The double-membrane vesicle (DMV): a virus-induced organelle dedicated to the replication of SARS-CoV-2 and other positive-sense single-stranded RNA viruses.

Positive single-strand RNA (+ RNA) viruses can remodel host cell membranes to induce a replication organelle (RO) isolating the replication of their genome from innate immunity mechanisms. Some of these viruses, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMVs) for this purpose. Viral non-structural proteins are essential for DMV biogenesis, but they cannot form without an original membrane from a host cell organelle and a significant supply of lipids. The endoplasmic reticulum (ER) and the initial mechanisms of autophagic processes have been shown to be essential for the biogenesis of SARS-CoV-2 DMVs. However, by analogy with other DMV-inducing viruses, it seems likely that the Golgi apparatus, mitochondria and lipid droplets are also involved. As for hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMVs have been identified. These pores presumably allow the supply of metabolites essential for viral replication within the DMV, together with the export of the newly synthesized viral RNA to form the genome of future virions. It remains unknown whether, as for HCV, DMVs with open pores can coexist with the fully sealed DMVs required for the storage of large amounts of viral RNA. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis and morphology between these two phylogenetically distant viruses. An understanding of the mechanisms of DMV formation and their role in the infectious cycle of SARS-CoV-2 may be essential for the development of new antiviral approaches against this pathogen or other coronaviruses that may emerge in the future.

Metabolic Profile and Pathological Alterations in the Muscle of Patients with Early-Stage Amyotrophic Lateral Sclerosis.

Diverse biomarkers and pathological alterations have been found in muscle of patients with Amyotrophic lateral sclerosis (ALS), but the relation between such alterations and dysfunction in energetic metabolism remains to be investigated. We established the metabolome of muscle and serum of ALS patients and correlated these findings with the clinical status and pathological alterations observed in the muscle. We obtained data from 20 controls and 17 ALS patients (disease duration: 9.4 ± 6.8 months). Multivariate metabolomics analysis identified a distinct serum metabolome for ALS compared to controls (p-CV-ANOVA < 0.035) and revealed an excellent discriminant profile for muscle metabolome (p-CV-ANOVA < 0.0012). Citramalate was discriminant for both muscle and serum. High lauroylcarnitine levels in muscle were associated with low Forced Vital Capacity. Transcriptomics analysis of key antioxidant enzymes showed an upregulation of SOD3 (p = 0.0017) and GLRX2(1) (p = 0.0022) in ALS muscle. Analysis of mitochondrial enzymatic activity in muscle revealed higher complex II/CS (p = 0.04) and lower LDH (p = 0.03) activity in ALS than in controls. Our study showed, for the first time, a global dysfunction in the muscle of early-stage ALS patients. Furthermore, we identified novel metabolites to be employed as biomarkers for diagnosis and prognosis of ALS patients.

A morphometric analysis of hepatitis B subviral particles shows no correlation of filament proportion and length with clinical stage and genotype.

It was recently suggested that the composition of circulating hepatitis B subviral particles (SVPs) could be used to differentiate the various stages in chronic hepatitis B virus (HBV) infection, with significantly lower proportions of L and M proteins in inactive carriers than in individuals with chronic hepatitis. L protein is abundant in virions and filamentous SVPs but almost absent from spherical SVPs. We, therefore, performed a morphometric analysis of SVPs in these two groups of patients, by conducting a retrospective analysis on sera from 15 inactive carriers and 11 patients with chronic hepatitis infected with various HBV genotypes. Subviral particles were concentrated by centrifugation on a sucrose cushion, with monitoring by transmission electron microscopy. The percentage of filamentous SVPs and filament length for 100 SVPs was determined with a digital camera. The L protein PreS1 promoter was sequenced from viral genomes by the Sanger method. No marked differences were found between patients, some of whom had only spherical SVPs, whereas others had variable percentages of filamentous SVPs (up to 28%), of highly variable length. High filament percentages were not associated with a particular sequence of the L protein promoter, HBV genotype or even disease stage. High levels of circulating filamentous SVPs are probably more strongly related to individual host factors than to viral strain characteristics or disease stage.

Pathology Assessments of Multiple Organs in Fatal COVID-19 in Intensive Care Unit vs. Non-intensive Care Unit Patients.

