Mid-density Gene Expression profiling of SARSCOV-2 infected samples using Applied BiosystemsTM TaqManTM Flexible Array Panels

Background/Objectives: The broad spectrum of clinical manifestations from SARS-COV-2 infection and observed risk factors for severe disease highlight the importance of understanding molecular mechanisms underlying SARS-CoV-2 associated disease pathogenesis. Research studies have identified a large number of host proteins that play roles in viral entry, innate immune response, or immune signalling during infection. The ability to interrogate subsets of these genes simultaneously within SARSCOV-2 infected samples is critical to understanding how their expression contribute to phenotypic variability of the disease caused by the pathogen. Method(s): 30 Nasopharyngeal swab were obtained and included SARS-CoV-2 infected and control samples. RNA was extracted, reverse transcribed and loaded onto flexible TaqMan array panels designed specifically for targeting the most cited genes related to SARS-COV-2 entry and restriction factors as well as cytokines, chemokines, and growth factors involved in the pathogenesis. Result(s): Our data indicated that not only were the levels of several of these host factors differentially modulated between the two study groups, but also that SARS-CoV-2 infected subjects presented with greater frequency of several important inflammatory cytokines and chemokines such as CCL2, CCL3, IFNG, entry receptors such as ACE2, TMRPS11A, and host restriction factors including LY6E and ZBP1. Conclusion(s): TaqMan array plates provide a fast, midthroughput solution to determine the levels of several virus and host-associated factors in various cell types and add to our understanding of how the pathogenesis may vary depending on gender, age, infection site etc.

SARS-CoV-2 infection disrupts trophoblast function and placental syncytial barrier integrity via its accessory protein, ORF3a

Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.

Antiviral activity of the human endogenous retrovirus-R envelope protein against SARS-CoV-2

Coronavirus-induced disease-19 (COVID-19), caused by SARS-CoV-2, is still a major global health challenge. Human endogenous retroviruses (HERVs) represent retroviral elements that were integrated into the ancestral human genome. HERVs are important in embryonic development as well as in the manifestation of diseases, including cancer, inflammation, and viral infections. Here, we analyze the expression of several HERVs in SARS-CoV-2-infected cells and observe increased activity of HERV-E, HERV-V, HERV-FRD, HERV-MER34, HERV-W, and HERV-K-HML2. In contrast, the HERV-R envelope is downregulated in cell-based models and PBMCs of COVID-19 patients. Overexpression of HERV-R inhibits SARS-CoV-2 replication, suggesting its antiviral activity. Further analyses demonstrate the role of the extracellular signal-regulated kinase (ERK) in regulating HERV-R antiviral activity. Lastly, our data indicate that the crosstalk between ERK and p38 MAPK controls the synthesis of the HERV-R envelope protein, which in turn modulates SARS-CoV-2 replication. These findings suggest the role of the HERV-R envelope as a prosurvival host factor against SARS-CoV-2 and illustrate a possible advantage of integration and evolutionary maintenance of retroviral elements in the human genome.Copyright © 2023 The Authors.

Impact of Covid-19 and Host Factors on the Humoral Immune Repertoire in Treated Hiv

