The Frequency of Hand-to-Eye/Nose Contact can Increase the Risk of Ocular Symptoms in SARS-CoV-2-Infected Patients (preprint)

Purpose: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the capacity to use the eye for transocular entry. The characteristics of lacrimal drainage remind us to pay attention to the transmission route passing through the nasolacrimal ducts and then into the respiratory tract. The aim of this study was to assess ocular symptoms and hand-to-eye/nose contact behaviour in SARS-CoV-2-infected patients. Methods: A questionnaire was designed by brain storing method according to practical requirements, then the survey of ocular symptoms and hand-to-eye/nose contact behaviour in SARS-CoV-2-infected patients was administered in person. The patients answered the items under the guidance of a nurse. The data of ocular symptoms and basic information was collected analysed. The correlation between ocular symptoms and hand-to-eye/nose contact behaviour was evaluated. Results: The most common ocular symptoms were increased discharge (in 53.19% of patients), foreign body sensation (44.68%), tearing (44.68%), conjunctival congestion (40.43%) and mild eye pain (40.43%). In total, 61.7% of patients had nasal obstruction and running. A total of 40.4% of patients blew their nose more frequently than usual because of nasal symptoms. A total of 63.8% of patients rubbed their eyes with their hands 1-5 times per day. Only 44.68% of patients washed their hands immediately when arriving at home more than 6 days per week. Increased frequencies of blowing noses (p=0.032), washing hands (p=0.025), and rubbing eyes (p=0.005) can affect ocular symptoms. The frequency and the way of face washing had no correlation with ocular symptoms. Conclusions: Ocular symptoms in SARS-CoV-2-infected patients are much more common than currently reported but nonspecific. This prompt us pay more attention to the true incidence of conjunctivitis in SARS-CoV-2-infected patients. The frequency of hand-to-eye/nose contact can increase the risk of presenting ocular symptoms. However, hand washing can decrease the risk. These results provide direct evidence of behaviour change and smooth the concerns for many people.

Cloning of LEA3 gene from two Festuca arundinacea cultivars Qiancao 1 and Qiancao 2 and their expression under drought stress

(Objective) The aim of this paper was to explore the function of LEA3 gene and the molecular mechanism of drought resistance of Festuca Arundinacea, for which LEA3 gene from two Guizhou native F. Arundinacea cultivars (Qiancao 1 and Qiancao 2) were cloned and the bioinformatics of LEA3 genes and their expression under drought stress at seedling stage were analyzed as well. (Method) In this experiment, we cloned LEA3 genes of Qiancao 1 and Qiancao 2 by RT-PCR, referring to the sequences recorded by NCBI. At the same time, the amino acid sequence was analyzed by relevant software, and the expression of amino acid under drought stress was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). (Result) In this study, we found that LEA3 genes in Qiancao 1 and Qiancao 2 consisted of open reading frame (ORF) of 609 by and 642 by respectively, which encoded proteins of 203 and 213 amino acids residues, respectively. Compared with Qiancao 2, the LEA3 protein of Qiancao 1 lacks the motif composed of 11 amino acids. At the same time, 11 amino acid differences were found between Qiancao I and Qiancao 2. Bioinformatics analysis showed that the molecular weight of Qiancao 1 LEA3 protein was 20.72 kDa, the isoelectric point was 8.55, and the grand average of hydropathicity (GRAVY) was -0.971. The LEA3 protein of Qiancao 2 has a molecular weight of 22 01 kDa, an isoelectric point of 8.99, and a GRAVY of - 1.069. The hydrophobicity analysis showed that both Qiancao 1 and Qiancao 2 LEA3 proteins had no transmembrane structure, and the hydrophilicity of Qiancao 2 LEA3 was stronger than that of Qiancao 1 LEA3. Alpha-helixes are dominant in Qiancao 1 and Qiancao 2 LEA3 proteins. LEA3 proteins of Qiancao 1 and Qiancao 2 have high homology with that of barley, goatgrass, wheatgrass and wheat. The phylogenetic tree showed that LEA3 proteins of Qiancao 1, Qiancao 2, wheat, ryegrass, barley, goatgrass, bromus inermis and wheatgeas were clustered into one group. The results of gRT-PCR analysis showed that the expression of Qiancao 1 LEA3 was higher than that of Qiancao 2 in the whole process of drought stress at seedling stage. Compared with mild stress, the response of LEA3 in Qiancao 1 and Qiancao 2 under severe stress was stronger. (Conclusion) It was found that the drought resistance of Qiancao 1 and Qiancao 2 was positively correlated with the expression of LEA3 gene. Under the same conditions of drought stress, the expression of Qiancao 1 LEA3 gene was higher than that of Qiancao 2. Therefore, it can be inferred that Qiancao 1 is more adaptable to arid environment than Qiancao 2.

