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 Ca 2+ 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

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

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

International audienceThe 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

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

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

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

MyCOVID project: Comparison of innate lung responses after ex vivo infection by mycobacteria from the M.tuberculosis complex and the SARSCoV-2

International audienceThe recent emergence of the new coronavirus SARS-Cov-2 reminds us of the importance of research on emerging and zoonotic pathogens. Most of the models used to study respiratory pathogens rely on cells lines, which do not represent the diversity of lung cells nor their organization1,2. We validated a model of Precision Cut Lung Slices (PCLS) to study the early steps of infection by Mycobacterium bovis (Mb) and tuberculosis (Mtb), respectively causing bovine and human tuberculosis. While Mb is zoonotic, Mtb is restricted to Humans but host specificities are not understood yet. The bovine PCLS study showed that type I interferon pathway was activated by Mb and not Mtb, with a significant contribution of resident alveolar macrophages3. By adapting the bovine protocol, we used biopsies obtained from surgical resection from lung adenocarcinoma, to obtain human PCLS. This enabled ex vivo infections in a preserved and functional lung micro-environment, with the presence of all resident cell-types and connective tissue architecture.Our project aims to investigate which cell types are infected by SARS-CoV-2, Mb and Mtb; and their contribution to physiopathology and more specifically to the IFN-I pathway, that is key to both antiviral and anti-mycobacterial response. Thus, the MyCOVID project will improve our understanding of the pathophysiology of top two deadliest respiratory diseases worldwide, COVID-19 and tuberculosis

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

International audienceIt 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

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

International audienceMany 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 membranecontaining 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 doublemembrane 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

Comparison of the innate immune response to infection by mycobacteria and SARS-CoV-2 in humans using an ex vivo physiological lung model

The recent emergence of the new coronavirus SARS-Cov-2 reminds us the importance of research on emerging and zoonotic pathogens. Most of the models used to study respiratory pathogens rely on cells lines, which do not represent the diversity of lung cells nor their tissular organization or cell-cell interactions 1,2.We validated a model of Precision Cut Lung Slices (PCLS) to study the early steps of infection by Mycobacterium bovis (Mb) and tuberculosis (Mtb), respectively causing bovine and human tuberculosis. While Mb is zoonotic, Mtb is restricted to Humans but host specificities are not understood yet. The bovine PCLS study showed that type I interferon pathway was activated by Mb and not Mtb, with a significant contribution of resident alveolar macrophages3. By adapting the bovine protocol, we used biopsies obtained from surgical resection from lung adenocarcinoma, to obtain human PCLS. This enabled ex vivo infections in a preserved and functional lung micro-environment.First, we set up the infection conditions (strains, timepoint, detection methods). We validated mycobacteria protocols but faced difficulty with SRAS-Cov-2 detection. We were able to observe infected cells with a neon green recombinant virus. Then we investigated which cell types were infected by our pathogens, by imaging (TEM, confocal microscopy). We focused on alveolar macrophage, a cell which play a key role in the development of both infections. Alveolar macrophages were numerously recruited in infected alveoli 48h after Mtb or Mb infection. The analysis of the transcriptomic signature of infected PCLS is ongoing. Our project will improve our understanding of the pathophysiology of top two deadliest respiratory diseases worldwide, COVID-19 and tuberculosis