Purpose: The objective of the present study was to provide a detailed histopathological description of fatal coronavirus disease 2019 (COVID 19), and compare the lesions in Intensive Care Unit (ICU) and non-ICU patients. Methods: In this prospective study we included adult patients who died in hospital after presenting with confirmed COVID-19. Multiorgan biopsies were performed. Data generated with light microscopy, transmission electron microscopy (TEM) and RT-PCR assays were reviewed. Results: 20 patients were enrolled in the study and the main pulmonary finding was alveolar damage, which was focal in 11 patients and diffuse in 8 patients. Chronic fibrotic and inflammatory lesions were observed in 18 cases, with acute inflammatory lesions in 12 cases. Diffuse lesions, collapsed alveoli and dystrophic pneumocytes were more frequent in the ICU group (62.5%, vs. 25%; 63%, vs. 55%; 87.5%, vs. 54%). Acute lesions (82%, vs. 37.5%; p = 0.07) with neutrophilic alveolitis (63.6% vs. 0%, respectively; p = 0.01) were observed more frequently in the non-ICU group. Viral RNA was detected in 12 lung biopsies (60%) up to 56 days after disease upset. TEM detected viral particles in the lung and kidney biopsy samples up to 27 days after disease upset. Furthermore, abundant networks of double-membrane vesicles (DMVs, a hallmark of viral replication) were observed in proximal tubular epithelial cells. Conclusion: Lung injury was different in ICU and non-ICU patients. Extrapulmonary damage consisting in kidney and myocardial injury were more frequent in ICU patients. Our TEM experiments provided the first description of SARS-CoV-2-induced DMVs in kidney biopsy samples-a sign of intense viral replication in this organ.

Crystalline podocytopathy and tubulopathy linked to kappa light chain deposits in a context of smoldering multiple myeloma.

A 42-year-old man with smoldering immunoglobulin G kappa multiple myeloma showed a heavy proteinuria composed of free light chain, prompting performance of a kidney biopsy. Electron microscopy revealed numerous rhomboid-shaped crystals labelled by the anti-kappa in immunogold, notably in the cytoplasm of podocytes, establishing the diagnosis of crystalline podocytopathy. This case illustrates a rare form of monoclonal gammopathy of renal significance, and highlights the key role of electron microscopy and immunogold to better elucidate the location and composition of crystals.

ELISA-Based Analysis Reveals an Anti-SARS-CoV-2 Protein Immune Response Profile Associated with Disease Severity.

Since the start of the COVID-19 pandemic, many studies have investigated the humoral response to SARS-CoV-2 during infection. Studies with native viral proteins constitute a first-line approach to assessing the overall immune response, but small peptides are an accurate and valuable tool for the fine characterization of B-cell epitopes, despite the restriction of this approach to the determination of linear epitopes. In this study, we used ELISA and peptides covering a selection of structural and non-structural SARS-CoV-2 proteins to identify key epitopes eliciting a strong immune response that could serve as a biological signature of disease characteristics, such as severity, in particular. We used 213 plasma samples from a cohort of patients well-characterized clinically and biologically and followed for COVID-19 infection. We found that patients developing severe disease had higher titers of antibodies mapping to multiple specific epitopes than patients with mild to moderate disease. These data are potentially important as they could be used for immunological profiling to improve our knowledge of the quantitative and qualitative characteristics of the humoral response in relation to patient outcome.

Antibody Responses after a Third Dose of COVID-19 Vaccine in Kidney Transplant Recipients and Patients Treated for Chronic Lymphocytic Leukemia.

The impact of a third dose of COVID-19 vaccine on antibody responses is unclear in immunocompromised patients. The objective of this retrospective study was to characterize antibody responses induced by a third dose of mRNA COVID-19 vaccine in 160 kidney transplant recipients and 20 patients treated for chronic lymphocytic leukemia (CLL). Prevalence of anti-spike IgG ≥ 7.1 and ≥ 30 BAU/mL after the third dose were 47% (75/160) and 39% (63/160) in kidney transplant recipients, and 57% (29/51) and 50% (10/20) in patients treated for CLL. Longitudinal follow-up identified a moderate increase in SARS-CoV-2 anti-spike IgG levels after a third dose of vaccine in kidney transplant recipients (0.19 vs. 5.28 BAU/mL, p = 0.03) and in patients treated for CLL (0.63 vs. 10.7 BAU/mL, p = 0.0002). This increase in IgG levels had a limited impact on prevalence of anti-spike IgG ≥ 30 BAU/mL in kidney transplant recipients (17%, 2/12 vs. 33%, 4/12, p = 0.64) and in patients treated for CLL (5%, 1/20 vs. 45%, 9/20, p = 0.008). These results highlight the need for vaccination of the general population and the importance of non-medical preventive measures to protect immunocompromised patients.

Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress.

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.

