental retardation proteins to disrupt UBAP2L interactions.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.

The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD ∼ 7-300 µM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

Type I interferon shapes brain distribution and tropism of tick-borne flavivirus.

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar-/- mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar-/-, the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs.

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.

At-home sampling to meet geographical challenges for serological assessment of SARS-CoV-2 exposure in a rural region of northern Sweden, March to May 2021: a retrospective cohort study.

BackgroundThe current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture for testing is usually performed by trained staff at healthcare centres. Long travel distances to healthcare centres in rural regions may introduce a bias of testing towards relatively large communities with closer access. Rural regions are therefore often not represented in population-based data.AimThe aim of this retrospective cohort study was to develop and implement a strategy for at-home testing in a rural region of Sweden during spring 2021, and to evaluate its role to provide equal health care for its inhabitants.MethodsWe developed a sensitive method to measure antibodies to the S-protein of SARS-CoV-2 and optimised this assay for clinical use together with a strategy of at-home capillary blood sampling.ResultsWe demonstrated that our ELISA gave comparable results after analysis of capillary blood or serum from SARS-CoV-2-experienced individuals. We demonstrated stability of the assay under conditions that reflected temperature and humidity during winter or summer. By assessment of capillary blood samples from 4,122 individuals, we could show both feasibility of the strategy and that implementation shifted the geographical spread of testing in favour of rural areas.ConclusionImplementation of at-home sampling enabled citizens living in remote rural areas access to centralised and sensitive laboratory antibody tests. The strategy for testing used here could therefore enable disease control authorities to get rapid access to information concerning immunity to infectious diseases, even across vast geographical distance.

Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein.

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

A Syntenin Inhibitor Blocks Endosomal Entry of SARS-CoV-2 and a Panel of RNA Viruses.

Viruses are dependent on host factors in order to efficiently establish an infection and replicate. Targeting the interactions of such host factors provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking and has previously been shown to be important for human papilloma virus (HPV) infection. Here, we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection and strongly reduced flavivirus infection, which is completely dependent on receptor-mediated endocytosis for their entry. In conclusion, we have identified a novel broad spectrum antiviral inhibitor that efficiently targets a broad range of RNA viruses.

Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections.

The coronavirus disease 2019, COVID-19, is a complex disease with a wide range of symptoms from asymptomatic infections to severe acute respiratory syndrome with lethal outcome. Individual factors such as age, sex, and comorbidities increase the risk for severe infections, but other aspects, such as genetic variations, are also likely to affect the susceptibility to SARS-CoV-2 infection and disease severity. Here, we used a human 3D lung cell model based on primary cells derived from multiple donors to identity host factors that regulate SARS-CoV-2 infection. With a transcriptomics-based approach, we found that less susceptible donors show a higher expression level of serine protease inhibitors SERPINA1, SERPINE1, and SERPINE2, identifying variation in cellular serpin levels as restricting host factors for SARS-CoV-2 infection. We pinpoint their antiviral mechanism of action to inhibition of the cellular serine protease, TMPRSS2, thereby preventing cleavage of the viral spike protein and TMPRSS2-mediated entry into the target cells. By means of single-cell RNA sequencing, we further locate the expression of the individual serpins to basal, ciliated, club, and goblet cells. Our results add to the importance of genetic variations as determinants for SARS-CoV-2 susceptibility and suggest that genetic deficiencies of cellular serpins might represent risk factors for severe COVID-19. Our study further highlights TMPRSS2 as a promising target for antiviral intervention and opens the door for the usage of locally administered serpins as a treatment against COVID-19. IMPORTANCE Identification of host factors affecting individual SARS-CoV-2 susceptibility will provide a better understanding of the large variations in disease severity and will identify potential factors that can be used, or targeted, in antiviral drug development. With the use of an advanced lung cell model established from several human donors, we identified cellular protease inhibitors, serpins, as host factors that restrict SARS-CoV-2 infection. The antiviral mechanism was found to be mediated by the inhibition of a serine protease, TMPRSS2, which results in a blockage of viral entry into target cells. Potential treatments with these serpins would not only reduce the overall viral burden in the patients, but also block the infection at an early time point, reducing the risk for the hyperactive immune response common in patients with severe COVID-19.

Molecular Organisation of Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions.

A Phase 2 Trial of the Effect of Antiandrogen Therapy on COVID-19 Outcome: No Evidence of Benefit, Supported by Epidemiology and In Vitro Data.

BACKGROUND: Men are more severely affected by COVID-19. Testosterone may influence SARS-CoV-2 infection and the immune response. OBJECTIVE: To clinically, epidemiologically, and experimentally evaluate the effect of antiandrogens on SARS-CoV-2 infection. DESIGNS, SETTINGS, AND PARTICIPANTS: A randomized phase 2 clinical trial (COVIDENZA) enrolled 42 hospitalized COVID-19 patients before safety evaluation. We also conducted a population-based retrospective study of 7894 SARS-CoV-2-positive prostate cancer patients and an experimental study using an air-liquid interface three-dimensional culture model of primary lung cells. INTERVENTION: In COVIDENZA, patients were randomized 2:1 to 5 d of enzalutamide or standard of care. OUTCOME MEASUREMENTS: The primary outcomes in COVIDENZA were the time to mechanical ventilation or discharge from hospital. The population-based study investigated risk of hospitalization, intensive care, and death from COVID-19 after androgen inhibition. RESULTS AND LIMITATIONS: Enzalutamide-treated patients required longer hospitalization (hazard ratio [HR] for discharge from hospital 0.43, 95% confidence interval [CI] 0.20-0.93) and the trial was terminated early. In the epidemiological study, no preventive effects were observed. The frail population of patients treated with androgen deprivation therapy (ADT) in combination with abiraterone acetate or enzalutamide had a higher risk of dying from COVID-19 (HR 2.51, 95% CI 1.52-4.16). In vitro data showed no effect of enzalutamide on virus replication. The epidemiological study has limitations that include residual confounders. CONCLUSIONS: The results do not support a therapeutic effect of enzalutamide or preventive effects of bicalutamide or ADT in COVID-19. Thus, these antiandrogens should not be used for hospitalized COVID-19 patients or as prevention for COVID-19. Further research on these therapeutics in this setting are not warranted. PATIENT SUMMARY: We studied whether inhibition of testosterone could diminish COVID-19 symptoms. We found no evidence of an effect in a clinical study or in epidemiological or experimental investigations. We conclude that androgen inhibition should not be used for prevention or treatment of COVID-19.