Background: People with HIV (PWH) on antiretroviral therapy (ART) appear to be at higher risk for worse COVID-19 outcomes, but the underlying mechanisms-including effects of COVID-19 and host factors on the broader humoral immune repertoire-are poorly understood. Method(s): REPRIEVE enrolled a global cohort of ART-treated PWH ages 40-75. COVID+ was defined by positive receptor binding domain IgG or IgA from annual visits 5/2020-2/2021. Antibody isotype, subclass, and Fc receptor Luminex arrays to SARS-CoV-2, CMV, EBV, HSV, HIV, influenza, pneumococcus, and RSV were assessed. Report of COVID diagnosis (collected every 4 months) was defined as mild, moderate, or severe (asymptomatic if no clinical diagnosis but IgG/ IgA+). FDR-corrected regression was used to assess effects of 1) COVID+ on non- SARS-CoV-2 repertoire in full cohort and 2) host factors on non-SARS-CoV-2 and SARS-CoV-2 repertoire in COVID- and COVID+ cohorts, respectively, adjusted for age, sex, region, nadir CD4, and HIV VL at entry. Result(s): Of 2,464 unvaccinated participants, 283 (11%) were COVID+;260 (92%) were asymptomatic. Median age was 53, 35% were women, 50% had nadir CD4 < 200, median current CD4 was 649, and 97% had HIV VL < 400. In the full cohort, COVID+ was associated with higher CMV PP65 IgG3 and FcgammaRIIA (P< 0.05);COVID severity was not associated with the non-SARS-CoV-2 repertoire. Among COVID-, older age, female sex, and lower nadir CD4 were associated with higher EBV and CMV responses;IgG1 levels were higher in women for all non-SARS-CoV-2 antigens assessed (P< 0.05). Among COVID+, higher BMI was associated with amplified SARS-CoV-2 IgG, IgA, IgM, and FcgammaRIIA responses (P< 0.05). Lower nadir CD4 was associated with a SARSCoV- 2 repertoire shift toward IgM and FcgammaRIIB (P< 0.05). Age and sex were not associated with SARS-CoV-2-related repertoire changes in COVID+. Conclusion(s): Our analysis presents a comprehensive view of host factors associated with the humoral immune repertoire among a global cohort of ART-treated PWH. COVID's association with higher CMV responses may suggest increased susceptibility to or a consequence of persistent inflammation after infection. The striking amplification of SARS-CoV-2 responses with higher BMI suggests an excessive inflammatory response. Lower nadir CD4 was related to uncontrolled extra-follicular and inhibitory SARS-CoV-2 responses, which are unlikely to be protective. These findings may suggest mechanisms underlying factors associated with worse COVID-19 outcomes among PWH. (Figure Presented).

Targeting virus-induced vulnerabilities using synthetic lethality as a new class of host-based antivirals

RNA viruses are the major class of human pathogens responsible for many global health crises, including the COVID-19 pandemic. However, the current repertoire of U.S. Food and Drug Administration (FDA)-approved antivirals is limited to only nine out of the known 214 human-infecting RNAviruses, and almost all these antivirals target viral proteins. Traditional antiviral development generally proceeds in a virus-centric fashion, and successful therapies tend to be only marginally effective as monotherapies, due to dose-limiting toxicity and the rapid emergence of drug resistance. Host-based antivirals have potential to alleviate these shortcomings, but do not typically discriminate between infected and uninfected cells, thus eliciting unintended effects. In infected cells where host proteins are repurposed by a virus, normal host protein functions are compromised;a situation analogous to a loss-of-function mutation, and cells harboring the hypomorph have unique vulnerabilities. As well-established in model systems and in cancer therapeutics, these uniquely vulnerable cells can be selectively killed by a drug that inhibits a functionally redundant protein. This is the foundation of synthetic lethality (SL). To test if viral induced vulnerabilities can be exploited for viral therapeutics, we selectively targeted synthetic lethal partners of GBF1, a Golgi membrane protein and a critical host factor for many RNA viruses including poliovirus, Coxsackievirus, Dengue, Hepatitis C and E virus, and Ebola virus. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. A genome-wide chemogenomic CRISPR screen identified synthetic lethal partners of GBF1 and revealed ARF1 as the top hit. Disruption of ARF1, selectively killed cells that synthesize poliovirus 3A alone or in the context of a poliovirus replicon. Combining 3A expression with sub-lethal amounts of GCA - a specific inhibitor of GBF1 further exacerbated the GBF1-ARF1 SL effect. Together our data demonstrate proof of concept for host-based SL targeting of viral infection. We are currently testing all druggable synthetic lethal partners of GBF1 from our chemogenomic CRISPR-screen, in the context of dengue virus infection for their abilities to selectively kill infected cells and inhibit viral replication and infection. Importantly, these SL gene partners of viral-induced hypomorphs only become essential in infected cells and in principle, targeting them will have minimal effects on uninfected cells. Our strategy to target SL interactions of the viral-induced hypomorph has the potential to change the current paradigm for host-based therapeutics that can lead to broad-spectrum antivirals and can be applied to other intracellular pathogens. This work is supported by National Institutes of Health grants R01 GM112108 and P41 GM109824, R21 AI151344 and foundation grant FDN-167277 from the Canadian Institutes of Health Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Intracellular "in silico microscopes"-fully 3D spatial Hepatitis C virus replication model simulations