A COVID-19 antibody curbs SARS-CoV-2 nucleocapsid protein-induced complement hyperactivation (preprint)

Although human antibodies elicited by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-reactive antibodies. Herein, we isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAbs) from a quick recovery coronavirus disease-19 (COVID-19) convalescent patient who had dominant antibody responses to the SARS-CoV-2 N protein rather than to the SARS-CoV-2 spike (S) protein. The complex structure of the N protein RNA binding domain with the mAb with the highest binding affinity (nCoV396) revealed changes in the epitopes and antigen’s allosteric regulation. Functionally, a virus-free complement hyper-activation analysis demonstrated that nCoV396 specifically compromises the N protein-induced complement hyper-activation, which is a risk factor for the morbidity and mortality of COVID-19 patients, thus laying the foundation for the identification of functional anti-N protein mAbs.

Large-scale single-cell analysis reveals critical immune characteristics of COVID-19 patients (preprint)

Dysfunctional immune response in the COVID-19 patients is a recurrent theme impacting symptoms and mortality, yet the detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 205 COVID-19 patients and controls to create a comprehensive immune landscape. Lymphopenia and active T and B cell responses were found to coexist and associated with age, sex and their interactions with COVID-19. Diverse epithelial and immune cell types were observed to be virus-positive and showed dramatic transcriptomic changes. Elevation of ANXA1 and S100A9 in virus-positive squamous epithelial cells may enable the initiation of neutrophil and macrophage responses via the ANXA1-FPR1 and S100A8/9-TLR4 axes. Systemic up-regulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis and designing effective therapeutic strategies for COVID-19.

Modeling the Opening SARS-CoV-2 Spike: an Investigation of its Dynamic Electro-Geometric Properties (preprint)

The recent COVID-19 pandemic has brought about a surge of crowd-sourced initiatives aimed at simulating the proteins of the SARS-CoV-2 virus. A bottleneck currently exists in translating these simulations into tangible predictions that can be leveraged for pharmacological studies. Here we report on extensive electrostatic calculations done on an exascale simulation of the opening of the SARS-CoV-2 spike protein, performed by the Folding@home initiative. We compute the electric potential as the solution of the non-linear Poisson-Boltzmann equation using a parallel sharp numerical solver. The inherent multiple length scales present in the geometry and solution are reproduced using highly adaptive Octree grids. We analyze our results focusing on the electro-geometric properties of the receptor-binding domain and its vicinity. This work paves the way for a new class of hybrid computational and data-enabled approaches, where molecular dynamics simulations are combined with continuum modeling to produce high-fidelity computational measurements serving as a basis for protein bio-mechanism investigations.

A comparative survey of betacoronavirus strain molecular dynamics identifies key ACE2 binding sites (preprint)

