Usefulness of real-time RT-PCR to understand the kinetics of SARS-CoV-2 in blood: a prospective study. (preprint)

Background SARS-CoV-2 viral load and kinetics assessed in serial blood samples from hospitalised COVID-19 patients by RT-PCR are poorly understood. Methods We conducted an observational, prospective case series study in hospitalised COVID-19 patients. Clinical outcome data (Intensive Care Unit admission and mortality) were collected from all patients until discharge. Viremia was determined longitudinally during hospitalisation, in plasma and serum samples using two commercial and standardised RT-PCR techniques approved for use in diagnosis of SARS-CoV-2. Viral load (copies/mL and log10) was determined with quantitative TaqPath TM COVID-19 test. Results SARS-CoV-2 viremia was studied in 57 hospitalised COVID-19 patients. Persistent viremia (PV) was defined as two or more quantifiable viral loads detected in blood samples (plasma/serum) during hospitalisation. PV was detected in 16 (28%) patients. All of them, except for one who rapidly progressed to death, cleared viremia during hospitalisation. Poor clinical outcome occurred in 62.5% of patients with PV, while none of the negative patients or those with sporadic viremia presented this outcome (p<0.0001). Viral load was significantly higher in patients with PV than in those with Sporadic Viremia (p<0.05). Patients presented PV for a short period of time: median time from admission was 5 days (Range=2-12) and 4.5 days (Range=2-8) for plasma and serum samples, respectively. Similar results were obtained with all RT-PCR assays for both types of samples. Conclusions Detection of persistent SARS-CoV-2 viremia, by real time RT-PCR, expressed as viral load over time, could allow identifying hospitalised COVID-19 patients at risk of poor clinical outcome.

SARS-CoV-2 viremia precedes an IL6 response in severe COVID-19 patients: results of a longitudinal prospective cohort (preprint)

Background: Interleukin 6 (IL6) levels and SARS-CoV-2 viremia have been correlated with COVID-19 severity. The association over time between them has not been assessed in a prospective cohort. Our aim was to evaluate the relationship between SARS-CoV-2 viremia and time evolution of IL6 levels in a COVID-19 prospective cohort. Methods: Secondary analysis from a prospective cohort including COVID-19 hospitalized patients from Hospital Universitario La Princesa between November 2020 and January 2021. Serial plasma samples were collected from admission until discharge. Viral load was quantified by Real-Time Polymerase Chain Reaction and IL6 levels with an enzyme immunoassay. To represent the evolution over time of both variables we used the graphic command twoway of Stata. Results: A total of 57 patients were recruited, with median age of 63 years (IQR [53-81]), 61.4% male and 68.4% caucasian. The peak of viremia appeared shortly after symptom onset in patients with persistent viremia (more than 1 sample with >1.3 log10 copies/ml) and also in those with at least one IL6>30 pg/ml, followed by a progressive increase in IL6 around 10 days later. Persistent viremia in the first week of hospitalization was associated with higher levels of IL6. Both IL6 and SARS-CoV-2 viral load were higher in males, with a quicker increase with age. Conclusions: In those patients with worse outcomes, an early peak of SARS-CoV-2 viral load precedes an increase in IL6 levels. Monitoring SARS-CoV-2 viral load during the first week after symptom onset may be helpful to predict disease severity in COVID-19 patients.

Circulating miRNAs and Cytokines Uncover Novel Physiopathological Mechanisms of COVID-19 (preprint)

Coronavirus Disease 2019 (COVID-19) pneumonia is a life-threatening infectious disease, especially for elderly patients with multiple comorbidities. Despite enormous efforts to understand its underlying etiopathogenic mechanisms, most of them remain elusive. In this study, we compared differential plasma miRNAs and cytokines profiles between COVID-19 and other community-acquired pneumonias (CAP). A first screening and subsequent validation assays in an independent cohort of patients revealed a signature of 15 dysregulated miRNAs between COVID-19 and CAP patients. Additionally, multivariate analysis displayed a combination of 4 miRNAs (miR-106b-5p, miR-221-3p, miR-25-3p and miR-30a-5p) that significantly discriminated between both pathologies. Search for targets of these miRNAs, combined with plasma protein measurements, identified a differential cytokine signature between COVID-19 and CAP that included EGFR, CXCL12 and IL-10. Significant differences were also detected in plasma levels of CXCL12, IL-17, TIMP-2 and IL-21R between mild and severe COVID-19 patients. These findings provide new insights into the etiopathological mechanisms underlying COVID-19.