Virus pandemics and endemics cause enormous pain and economic, political, and social costs and turmoil. While the Covid19 pandemics induced obvious damages, the "silent" Hepatitis C virus (HCV) infection induced liver damages are the main reason for liver transplantations. HCV-generated virus genome replication factories are housed within virus-induced intracellular structures termed membranous webs (MW) which are derived from the Endoplasmatic Reticulum (ER). Up to now, very advanced experimental data such as highly spatially resolved fuorescence and electron-tomography data often do not enter computational HCV viral RNA (vRNA) cycle models. Based upon difusion-reaction partial differential equation (PDE) models, we are developing fully 3D resolved "in silico microscopes" to mirror in vitro / in vivo experiments of the intracellular vRNA cycle dynamics. Our first models described the major components (vRNA, non-structural viral proteins-NSPs-and a host factor). The next steps incorporated additional parameters: Different aggregate states of vRNA and NSPs, and population dynamics inspired difusion and reaction co-Effcients instead of multilinear ones. Our work in progress framework presently is merging effects restricted to 2D manifold surface grids (e.g. ER surface, NSP difusion) with others occurring in 3D volume meshes (e.g. cytosol, host factor supply). We estimate and incorporate realistic parameters such as NSP difusion constants. The simulations are performed upon experimental data based reconstructed cell geometries and help understanding the relation of form and function of virus replication. In the long run, our framework might help to facilitate the systematic development of Effcient direct antiviral agents and vaccines.

Effect of miRNAs, Proinflammatory Cytokines and ACE2 in COVID-19 Pathophysiology

Background: Angiotensin-converting enzyme 2 (ACE2) is the main cellular receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and acts as a pro-inflammatory mediator of Coronavirus disease (COVID-19). The clinical outcome of SARS-CoV-2 infection is influenced by the pro-inflammatory mediators. The specific microRNAs (miRNAs) influence the ACE2 expression and are accountable for the increased circulatory pro-inflammatory mediator levels. Thus, host factors play a crucial role in COVID-19 pathophysiology. The pathogenesis of COVID-19 disease is not well understood. Hence we comprehended the role of miRNAs, pro-inflammatory cytokines, and ACE2 genes in COVID-19 pathophysiology. Method(s): We utilized multiple databases, specifically EMBASE, PubMed (Medline), and Google Scholar, for our search. Discussion(s): SARS-CoV-2 genes could be the target of host miRNAs. The miRNAs regulate the expression of ACE2 in various organs, including the kidney, heart, blood vessels, and lung. ACE2 acts as a pro-inflammatory mediator of SARS-CoV-2 associated disease. Pro-inflammatory cytokines (IL-6, IL-1beta, and TNF) have been associated with severe COVID-19 disease. Hence variation in expression of miRNAs would influence the regulation of COVID-19 pathophysiology. The clinical outcomes of COVID-19 are variable which could be linked with the difference in binding of host miRNA to the target genes. Conclusion(s): Correlation of these genes with severe or critical stages of patients will provide bio-markers for the severity of lung inflammation which would be useful in the rapid identification of patients in need of hospital admission. Analysis of the relationship between the miRNAs and ACE2 will be helpful in designing anti-miR therapy for ACE2-related SARS-CoV-2 infection.Copyright © 2021 Bentham Science Publishers.

Neuropilins: C-end rule peptides and their association with nociception and COVID-19.

