Preferential apical infection of intestinal cell monolayers by SARS-CoV-2 is associated with damage to cellular barrier integrity: Implications for the physiopathology of COVID-19 (preprint)

SARS-CoV-2 can infect different organs, including the intestine. In Caco-2 intestinal cell line, SARS-CoV-2 modulates the ACE2 receptor expression and affects the expression of molecules involved in intercellular junctions. To further explore the possibility that the intestinal epithelium serves as an alternative infection route for SARS-CoV-2, we used a model of polarised intestinal cell monolayers grown on the polycarbonate membrane of Transwell inserts, inoculated with the virus either in the upper or lower chamber of culture. In both polarised Caco-2 cell monolayers and co-culture Caco-2/HT29 cell monolayer, apical SARS-CoV-2 inoculation was found to be much more effective in establishing infection than basolateral inoculation. In addition, apical SARS-CoV-2 infection triggers monolayer degeneration, as shown by histological examination, measurement of trans-epithelial electronic resistance, and cell adhesion molecule expression. During this process, the infectious viruses reach the lower chamber, suggesting either a transcytosis mechanism from the apical side to the basolateral side of cells, a paracellular trafficking of the virus after damage to intercellular junctions in the epithelial barrier, or both. Taken together, these data highlight a preferential tropism of SARS-CoV-2 for the apical side of the human intestinal tract and suggests that infection via the intestinal lumen leads to a systemic infection.

Early proximal tubular injury in COVID-19 related ARDS: the URICOV study (preprint)

Background: During COVID-19, renal impairment is the most frequent after lung impairment and is associated with a poor prognosis particularly in intensive care unit (ICU). In this work we aimed to assess the existence and incidence of early renal dysfunction and its prognostic value in patients with COVID-19-related acute respiratory distress syndrome (ARDS) and to compare them with patients with non-COVID-19-related ARDS. Methods: This prospective multicenter study was conducted in 3 ICUs. Patients aged 18 years and older with invasive mechanical ventilation for ARDS were enrolled. Precise evaluation of renal dysfunction markers including urinary proteins electrophoresis (UPE) and quantification, was performed within 24 hours after mechanical ventilation onset. Results: From March 2020 to December 2021, 135 patients in ICU for ARDS were enrolled: 100 COVID-19 ARDS and 35 non-COVID-19 ARDS. UPE found more tubular dysfunction in COVID-19 patients (68% vs. 21.4%, p<0.0001) and more normal profiles in non-COVID-19 patients (65.0% vs. 11.2%, p=0.0003). COVID-19 patients significantly displayed early urinary leakage of tubular proteins like beta-2-microglobulin and free-light chains, tended to display more frequently acute kidney injury (AKI) (51.0% vs 34.3%, p=0.088), and had longer mechanical ventilation (20 vs. 9 days, p<0.0001) and longer ICU length of stay (26 vs. 15 days, p<0.0001). In COVID-19 ARDS, leakage of free lambda light chain was significantly associated with the onset of KDIGO ≥2 AKI (OR: 1.014, 95%CI [1.003-1.025], p=0.011). Conclusion: Patients admitted to the ICU for COVID-19-related ARDS display a proximal tubular dysfunction, prior to the onset of AKI, which predicts AKI. Proximal tubular damage seems an important mechanism of COVID-19-induced nephropathy. Analysis of urinary proteins is a reliable and non-invasive tool to assess proximal tubular dysfunction in the ICU. Trial Registration: Registered retrospectively with www.clinicaltrials.gov (NCT05699889) 26 January 2023.

Vγ9Vδ2 T cells are potent inhibitors of SARS-CoV-2 replication and exert effector phenotypes in COVID-19 patients (preprint)

V{gamma}9V{delta}2 T cells play a key role in the innate immune response to viral infections, including SARS-CoV-1 and 2, and are activated through butyrophilin (BTN)-3A. Here, the objectives were to: 1) characterize the effects of SARS-CoV-2 infection on the number, phenotype, and activation of V{gamma}9V{delta}2 T cells in infected patients, and 2) assess the effects of in vitro SARS-CoV-2 infection on the expression of BTN3A and its impact on the activation and response of V{gamma}9V{delta}2 T cells to an anti-BTN3A antibody. Blood V{gamma}9V{delta}2 T cells decreased in clinically mild SARS-CoV-2 infections compared to healthy volunteers (HV). This decrease was maintained up to 28 days and in the recovery period. Terminally differentiated V{gamma}9V{delta}2 T cells tend to be enriched on the day of diagnosis, 28 days after and during the recovery period compared to HV. Furthermore, these cells showed cytotoxic and inflammatory activities as shown by TNF, IFN{gamma} and CD107a/b increase following anti-BTN3A activation. Moreover, BTN3A upregulation and V{gamma}9V{delta}2 T cell infiltration were observed in a lung biopsy from a fatal SARS-CoV-2 infection, as compared to HV. In vitro, SARS-CoV-2 infection significantly increased BTN3A expression in macrophages and lung cell lines. The activation via BTN3A enhanced the anti-SARS-CoV-2 V{gamma}9V{delta}2 T cells cytotoxicity and IFN-{gamma} and TNF in SARS-CoV-2 infected patient. Increasing concentrations of anti-BTN3A were accompanied by an inhibition of viral replication. Altogether, these data suggest that V{gamma}9V{delta}2 T cells are important in the immune response against SARS-CoV-2 infection and that activation by an anti-BTN3A antibody may enhance their response.