Comparative functional analysis of the binding interactions between various betacoronavirus strains and their potential human target proteins, such as ACE1, ACE2 and CD26, is critical to our future understanding and combating of COVID-19. Here, employing large replicate sets of GPU accelerated molecular dynamics simulations, we statistically compare atom fluctuations of the known human target proteins in both the presence and absence of different strains of the viral receptor binding domain (RBD) of the S spike glycoprotein. We identify a common interaction site between the N-terminal helices of ACE2 and the viral RBD in all strains (hCoV-OC43, hCoV-HKU1, MERS-CoV, SARS-CoV1, and SARS-CoV-2) and a second more dynamically complex RBD interaction site involving the ACE2 amino acid sites K353, Q325, and a novel motif, AAQPFLL (386-392) in the more recent cross-species spillovers (i.e. absent in hCoV-OC43). We use computational mutagenesis to further confirm the functional relevance of these sites. We propose a "one touch/two touch" model of viral evolution potentially involved in functionally facilitating binding interactions in zoonotic spillovers. We also observe these two touch sites governing RBD binding activity in simulations on hybrid models of the suspected viral progenitor, batCoV-HKU4, interacting with both the human SARS target, ACE2, and the human MERS target, CD26. Lastly, we confirm that the presence of a common hypertension drug (lisinopril) within the target site of SARS-CoV-2 bound models of ACE1 and ACE2 acts to enhance the RBD interactions at the same key sites in our proposed model. In the near future, we recommend that our comparative computational analysis identifying these key viral RBD-ACE2 binding interactions be supplemented with comparative studies of site-directed mutagenesis in order to screen for current and future coronavirus strains at high risk of zoonotic transmission to humans. STATEMENT OF SIGNIFICANCEWe generated structural models of the spike glycoprotein receptor binding domain from recent and past betacoronavirus outbreak strains aligned to the angiotensin 1 converting enzyme 2 protein, the primary target protein of the SARS-CoV-2 virus causing COVID 19. We then statistically compared computer simulated molecular dynamics of viral bound and unbound versions of each model to identify locations where interactions with each viral strain have dampened the atom fluctuations during viral binding. We demonstrate that all known strains of betacoronavirus are strongly interactive with the N-terminal helix region of ACE2. We also identify a more complex viral interaction with three novel sites that associates with more recent and deadly SARS strains, and also a bat progenitor strain HKU4.

A COVID-19 antibody curbs SARS-CoV-2 nucleocapsid protein-induced complement hyper-activation (preprint)

Although human antibodies elicited by severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-directed antibodies. Herein, we isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAb) from a quick recovery coronavirus disease-19 (COVID-19) convalescent, who had dominant antibody responses to SARS-CoV-2 N protein rather than to Spike protein. The complex structure of N protein RNA binding domain with the highest binding affinity mAb nCoV396 reveals the epitopes and antigens allosteric changes. Functionally, a virus-free complement hyper-activation analysis demonstrates that nCoV396 specifically compromises N protein-induced complement hyper-activation, a risk factor for morbidity and mortality in COVID-19, thus paving the way for functional anti-N mAbs identification. One Sentence SummaryB cell profiling, structural determination, and protease activity assays identify a functional antibody to N protein.

Immunoreactive peptide maps of SARS-CoV-2 and other human coronaviruses (preprint)

Serodiagnosis of SARS-CoV-2 infection is impeded by immunological cross-reactivity to the human coronaviruses (HCoV) SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, 229E, HKU1, and NL63. Here we report the identification of humoral immune responses to SARS-CoV-2 and other HCoV peptides that can be used to detect asymptomatic, mild and, severe SARS-CoV-2 infections, and may enable the discovery of biomarkers for immunity following infection or vaccination.

A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy (preprint)

In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provides a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain (NTD) of the viral S glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain (RBD) of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic virus infection in a standard assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.

Thymosin alpha-1 Protected T Cells from Excessive Activation in Severe COVID-19 (preprint)

Two typical features of uncontrolled inflammation, cytokine storm and lymphopenia, are associated with the severity of coronavirus disease 2019 (COVID-19), demonstrating that both innate and adaptive immune responses are involved in the development of this disease. Recent studies have explored the contribution of innate immune cells to the pathogenesis of the infection. However, the impact of adaptive immunity on this disease remains unknown. In order to clarify the role of adaptive immune response in COVID-19, we characterized the phenotypes of lymphocytes in PBMCs from patients at different disease stages using single-cell RNA sequencing (scRNA-seq) technology. Dynamics of the effector cell levels in lymphocytes revealed a distinct feature of adaptive immunity in severely affected patients, the coincidence of impaired cellular and enhanced humoral immune responses, suggesting that dysregulated adaptive immune responses advanced severe COVID-19. Excessive activation and exhaustion were observed in CD8 T effector cells, which might contribute to the lymphopenia. Interestingly, expression of Prothymosin alpha (PTMA), the proprotein of Tα1, was significantly increased in a group of CD8 T memory stem cells, but not in excessively activated T cells. We further showed that Tα1 significantly promoted the proliferation of activated T cells in vitro and relieved the lymphopenia in COVID-19 patients. Our data suggest that protection of T cells from excessive activation might be critical for the prevention of severe COVID-19.