Viral internalization is aided by host cell surface receptors. In the case of SARS-CoV-2 and SARS-CoV, the primary host receptor is the angiotensin-converting enzyme 2 (ACE2). Considering the disparities in the transmission rate and viral tropism of the two coronaviruses, additional host factors were suspected. Recently, a novel host factor for SARS-CoV-2 entry, neuropilin-1 (NRP-1) has been identified. These receptors potentiate viral infection in the presence of other host factors like ACE2. Through its C-end rule (CendR) motif exposed following furin processing, the SARS-CoV-2 spike protein binds to the CendR pocket of NRP-1 and achieves cell entry through endocytosis. The binding of SARS-CoV-2 spike protein to the NRP-1 receptor interferes with the docking of its endogenous ligand VEGF-A, signaling that would otherwise promote nociception. This review looks at the function of neuropilins and how it contributes to SARS-CoV-2 infection and nociception.

Invasive fungal infections in COVID-19 patients from Pakistan

Objective: The objective of this study is to report the frequency and clinical characteristic of IFI in COVID-19 patients. Method(s): This observational study was conducted in Karachi, Pakistan from March 2020-April 2021. Patients with COVID-19 associated aspergillosis (CAPA) were diagnosed using ECMM/ISHAM criteria modified to include tracheal aspirate culture and/or Galactomannan Index (GMI) >4.5 in the possible CAPA category. COVID-19 associated candidemia (CAC) was defined by isolation of Candida species from blood cultures. COVID-19 associated mucormycosis (CAM) was defined as updated EORTC/MSG criteria with inclusion of COVID-19 as host factor. Pneumocystis jirovecii pneumonia (PJP) was defined by consistent clinical and radiological features and PCR positivity. Result(s): During the study period a total of 123 (3.3%) IFI in 3506 hospitalized COVID-19 patients were identified. This included 78 (2.2%) CAPA patients (42 probable;36 possible), 29 (0.8%) CAC (5 C. auris;24 non-C. auris), 10 (0.3%) CAM (7 pulmonary;3 rhinocerebral), 3 (0.08%) PJP and three (0.08%) cases of rare invasive fungal infections (2 C. neoformans;1 Trichosporon asahii). Outcome data was available on 117/123 patients. Of these 117 patients, 78 expired (66.7%). These include 52/74 (70%) CAPA patients, 17/27 (63%) CAC patients, 7/10 (70%) CAM patients and 2/3 (67%) PJP patients. Conclusion(s): We report a rate of 3.3% IFI amongst hospitalized COVID-19 patients at our center. We consider this rate to be an underestimate due to less bronchoscopic procedures and inclusion of only candidemia cases. We also report higher mortality rate with IFI in our patients than global data probably due to delayed diagnosis, co-infections and limited therapeutic options.

Conserved requirement of autophagy-related effectors during coronavirus replication.

The recurrence of zoonotic transmission events highlights the need for novel treatment strategies against emerging coronaviruses (CoVs), namely SARS-CoV, MERS-CoV and most notably SARS-CoV-2. Our recently performed genome-wide CRISPR knockout screen revealed a list of conserved pan-coronavirus as well as MERS-CoV or HCoV-229E-specific host dependency factors (HDF) essential during the viral life cycle. Intriguingly, we identified the macroautophagy/autophagy pathway-regulating immunophilins FKBP8, TMEM41B, and MINAR1 as conserved MERS-CoV, HCoV-229E, SARS-CoV, and SARS-CoV-2 host factors, which further constitute potential targets for therapeutic intervention by clinically approved drugs.

Prevalence of Olfactory Dysfunction with the Omicron Variant of SARS-CoV-2: A Systematic Review and Meta-Analysis.

The omicron variant is thought to cause less olfactory dysfunction than previous variants of SARS-CoV-2, but the reported prevalence differs greatly between populations and studies. Our systematic review and meta-analysis provide information regarding regional differences in prevalence as well as an estimate of the global prevalence of olfactory dysfunction based on 62 studies reporting information on 626,035 patients infected with the omicron variant. Our estimate of the omicron-induced prevalence of olfactory dysfunction in populations of European ancestry is 11.7%, while it is significantly lower in all other populations, ranging between 1.9% and 4.9%. When ethnic differences and population sizes are considered, the global prevalence of omicron-induced olfactory dysfunction in adults is estimated to be 3.7%. Omicron's effect on olfaction is twofold to tenfold lower than that of the alpha or delta variants according to previous meta-analyses and our analysis of studies that directly compared the prevalence of olfactory dysfunction between omicron and previous variants. The profile of the prevalence differences between ethnicities mirrors the results of a recent genome-wide association study that connected a gene locus encoding an odorant-metabolizing enzyme, UDP glycosyltransferase, to the extent of COVID-19-related loss of smell. Our analysis is consistent with the hypothesis that this enzyme contributes to the observed population differences.