Expression of ACE2 receptor, soluble ACE2, Angiotensin I, Angiotensin II and Angiotensin (1-7), is modulated in COVID-19 patients (preprint)

Although SARS-CoV-2 is primarily a pulmonary-tropic virus, it is nonetheless responsible for multi-organ failure in patients with severe forms of COVID-19, particularly those with hypertension or cardiovascular disease. Infection requires virus binding to the angiotensin I converting enzyme 2 (ACE2) monocarboxypeptidase, a regulator of blood pressure homeostasis through its ability to catalyze the proteolysis of Angiotensin II (AngII) into Ang(1-7). Although assumed, it had not been proven so far whether the SARS-CoV-2 replication in COVID-19 patients could modulate the expression of the ACE2 receptor and/or the AngII plasma levels. We demonstrate here, that in COVID-19 patients the ACE2 mRNA expression is markedly reduced in circulating blood cells. This ACE2 gene dysregulation mainly affects the monocytes which also show a lower expression of membrane ACE2 protein. Moreover, a significant decrease in soluble ACE2 plasma levels is observed in COVID-19 patients, whereas the concentration of sACE2 returns to normal levels in patients recovered from COVID-19. In the plasma of COVID-19 patients, we also found an increase in AngI and AngII. On the other hand, the plasma levels of Ang(1-7) remains almost stable in COVID-19 patients. Despite the Ang(1-7) presence in the plasma of COVID-19 patients it seems insufficient to prevent the effects of massive AngII accumulation. These are the first direct evidence that the SARS-CoV-2 may affect the expression of blood pressure regulators with possible harmful consequences on COVID-19 outcome.

An ultra-sensitive, ultra-fast whole blood monocyte CD169 assay for COVID-19 screening (preprint)

CoVID-19 is an unprecedented epidemic, globally challenging health systems, societies, and economy. Its diagnosis relies on molecular methods, with drawbacks revealed by current use as mass screening. Monocyte CD169 upregulation has been reported as a marker of viral infections, we evaluated a flow cytometry three-color rapid assay of whole blood monocyte CD169 for CoVID-19 screening. Outpatients (n=177) with confirmed CoVID-19 infection, comprising 80 early-stage ([≤]14 days after symptom onset), 71 late-stage ([≥]15 days), and 26 asymptomatic patients received whole blood CD169 testing in parallel with SARS-CoV-2 RT-PCR. Upregulation of monocyte CD169 without polymorphonuclear neutrophil CD64 changes was the primary endpoint. Sensitivity was 98% and 100% in early-stage and asymptomatic patients respectively, specificity was 50% and 84%. Rapid whole blood monocyte CD169 evaluation was highly sensitive when compared with RT-PCR, especially in early-stage, asymptomatic patients whose RT-PCR tests were not yet positive. Diagnostic accuracy, easy finger prick sampling and minimal time-to-result (15-30 minutes) rank whole blood monocyte CD169 upregulation as a potential screening and diagnostic support for CoVID-19. Secondary endpoints were neutrophil CD64 upregulation as a marker of bacterial infections and monocyte HLA-DR downregulation as a surrogate of immune fitness, both assisting with adequate and rapid management of non-CoVID cases.

A theoretical analysis of the putative ORF10 protein in SARS-CoV-2 (preprint)

Found just upstream of the 3'-untranslated region in the SARS-CoV-2 genome is the putative ORF10 which has been proposed to encode for the hypothetical ORF10 protein. Even though current research suggests this protein is not likely to be produced, further investigations into this protein are still warranted. Herein, this study uses multiple bioinformatic programs to theoretically characterize and construct the ORF10 protein in SARS-CoV-2. Results indicate this protein is mostly ordered and hydrophobic with high protein-binding propensity, especially in the N-terminus. Although minimal, an assessment of twenty-two missense mutations for this protein suggest slight changes in protein flexibility and hydrophobicity. When compared against two other protein models, this study's model was found to possess higher quality. As such, this model suggests the ORF10 protein contains a {beta}--{beta} motif with a {beta}-molecular recognition feature occurring as the first {beta}-strand. Furthermore, this protein also shares a strong phylogenetic relationship with other putative ORF10 protein's in closely related coronaviruses. Despite not yielding evidence for the existence of this protein within SARS-CoV-2, this study does present theoretical examinations that can serve as platforms to drive additional experimental work that assess the biological relevance of this hypothetical protein in SARS-CoV-2.