Serological assessment of SARS-CoV-2 exposure in northern Sweden by the use of at-home sampling to meet geographical challenges in rural regions.

The current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture is usually performed by trained staff at health care centers. Long travel distances may introduce a bias of testing towards relatively large communities with close access to health care centers. Rural regions may thus be overlooked. Here, we demonstrate a sensitive method to measure antibodies to the S-protein of SARS-CoV-2. We adapted and optimized this assay for clinical use together with capillary blood sampling to meet the geographical challenges of serosurveillance. Finally, we tested remote at-home capillary blood sampling together with centralized assessment of S-specific IgG in a rural region of northern Scandinavia that encompasses 55,185 sq kilometers. We conclude that serological assessment from capillary blood sampling gives comparable results as analysis of venous blood. Importantly, at-home sampling enabled citizens living in remote rural areas access to centralized and sensitive laboratory antibody tests.

Convalescent plasma treatment in severely immunosuppressed patients hospitalized with COVID-19: an observational study of 28 cases.

BACKGROUND: Immunosuppressed patients are particularly vulnerable to severe infection from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), risking prolonged viremia and symptom duration. In this study we describe clinical and virological treatment outcomes in a heterogeneous group of patients with severe immunosuppression due to various causes suffering from COVID-19 infection, who were all treated with convalescent plasma (CCP) along with standard treatment. METHODS: We performed an observational, retrospective case series between May 2020 to March 2021 at three sites in Skåne, Sweden, with a population of nearly 1.4 million people. All patients hospitalized for COVID-19 who received CCP with the indication severe immunosuppression as defined by the treating physician were included in the study (n = 28). RESULTS: In total, 28 severely immunocompromised patients, half of which previously had been treated with rituximab, who had received in-hospital convalescent plasma treatment of COVID-19 were identified. One week after CCP treatment, 13 of 28 (46%) patients had improved clinically defined as a decrease of at least one point at the WHO-scale. Three patients had increased score points of whom two had died. For 12 patients, the WHO-scale was unchanged. CONCLUSION: As one of only few studies on CCP treatment of COVID-19 in hospitalized patients with severe immunosuppression, this study adds descriptive data. The study design prohibits conclusions on safety and efficacy, and the results should be interpreted with caution. Prospective, randomized trials are needed to investigate this further.

Convalescence plasma treatment of COVID-19: results from a prematurely terminated randomized controlled open-label study in Southern Sweden.

OBJECTIVE: Convalescent plasma has been tried as therapy for various viral infections. Early observational studies of convalescent plasma treatment for hospitalized COVID-19 patients were promising, but randomized controlled studies were lacking at the time. The objective of this study was to investigate if convalescent plasma is beneficial to hospitalized patients with COVID-19. RESULTS: Hospitalized patients with confirmed COVID-19 and an oxygen saturation below 94% were randomized 1:1 to receive convalescent plasma in addition to standard of care or standard of care only. The primary outcome was number of days of oxygen treatment to keep saturation above 93% within 28 days from inclusion. The study was prematurely terminated when thirty-one of 100 intended patients had been included. The median time of oxygen treatment among survivors was 11 days (IQR 6-15) for the convalescent plasma group and 7 days (IQR 5-9) for the standard of care group (p = 0.4, median difference -4). Two patients in the convalescent plasma group and three patients in the standard of care group died (p = 0.64, OR 0.49, 95% CI 0.08-2.79). Thus no significant differences were observed between the groups. Trial registration ClinicalTrials NCT04600440, retrospectively registered Oct 23, 2020.

Fluvastatin mitigates SARS-CoV-2 infection in human lung cells.

Clinical data of patients suffering from COVID-19 indicates that statin therapy, used to treat hypercholesterolemia, is associated with a better disease outcome. Whether statins directly affect virus replication or influence the clinical outcome through modulation of immune responses is unknown. We therefore investigated the effect of statins on SARS-CoV-2 infection in human lung cells and found that only fluvastatin inhibited low and high pathogenic coronaviruses in vitro and ex vivo in a dose-dependent manner. Quantitative proteomics revealed that fluvastatin and other tested statins modulated the cholesterol synthesis pathway without altering innate antiviral immune responses in infected lung epithelial cells. However, fluvastatin treatment specifically downregulated proteins that modulate protein translation and viral replication. Collectively, these results support the notion that statin therapy poses no additional risk to individuals exposed to SARS-CoV-2 and that fluvastatin has a moderate beneficial effect on SARS-CoV-2 infection of human lung cells.