GRP78, a Novel Host Factor for SARS-CoV-2: The Emerging Roles in COVID-19 Related to Metabolic Risk Factors.

The outbreak of coronavirus disease 19 (COVID-19), caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in an unprecedented amount of infection cases and deaths, leading to the global health crisis. Despite many research efforts, our understanding of COVID-19 remains elusive. Recent studies have suggested that cell surface glucose-regulated protein 78 (GRP78) acts as a host co-receptor for SARS-CoV-2 infection and is related to COVID-19 risks, such as older age, obesity, and diabetes. Given its significance in a wide range of biological processes, such as protein homeostasis and cellular signaling, GRP78 might also play an important role in various stages of the viral life cycle and pathology of SARS-CoV-2. In this perspective, we explore the emerging and potential roles of GRP78 in SARS-CoV-2 infection. Additionally, we discuss the association with COVID-19 risks and symptoms. We hope this review article will be helpful to understand COVID-19 pathology and promote attention and study of GRP78 from many clinical and basic research fields.

EXPRESSION OF EPITHELIAL PROTEASES PREDICTS SARS-COV-2 ENTRY IN HUMAN ENTEROIDS

Introduction: Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium and can persist long after the respiratory infection has cleared. We previously observed that intestinal SARS-CoV-2 infection levels varied by individual donors and did not correlate positively with ACE2, the cognate SARS-CoV-2 receptor. Therefore we aimed to delineate host factors that influence viral infection in the intestine. Methods: Published dataset GSE75214 was downloaded and expression levels of select genes were querried. Primary human ileal spheroids (enteroids), derived from healthy donors and patients with Crohn's disease (CD), were grown on 2D transwells until confluent. Cells were differentiated for 3d before infection with a modified vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and GFP for 1h at a multiplicity of infection of ~0.5. Cells were harvested pre-infection and 24h after infection and expression of select genes was performed by qRT-PCR. Expression data were fit to a linear regression model to predict viral RNA levels. Results: Small intestine biopsy samples from CD patients demonstrated a reduction in ACE and an increase in CTSB and CTSL expression during active inflammation compared to healthy controls. Viral RNA expression did not correlate with ACE2 expression in CD enteroids. A subset of CD enteroids exhibited enhanced protease expression (TMPRSS2, TMPRSS4, CTSL), each of which correlated with higher viral RNA levels (P=0.04, P=0.002, P=0.006, respectively). Expression of these proteases was higher in the pre-infection for the sample subset. Principle component analysis of uninfected expression data demonstrated these samples clustered separately from the others, with the difference driven by TMPRSS2, TMPRSS4, and CTSL. Modeling viral RNA levels based on gene expression revealed expression levels of these proteases are a predictive expression signature. Conclusions: Host protease expression can predict SARS-CoV-2 infection and represent potential therapeutic targets for COVID-19. This is consistent with the recent report showing that cathepsin inhibition reduces SARS-CoV-2 spike-mediated syncytia formation. High expression of these proteases in the intestine may also be a novel biomarker for the risk of intestinal complications associated with COVID-19.(Figure Presented)RNA data from dataset GSE75214 demonstrating reduced ACE2 and increased CTSB and CTSL in patients with Crohn's disease during active inflammation compared to healthy controls. (Figure Presented) Enteroids from healthy control donors and patients with Crohn's disease were grown in 2D transwells and expression of indicated genes was assessed in pre-infection (A) and after infection with VSV-SARS-CoV-2 (B)