GNS561 exhibits potent in vitro antiviral activity against SARS-CoV-2 through autophagy inhibition (preprint)

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) has spread quickly worldwide, with more than 29 million cases and 920,000 deaths. Interestingly, coronaviruses were found to subvert and hijack the autophagic process to allow their viral replication. One of the spotlights had been focused on the autophagy inhibitors as a target mechanism effective in the inhibition of SARS-CoV-2 infection. Consequently, chloroquine (CQ) and hydroxychloroquine (HCQ), a derivative of CQ, was suggested as the first potentially be therapeutic strategies as they are known to be autophagy inhibitors. Then, they were used as therapeutics in SARS-CoV-2 infection along with remdesivir, for which the FDA approved emergency use authorization. Here, we investigated the antiviral activity and associated mechanism of GNS561, a small basic lipophilic molecule inhibitor of late-stage autophagy, against SARS-CoV-2. Our data indicated that GNS561 showed the highest antiviral effect for two SARS-CoV-2 strains compared to CQ and remdesivir. Focusing on the autophagy mechanism, we showed that GNS561, located in LAMP2-positive lysosomes, together with SARS-CoV-2, blocked autophagy by increasing the size of LC3-II spots and the accumulation of autophagic vacuoles in the cytoplasm with the presence of multilamellar bodies characteristic of a complexed autophagy. Finally, our study revealed that the combination of GNS561 and remdesivir was associated with a strong synergistic antiviral effect against SARS-CoV-2. Overall, our study highlights GNS561 as a powerful drug in SARS-CoV-2 infection and supports that the hypothesis that autophagy inhibitors could be an alternative strategy for SARS-CoV-2 infection.

Monocytes and macrophages, targets of SARS-CoV-2: the clue for Covid-19 immunoparalysis (preprint)

To date, the Covid-19 pandemic affected more than 18 million individuals and caused more than 690, 000 deaths. Its clinical expression is pleiomorphic and severity is related to age and comorbidities such as diabetes and hypertension. The pathophysiology of the disease relies on aberrant activation of immune system and lymphopenia that has been recognized as a prognosis marker. We wondered if the myeloid compartment was affected in Covid-19 and if monocytes and macrophages could be infected by SARS-CoV-2. We show here that SARS-CoV-2 efficiently infects monocytes and macrophages without any cytopathic effect. Infection was associated with the secretion of immunoregulatory cytokines (IL-6, IL-10, TGF-{beta}) and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In addition, we found that in vitro macrophage polarization did not account for the permissivity to SARS-CoV-2, since M1-and M2-type macrophages were similarly infected. Finally, in a cohort of 76 Covid-19 patients ranging from mild to severe clinical expression, all circulating monocyte subsets were decreased, likely related to massive emigration into tissues. Monocytes from Covid-19 patients exhibited decreased expression of HLA-DR and increased expression of CD163, irrespective of the clinical status. Hence, SARS-CoV-2 drives circulating monocytes and macrophages inducing immunoparalysis of the host for the benefit of Covid-19 disease progression.

Daytime variation in SARS-CoV-2 infection and cytokine production (preprint)

S. Ray and A. Reddy recently anticipated the implication of circadian rhythm in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of the coronavirus disease (Covid-19). In addition to its key role in the regulation of biological functions, the circadian rhythm has been suggested as a regulator of viral infections. Specifically, the time of day of infection was found critical for illness progression, as has been reported for influenza, respiratory syncytial and parainfluenza type 3 viruses. We analyzed circadian rhythm implication in SARS-CoV-2 virus infection of isolated human monocytes, key actor cells in Covid-19 disease, from healthy subjects. The circadian gene expression of Bmal1 and Clock genes was investigated with q-RTPCR. Monocytes were infected with SARS-CoV-2 virus strain and viral infection was investigated by One-Step qRT-PCR and immunofluorescence. Interleukin (IL)-6, IL-1{beta} and IL-10 levels were also measured in supernatants of infected monocytes. Using Cosinor analysis, we showed that Bmal1 and Clock transcripts exhibited circadian rhythm in monocytes with an acrophase and a bathyphase at Zeitgeber Time (ZT)6 and ZT17. After forty-eight hours, the amount of SARS-CoV-2 virus increased in the monocyte infected at ZT6 compared to ZT17. The high virus amount at ZT6 was associated with significant increased release in IL-6, IL-1{beta} and IL-10 compared to ZT17. Our results suggest that time day of SARS-CoV-2 infection affects viral infection and host immune response. They support consideration of circadian rhythm in SARS-CoV-2 disease progression and we propose circadian rhythm as a novel target for managing viral progression. ImportanceThe implication of circadian rhythm (CR) in pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been recently anticipated. The time of day of infection is critical for illness progression as reported for influenza, respiratory syncytial and parainfluenza type 3 viruses. In this study, we wondered if SARS-CoV-2 infection and cytokine production by human monocytes, innate immune cells affected by Covid-19, were regulated by CR. Our results suggest that time day of SARS-CoV-2 infection affects viral infection and host immune response. They support consideration of circadian rhythm in SARS-CoV-2 disease progression and we propose circadian rhythm as a novel target for managing viral progression.