Alternative splicing creates a pseudo-strictosidine ß-d-glucosidase modulating alkaloid synthesis in Catharanthus roseus.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by ß-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine ß-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks ß-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of ß-glucosidase multimerization, an organization common to many defensive GH1 members.

Annulate lamellae and intracellular pathogens.

Annulate lamellae (AL) have been observed many times over the years on electron micrographs of rapidly dividing cells, but little is known about these unusual organelles consisting of stacked sheets of endoplasmic reticulum-derived membranes with nuclear pore complexes (NPCs). Evidence is growing for a role of AL in viral infection. AL have been observed early in the life cycles of the hepatitis C virus (HCV) and, more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggesting a specific induction of mechanisms potentially useful to these pathogens. Like other positive-strand RNA viruses, these viruses induce host cells membranes rearrangements. The NPCs of AL could potentially mediate exchanges between these partially sealed compartments and the cytoplasm. AL may also be involved in regulating Ca2+ homeostasis or cell cycle control. They were recently observed in cells infected with Theileria annulata, an intracellular protozoan parasite inducing cell proliferation. Further studies are required to clarify their role in intracellular pathogen/host-cell interactions.

Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69-V70, France, August to December 2020.

We report the strategy leading to the first detection of variant of concern 202012/01 (VOC) in France (21 December 2020). First, the spike (S) deletion H69-V70 (ΔH69/ΔV70), identified in certain SARS-CoV-2 variants including VOC, is screened for. This deletion is associated with a S-gene target failure (SGTF) in the three-target RT-PCR assay (TaqPath kit). Subsequently, SGTF samples are whole genome sequenced. This approach revealed mutations co-occurring with ΔH69/ΔV70 including S:N501Y in the VOC.

Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release.

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.

Controlled Heat and Humidity-Based Treatment for the Reuse of Personal Protective Equipment: A Pragmatic Proof-of-Concept to Address the Mass Shortage of Surgical Masks and N95/FFP2 Respirators and to Prevent the SARS-CoV2 Transmission.

Background: The coronavirus infectious disease-2019 (COVID-19) pandemic has led to an unprecedented shortage of healthcare resources, primarily personal protective equipment like surgical masks, and N95/filtering face piece type 2 (FFP2) respirators. Objective: Reuse of surgical masks and N95/FFP2 respirators may circumvent the supply chain constraints and thus overcome mass shortage. Methods, design, setting, and measurement: Herein, we tested the effects of dry- and moist-air controlled heating treatment on structure and chemical integrity, decontamination yield, and filtration performance of surgical masks and FFP2 respirators. Results: We found that treatment in a climate chamber at 70°C during 1 h with 75% humidity rate was adequate for enabling substantial decontamination of both respiratory viruses, oropharyngeal bacteria, and model animal coronaviuses, while maintaining a satisfying filtering capacity. Limitations: Further studies are now required to confirm the feasibility of the whole process during routine practice. Conclusion: Our findings provide compelling evidence for the recycling of pre-used surgical masks and N95/FFP2 respirators in case of imminent mass shortfall.

Clinicopathologic features of infection-related glomerulonephritis with IgA deposits: a French Nationwide study.

BACKGROUND: Infection-related glomerulonephritis with IgA deposits (IRGN-IgA) is a rare disease but it is increasingly reported in the literature. Data regarding epidemiology and outcome are lacking, especially in Europe. We aimed to assess the clinical, pathologic and outcome data of IRGN-IgA. METHODS: Clinical and outcome data from patients from 11 French centers over the 2007-2017 period were collected retrospectively. We reviewed pathologic patterns and immunofluorescence of renal biopsies and evaluated C4d expression in IRGN-IgA. We analyzed the correlation between histological presentation and outcome. RESULTS: Twenty-seven patients (23 men, mean age: 62 ± 15 years) were included. Twenty-one (78%) had Staphylococcus aureus infection and twelve (44%) were diabetic. At the time of biopsy, 95.2% had haematuria, 48.1% had a serum creatinine level of > 4 mg/dL, and 16% had hypocomplementemia. The most common pathologic presentation included mesangial (88.9%) and endocapillary proliferative glomerulonephritis (88.9%) with interstitial fibrosis and tubular atrophy (IF/TA) (85.1%). Diffuse and global glomerular C4d expression was found in 17.8%, mostly in biopsies with acute or subacute patterns, and was associated with a short delay between infection and renal biopsy compared to segmental and focal staining. After median follow-up of 13.2 months, 23.1% died, 46.2% had persistent renal dysfunction and 15.4% reached end-stage renal disease. Renal outcome was correlated to IF/TA severity. CONCLUSIONS: Infection-related glomerulonephritis with IgA deposits is usually associated with Staphylococcus infections and mainly affects adult men. This entity has a poor prognosis which is correlated to interstitial fibrosis and tubular atrophy severity.