Detection of SARS-Cov-2 RNA in serum is associated with increased mortality risk in hospitalized COVID-19 patients. (preprint)

BackgroundCOVID-19 has overloaded national health services worldwide. Thus, early identification of patients at risk of poor outcomes is critical. Our objective was to analyse SARS-CoV-2 RNA detection in serum as a severity biomarker in COVID-19. Methods and FindingsRetrospective observational study including 193 patients admitted for COVID-19. Detection of SARS-CoV-2 RNA in serum (CoVemia) was performed with samples collected at 48-72 hours of admission by two techniques from Roche and Thermo Fischer Scientific (TFS). Main outcome variables were mortality and need for ICU admission during hospitalization for COVID-19. CoVemia was detected in 50-60% of patients depending on technique. The correlation of Ct in serum between both techniques was good (intraclass correlation coefficient: 0.612; p < 0.001). Patients with CoVemia were older (p = 0.006), had poorer baseline oxygenation (PaO2/FiO2; p < 0.001), more severe lymphopenia (p < 0.001) and higher LDH (p < 0.001), IL-6 (p = 0.021), C-reactive protein (CRP; p = 0.022) and procalcitonin (p = 0.002) serum levels. We defined "relevant CoVemia" when detection Ct was < 34 with Roche and < 31 for TFS. These thresholds had 95% sensitivity and 35 % specificity. Relevant CoVemia predicted death during hospitalization (OR 9.2 [3.8 - 22.6] for Roche, OR 10.3 [3.6 - 29.3] for TFS; p < 0.001). Cox regression models, adjusted by age, sex and Charlson index, identified increased LDH serum levels and relevant CoVemia (HR = 9.87 [4.13-23.57] for TFS viremia and HR = 7.09 [3.3-14.82] for Roche viremia) as the best markers to predict mortality. ConclusionsCoVemia assessment at admission is the most useful biomarker for predicting mortality in COVID-19 patients. CoVemia is highly reproducible with two different techniques (TFS and Roche), has a good consistency with other severity biomarkers for COVID-19 and better predictive accuracy. AUTHOR SUMMARYCOVID-19 shows a very heterogeneous clinical picture. In addition, it has overloaded national health services worldwide. Therefore, early identification of patients with poor prognosis is critical to improve the use of limited health resources. In this work, we evaluated whether baseline SARS-CoV2 RNA detection in blood (CoVemia) is associated with worse outcomes. We studied almost 200 patients admitted to our hospital and about 50-60% of them showed positive CoVemia. Patients with positive CoVemia were older and had more severe disease; CoVemia was also more frequent in patients requiring admission to the ICU. Moreover, we defined "relevant CoVemia", as the amount of viral load that better predicted mortality obtaining 95% sensitivity and 35% specificity. In addition, relevant CoVemia was a better predictor than other biomarkers such as LDH, lymphocyte count, interleukin-6, and indexes used in ICU such as qSOFA and CURB65. In summary, detection of CoVemia is the best biomarker to predict death in COVID-19 patients. Furthermore, it is easy to be implemented and is reproducible with two techniques (Roche and Thermo Fisher Scientific) that are currently used for diagnosis in nasopharyngeal swabs samples.

In vivo structure and dynamics of the RNA genome of SARS-Cov-2 (preprint)

The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3-UTR and 5-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide direct evidence of interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE, forming a "high-order pseudoknot" embedding FSE, which might help ribosome stalling at frameshift sites. More importantly, we found that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome cyclization is weakened and genome domains remain stable. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, describes dynamics of RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies.

Structural characterization of cocktail-like targeting polysaccharides from Ecklonia kurome Okam and their anti-SARS-CoV-2 activities in vitro (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 {micro}M). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 x 10-6). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 {micro}M). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 {micro}g/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio (preprint)

Following the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa, respectively) that illustrate multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20C/G variants. One isolate, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His, membrane glycoprotein (M) p.Ala85Ser (Q677H) and nucleocapsid (N) p.Asp377Tyr (D377Y) mutations. The other isolate contains S N501Y and ORF8 Arg52Ile (R52I), which are two markers of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. It is also from a different clade and shares multiple mutations with the clade 20C/G viruses circulating in Ohio prior to December 2020. These two SARS-CoV-2 viruses emerging now in the United States add to the diversity of S gene shifts occurring worldwide and support multiple independent acquisition of S N501Y (in likely contrast to the unitary S D614G shift) occurring first during this period of the pandemic.

CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating off-the-shelf S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Enhanced Cholesterol-dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to its N-terminal Counterpart (preprint)

Membrane fusion is an important step for the entry of the lipid-sheathed viruses into the host cells. The fusion process is being carried out by fusion proteins present in the viral envelope. The class I viruses contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion, and is termed as fusion peptide. However, Severe Acute Respiratory Syndrome Coronavirus (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV is far more efficient than its N-terminal counterpart (FP) to induce hemifusion between small unilamellar vesicles. Moreover, the ability of IFP1 to induce hemifusion formation increases dramatically with growing cholesterol content in the membrane. Interestingly, IFP1 is capable of inducing hemifusion, but fails to open pore.

COVATOR: A Software for Chimeric Coronavirus Identification (preprint)

The term chimeric virus was not popular in the last decades. Recently, according to current sequencing efforts in discovering COVID-19 Secrets, the generated information assumed the presence of 6 Coronavirus main strains, but coronavirus diverges into hundreds of sub-strains. the bottleneck is the mutation rate. With two mutation/month, humanity will meet a new sub-strain every month. Tracking new sequenced viruses is urgently needed because of the pathogenic effect of the new sub-strains. here we introduce COVATOR, A user-friendly and python-based software that identifies viral chimerism. COVATOR aligns input genome and protein that has no known source, against genomes and protein with known source, then gives the user a graphical summary.