UNRAVELING the ANTIVIRAL ACTIVITY of PLITIDEPSIN by ULTRASTRUCTURAL ANALYSIS

Background: The use of compounds against highly conserved cellular host factors required to complete the replication cycle of distinct viruses such as SARS-CoV-2 offers a common solution to diverse viral threats. This approach is especially relevant for pan-antiviral effects given that viruses converge at intracellular steps such as viral genome replication and protein production. Currently, there are only a limited number of approved drugs involved in targeting intracellular host factors. One of these compounds is plitidiepsin, which has shown a potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A. Plitidepsin inhibits nucleocapsid viral protein expression and viral induced cytopathic effect in vitro. In addition, it also reduces genomic and subgenomic RNA expression. However, how plitidepsin exerts its antiviral activity remains unknown. Methods: Current models of SARS-CoV-2 replication propose that upon viral fusion, non-structural viral proteins form a replication-transcription complex that associates to compartments with a double membrane vesicle (DMV) morphology that shelters the viral genome replication. Here we have used an electron microcopy analysis to explore the antiviral effect of plitidepsin and its impact on SARS-CoV-2 replication and DMV formation on target Vero E6 cells. Results: This ultrastructural analysis allowed to recapitulate the SARS-CoV-2 infectious life cycle, where evident viral DMV formation was observed as well as viral budding events along with cell-associated viruses. However, in cells treated with plitidepsin at different non-toxic concentrations (0.2 and 0.05 μ M) there was a lack of viral DMV formation and a complete absence of viral particles. Complementary SARS-CoV-2 nucleocapsid and dsRNA immunogold labelling unambiguously confirmed the lack of viral replication in plitidepsin-treated cells. Overall, these data indicate that plitidepsin treatment abrogated the formation of DMVs, and the detection of nucleocapsid or dsRNA viral products. Conclusion: Electron microscopy ultrastructural analysis coupled to immunogold labelling of SARS-CoV-2 products offer a unique approach to understand how antivirals work. This knowledge is key to identify the mechanism of action of promising compounds interfering with host factors whose implication in strategic biological processes can be applied as pan-antiviral strategies.

SARS-CoV-2 PLASMA VIREMIA and DYSREGULATED IMMUNITY in SEVERE COVID-19

Background: Critical COVID-19 occurs ca. 7d from symptoms onset, and is associated to immune dysregulation as well as SARS-CoV-2 detection in plasma (i.e. viremia). We hereby sought to detail the association between SARS-CoV-2 viremia measured at the end of the first week of disease and immune phenotypes/function in COVID-19 patients. Methods: We consecutively enrolled patients hospitalized in the acute phase of ascertained SARS-CoV-2 pneumonia. In this disease stage, we studied SARS-CoV-2 viremia (RT-PCR) and cytokines (MACSPlex), HLA-DR+CD38+ activated, GRZB+PRF+ pro-cytolitic T-cells, intracellular cytokine production (IL-2, IFNγ, TNFα, IL-4, IL-17A) after SARS-CoV-2 challenge (S-N-M-peptide pool). Simultaneous Th1-cytokines production (polyfunctionality) and amount (iMFI) was assessed. Humoral response: anti-S1/S2 IgG, anti-RBD total-Ig, IgM, IgA, IgG1 and IgG3 (ELISA), pseudoviruses neutralization (ID50) and Fc-mediated functions (%ADCC). Results: Out of 54 patients, 27 had detectable viremia (V+). Albeit comparable age and co-morbidities, V+ patients more frequently required non-invasive/invasive ventilatory support (p=0.035), with a trend to higher death (p=0.099) vs patients with undetectable viremia (V-)(Fig.1A). V+ displayed higher circulating IFN-α (p=0.002) and IL-6 (0.003), lower activated HLA-DR+CD38+CD4 (p=0.01) and CD8 (p=0.02), with no differences in GRZB+PRF+ T-cells. V+ featured reduced SARS-CoV-2-specific cytokine-producing T-cells, reaching significance for IFNγ+CD4 (p=0.02), TNFα+CD8, IL-4+CD8 (p=0.04) (Fig.1B-C), with lower bi-and tri-functional SARS-CoV-2-specific CD4 Th1, reaching significance for IL-2+TNFα+CD4 (p=0.03) (Fig.1D). A trend towards lower cytokines iMFI in bi-and tri-functional SARS-CoV-2-specific CD4 Th1 was observed in V+, reaching significance for IL-2+TNFα+CD4, p=0.004. V+ displayed lower anti-S IgG, anti-RBD total-Ig, IgM, IgG1 and IgG3 (Fig.1E), with lower ID50 and %ADCC vs V-(Fig.1F-G). Conclusion: Hospitalized COVID-19 patients with detectable plasma SARS-CoV-2 RNA in the acute phase of disease present worse outcome, higher inflammatory cytokines, fewer activated and SARS-CoV-2-specific polyfunctional T-cells, suggesting a link between SARS-CoV-2 viremia at the end of the first stage of disease and immune dysregulation. Whether high ab initium viral burden and/or intrinsic host factors contribute to a delayed and/or exhausted immune response in severe COVID-19 remains to be elucidated, to further inform strategies of targeted therapeutic interventions.