Hepatitis B virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis.

Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP-overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2-NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae-mediated endocytosis. Instead, entry occurred via the clathrin-mediated endocytosis pathway. HBV internalisation was inhibited by pitstop-2 treatment and RNA-mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP-2 and dynamin-2. We were able to visualise HBV entry in clathrin-coated pits and vesicles by electron microscopy (EM) and cryo-EM with immunogold labelling. These data demonstrating that HBV uses a clathrin-mediated endocytosis pathway to enter HepG2-NTCP cells increase our understanding of the complete HBV life cycle.

Direct interaction between the hepatitis B virus core and envelope proteins analyzed in a cellular context.

Hepatitis B virus (HBV) production requires intricate interactions between the envelope and core proteins. Analyses of mutants of these proteins have made it possible to map regions involved in the formation and secretion of virions. Tests of binding between core and envelope peptides have also been performed in cell-free conditions, to study the interactions potentially underlying these mechanisms. We investigated the residues essential for core-envelope interaction in a cellular context in more detail, by transiently producing mutant or wild-type L, S, or core proteins separately or in combination, in Huh7 cells. The colocalization and interaction of these proteins were studied by confocal microscopy and co-immunoprecipitation, respectively. The L protein was shown to constitute a molecular platform for the recruitment of S and core proteins in a perinuclear environment. Several core amino acids were found to be essential for direct interaction with L, including residue Y132, known to be crucial for capsid formation, and residues L60, L95, K96 and I126. Our results confirm the key role of L in the tripartite core-S-L interaction and identify the residues involved in direct core-L interaction. This model may be valuable for studies of the potential of drugs to inhibit HBV core-envelope interaction.

Combination of ß-(1, 3)-D-glucan testing in serum and qPCR in nasopharyngeal aspirate for facilitated diagnosis of Pneumocystis jirovecii pneumonia.

BACKGROUND: Currently, the biological diagnosis of Pneumocystis jirovecii pneumonia (PjP infection) usually relies on microbiological investigations in bronchial-alveolar lavage fluid (BALF) by conventional staining methods and/or molecular biology. However, bronchial-alveolar lavage is sometimes complicated to manage, especially in weakened patients. Therefore, alternative clinical samples-easier to collect-are warranted in such specific contexts. OBJECTIVE: Over a four-year period, diagnostic performance of an original method based on combination of quantitative real-time polymerase chain reaction (qPCR) in nasopharyngeal aspirate (NPA) with measurement of ß-(1, 3)-D-glucan antigen (BDG) in serum was prospectively assessed in a single centre. PATIENTS/METHODS: Results were compared with those obtained in BALF through direct staining methods and qPCR. True positives were defined by an independent committee based on clinical, radiological and biological data. Overall, 48 individuals with a definitive diagnosis of PjP infection were included, and 48 controls were selected upon matching for age, sex and underlying disease(s). RESULTS: qPCR results were strongly correlated between BALF and NPA (P < .0001). Altogether, greater diagnostic performance was achieved when establishing the positive cut-off of BDG antigen at 143 pg/mL. In such conditions, sensitivity of the testing based on either positive BDG measurement or positive qPCR in NPA was then calculated at 93.75%, 95% CI [82.37%-98.40%], and specificity at 97.87%, 95% CI [87.66%-100.00%]. CONCLUSIONS: Further validation through multicentre studies is now required, especially for establishing clear cut-offs. However, one could already state that combination of qPCR in the NPA with BDG measurement in serum may be a valuable substitute for BALF examination.

Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety.

Transmission electron microscopy (TEM) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer-scale resolution. Between the 1960s and 1990s, TEM contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. The diagnosis of viral infections improved considerably during the 1990s, with the advent of highly sensitive techniques, such as enzyme-linked immunosorbent assay (ELISA) and PCR, rendering TEM obsolete for this purpose. However, the last 20 years have demonstrated the utility of this technique in particular situations, due to its "catch-all" nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. Thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, TEM provided the answer. TEM is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. It is also used to check the microbiological safety of biological products. Many biopharmaceuticals are produced in animal cells that might contain little-known, difficult-to-detect viruses. In this context, the "catch-all" properties of TEM make it possible to document the presence of viruses or virus-like particles in these products.