Evaluation of two RT-PCR techniques for SARS-CoV-2 RNA detection in serum for microbiological diagnosis (preprint)

Presence of SARS-CoV-2 RNA in serum (viraemia) in COVID-19 patients has been related to poor prognosis and death. The aim of this study was to evaluate the ability of two commercial reverse real-time-PCR (rRT-PCR) kits, cobas SARS-CoV-2 (Cobas test) and TaqPath COVID-19 CE-IVD RT-PCR Kit (Taqpath test), to detect viraemia in COVID-19 patients and their implementation as routine diagnosis in microbiology laboratory. This retrospective cohort study was conducted with 203 adult patients admitted to Hospital Universitario de La Princesa, (89 Intensive Care Unit and 114 ward) with at least one serum sample collected in the first 48 hours from admission. A total 265 serum samples were included for study. Evaluation of both rRT-PCR techniques was performed comparing with the gold standard, a Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit; considering at least one target as a positive result. Comparison of Cobas test and Taqpath test with the gold standard method, showed high values of specificity (93.75 and 92.19 respectively) and Positive Predictive Value (92.92 and 99.88 respectively). Nevertheless, sensitivity (53.72 and 73.63 respectively) and Negative Predictive Value (32.53 and 42.99 respectively) were lower; Kappa values were 0.35 for cobas test and 0.56 for Taqpath test. For both techniques, differences of viraemia detection between the ICU and non-ICU patients were significant (p<0.001). Consequently, SARS-CoV-2 viraemia positive results obtained by both rRT-PCR should be considered good tools and may help in handling COVID-19 patients. Moreover, these methods could be easily integrated in the routine laboratory COVID-19 diagnosis and may open new strategies based on an early COVID-19 treatment.

SARS-Cov-2 cysteine-like protease (Mpro) is immunogenic and can be detected in serum and saliva of COVID-19-seropositive individuals (preprint)

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2 specific antibodies are based on serum detection of antibodies to either the viral spike glycoprotein (the major target for neutralising antibodies) or the viral nucleocapsid protein that are known to be highly immunogenic in other coronaviruses. Conceivably, exposure of antigens released from infected cells could stimulate antibody responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other non-structural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titre IgG, IgM and IgA antibody responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher antibody titres in these assays associated with more severe disease and no cross-reactive antibodies against prior betacoronavirus were found. Remarkably, IgG antibodies specific for Mpro and other SARS-CoV-2 antigens can also be detected in saliva. In conclusion, Mpro is a potent antigen in infected patients that can be used in serological tests and its detection in saliva could be the basis for a rapid, non-invasive test for COVID-19 seropositivity.

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 (preprint)

Background: SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the pathogenesis and outcome of COVID-19. We aimed to investigate which specific responses from either cellular or humoral immunity associate to severity and progression of COVID-19. Methods: A cohort of 276 patients classified in mild, moderate and severe, was studied. Peripheral blood lymphocyte subpopulations were quantified by flow cytometry, and immunoglobulins and complement proteins by nephelometry. Results: At admission, dramatic lymphopenia of T, B and NK cells associated to severity. However, only the proportion of B cells increased, while T and NK cells appeared unaffected. Accordingly, the number of plasma cells and circulating follicular helper T cells (cTfh) increased, but levels of IgM, IgA and IgG were unaffected. When degrees of severity were considered, IgG was lower in severe patients, suggesting an IgG consumption by complement activation or antibody-dependent cellular cytotoxicity (ADCC). Activated CD56-CD16+ NK-cells, which mediate ADCC, were increased. Regarding complement, C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, compared to healthy donors. Moreover, IgG and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Conclusion: Our study provides important clues to understand the immune response observed in COVID-19 patients, which is probably related to viral clearance, but also underlies its pathogenesis and severity. This study associates for the first time COVID-19 severity with an imbalanced humoral immune response characterized by excessive consumption of IgG and C4, identifying new targets for therapeutic intervention.

Differential Redistribution of Activated Monocyte and Dendritic Cell Subsets to the Lung Associates with Severity of COVID-19 (preprint)

The SARS-CoV-2 is responsible for the pandemic COVID-19 in infected individuals, who can either exhibit mild symptoms or progress towards a life-threatening acute respiratory distress syndrome (ARDS). It is known that exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8+ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. With the aim to improve the knowledge in this area, we developed a cross-sectional study, in which we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood of COVID-19 patients with different clinical severity in comparison with healthy control individuals. Furthermore, these subpopulations and their association with antiviral effector CD8+ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Collectively, our results suggest that inflammatory transitional and non-classical monocytes preferentially migrate from blood to lungs in patients with severe COVID-19. CD1c+ conventional dendritic cells also followed this pattern, whereas CD141+ conventional and CD123hi plasmacytoid dendritic cells were depleted from blood but were absent in the lungs. Thus, this study increases the knowledge on the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies to fight SARS-CoV-2 infection.