Insights into potential mechanisms of asthma patients with COVID-19: A study based on the gene expression profiling of bronchoalveolar lavage fluid.

BACKGROUND: The 2019 novel coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a major challenge threatening the global healthcare system. Respiratory virus infection is the most common cause of asthma attacks, and thus COVID-19 may contribute to an increase in asthma exacerbations. However, the mechanisms of COVID-19/asthma comorbidity remain unclear. METHODS: The "Limma" package or "DESeq2" package was used to screen differentially expressed genes (DEGs). Alveolar lavage fluid datasets of COVID-19 and asthma were obtained from the GEO and GSV database. A series of analyses of common host factors for COVID-19 and asthma were conducted, including PPI network construction, module analysis, enrichment analysis, inference of the upstream pathway activity of host factors, tissue-specific analysis and drug candidate prediction. Finally, the key host factors were verified in the GSE152418 and GSE164805 datasets. RESULTS: 192 overlapping host factors were obtained by analyzing the intersection of asthma and COVID-19. FN1, UBA52, EEF1A1, ITGB1, XPO1, NPM1, EGR1, EIF4E, SRSF1, CCR5, PXN, IRF8 and DDX5 as host factors were tightly connected in the PPI network. Module analysis identified five modules with different biological functions and pathways. According to the degree values ranking in the PPI network, EEF1A1, EGR1, UBA52, DDX5 and IRF8 were considered as the key cohost factors for COVID-19 and asthma. The H2O2, VEGF, IL-1 and Wnt signaling pathways had the strongest activities in the upstream pathways. Tissue-specific enrichment analysis revealed the different expression levels of the five critical host factors. LY294002, wortmannin, PD98059 and heparin might have great potential to evolve into therapeutic drugs for COVID-19 and asthma comorbidity. Finally, the validation dataset confirmed that the expression of five key host factors were statistically significant among COVID-19 groups with different severity and healthy control subjects. CONCLUSIONS: This study constructed a network of common host factors between asthma and COVID-19 and predicted several drugs with therapeutic potential. Therefore, this study is likely to provide a reference for the management and treatment for COVID-19/asthma comorbidity.

Impact of Overall Dexamethasone Exposure on Development of Invasive Pulmonary Aspergillosis in Hospitalized Patients with COVID-19

Background. Abbreviated courses of corticosteroids, such as dexamethasone, have demonstrated significant improvements in clinical outcomes among patients infected with COVID-19, although chronic corticosteroid use can predispose patients to opportunistic infections. The RECOVERY trial investigators showed reduced 28-day mortality among patients treated with 6 mg/day dexamethasone for up to 10 days, however in clinical practice the dosage and duration of dexamethasone therapy can vary widely based on severity of disease and provider discretion. Upon observing an anecdotal increase in the number of patients presenting with potential invasive aspergillosis during the third wave of COVID-19, we sought to evaluate the impact of overall dexamethasone exposure on the development of invasive pulmonary aspergillosis. Methods. Patients presenting to our institution from Dec. 2020 - Jan. 2021 with positive PCR for SARS-CoV-2 were screened for dexamethasone therapy. Assignment of high vs low dose dexamethasone groups were retrospectively made based on overall dexamethasone exposure. Low dose dexamethasone assignment was restricted to a total exposure of no more than 78 mg during a patient's hospitalization. Adjudication of invasive pulmonary aspergillosis was made based on criteria that included host factors, radiologic findings, clinical factors, and mycological evidence. Results. Dexamethasone therapy was provided to 202 patients admitted to the hospital with COVID-19. Invasive pulmonary aspergillosis was determined to be probable in n=7 patients based on European Organization for Research and Treatment of Cancer (EORTC) criteria, and in n=13 patients based on expanded criteria. Patients in the low dose dexamethasone group were less likely to be diagnosed with probable IPA based on EORTC criteria (n=0, 0% on low dose vs. n=7, 11% on high dose) as well as expanded criteria (n=9, 5% on low dose vs. n=11, 17% on high dose), p< 0.001. Conclusion. Patients hospitalized with COVID-19 receiving high-dose dexamethasone may be at a higher risk of opportunistic infections such as invasive pulmonary aspergillosis compared to patients who receive low-dose dexamethasone therapy. Further investigation is needed to obtain higher certainty of IPA diagnosis.

Investigation of patient and viral characteristics associated with SARS-CoV-2 vaccine breakthrough infections in Atlanta, GA

Purpose of Study Despite the tremendous success of SARSCoV- 2 vaccines, breakthrough infections occur and are being recognized with increasing frequency. It is unclear whether breakthrough infections are the result of host and/or viral factors. We examined clinical and viral genomic data from patients with SARS-CoV-2 infection after vaccination to elucidate factors contributing to breakthrough. Methods Used This study was conducted in the Emory Healthcare (EHC) System. Patients with vaccine breakthrough infection, defined as a positive PCR test ≥14 days after the final dose of an FDA approved vaccine, were identified by both routine surveillance and notification by treating clinicians. Vaccination status was obtained from the Georgia Registry of Immunization Transactions and Services records by the Georgia Emerging Infections Program. Clinical information was derived from electronic medical records and was compared to data from 2-3 matched controls per case. Residual SARS-CoV-2 positive nasopharyngeal (NP) samples were collected and underwent RNA extraction. SARSCoV- 2 genome sequencing was performed using random-primer cDNA synthesis, Nextera XT library preparation, and Illumina sequencing. Summary of Results Forty vaccine breakthrough cases were identified between March 22 and July 16, 2021. The median time from final vaccine dose to positive COVID-19 test was 91 days (range 15-163). Compared to 94 controls, vaccine breakthrough cases were significantly older (median 57.5 years vs 42.0 years, p<.0001). Individuals over 60 accounted for half of all breakthrough cases, and individuals over 40 accounted for 80%. Immunosuppressed individuals represented 37.5% of breakthrough cases compared to 25% of unvaccinated controls. Rates of symptomatic infection and severe disease leading to hospitalization were similar between cases and controls. There was no difference in SARS-CoV-2 RT-PCR cycle threshold (Ct) between cases (n=32, median Ct=20.7, interquartile range (IQR)- 10.3) and controls (n=94, median Ct=24.0, IQR= 7.0;p=0.34). SARS-CoV-2 genome sequences from 24 cases were compared to 116 baseline surveillance sequences from unvaccinated EHC patients. There was no distinct phylogenetic clustering of vaccine breakthrough cases, and their sequences belonged to the predominant lineage of the time. From March 22-June 19, B.1.1.7 (alpha) accounted for 78% of breakthrough infections and 77% of surveillance sequences. From June 20-July 16, B.1.617.2 (delta) accounted for 86% of breakthrough infections and 72% of surveillance sequences. No spike mutations or deletions were associated with vaccine breakthrough infections. Conclusions Overall, our findings suggest that host factors, such as older age and immunosuppression, play a more important role than viral factors in SARS-CoV-2 vaccine breakthrough infections. Further studies are needed to understand the potential impacts of waning immunity or poor immunogenicity in individuals who experience vaccine breakthrough infections.