Repurposing CRISPR/Cas to Discover SARS-CoV-2 Detecting and Neutralizing Aptamers.

RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.

Inactivated vaccine-elicited potent antibodies can broadly neutralize SARS-CoV-2 circulating variants.

A full understanding of the inactivated COVID-19 vaccine-mediated antibody responses to SARS-CoV-2 circulating variants will inform vaccine effectiveness and vaccination development strategies. Here, we offer insights into the inactivated vaccine-induced antibody responses after prime-boost vaccination at both the polyclonal and monoclonal levels. We characterized the VDJ sequence of 118 monoclonal antibodies (mAbs) and found that 20 neutralizing mAbs showed varied potency and breadth against a range of variants including XBB.1.5, BQ.1.1, and BN.1. Bispecific antibodies (bsAbs) based on nonoverlapping mAbs exhibited enhanced neutralizing potency and breadth against the most antibody-evasive strains, such as XBB.1.5, BQ.1.1, and BN.1. The passive transfer of mAbs or their bsAb effectively protected female hACE2 transgenic mice from challenge with an infectious Delta or Omicron BA.2 variant. The neutralization mechanisms of these antibodies were determined by structural characterization. Overall, a broad spectrum of potent and distinct neutralizing antibodies can be induced in individuals immunized with the SARS-CoV-2 inactivated vaccine BBIBP-CorV, suggesting the application potential of inactivated vaccines and these antibodies for preventing infection by SARS-CoV-2 circulating variants.

Temporal dynamics of SARS-CoV-2 genome mutations that occurred in vivo on an aircraft.

We analyzed variations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome during a flight-related cluster outbreak of coronavirus disease 2019 (COVID-19) in Shenzhen, China, to explore the characteristics of SARS-CoV-2 transmission and intra-host single nucleotide variations (iSNVs) in a confined space. Thirty-three patients with COVID-19 were sampled, and 14 were resampled 3-31 days later. All 47 nasopharyngeal swabs were deep sequenced. iSNVs and similarities in the consensus genome sequence were analyzed. Three SARS-CoV-2 variants of concern, Delta (n=31), Beta (n=1), and C.1.2 (n=1), were detected among the 33 patients. The viral genome sequences from 30 Delta-positive patients had similar SNVs; 14 of these patients provided two successive samples. Overall, the 47 sequenced genomes contained 164 iSNVs. Of the 14 paired (successive) samples, the second samples (T2) contained more iSNVs (median: 3; 95% confidence interval [95%CI]: 2.77-10.22) than did the first samples (T1; median: 2; 95%CI: 1.63-3.74; Wilcoxon test, P=0.021). 38 iSNVs were detected in T1 samples, and only seven were also detectable in T2 samples. Notably, T2 samples from two of the 14 paired samples had additional mutations than the T1 samples. The iSNVs of the SARS-CoV-2 genome exhibited rapid dynamic changes during a flight-related cluster outbreak event. Intra-host diversity increased gradually with time, and new site mutations occurred in vivo without a population transmission bottleneck. Therefore, we could not determine the generational relationship from the mutation site changes alone.

Characterization of viral shedding of Severe acute respiratory syndrome Coronavirus-2

Corona virus disease 2019 (COVID-19) is an emerging pandemic of highly contagious caused by severe acute respiratory syndrome coronavirus -2 (SARS-CoV-2). Understanding the infectivity of various clinical samples and its transmission routes have been the main focus of current researches since the causative pathogens was identified. In this comprehensive review, we discuss the viral shedding from different clinical samples and reveal that infectious virus may be mainly discharged through respiratory and digestive systems. Also, SARS-CoV-2 showed a potential tropism for eyes, kidney, testis, placenta and other extrapulmonary tissues and high viral loads correlated with severe conditions. A better understanding of viral shedding may help the studies on pathogenesis and transmission of SARS-CoV-2 and provide suggestions for the disease control.

IgM, IgG and IgA antibody responses to SARS-CoV-2 infection in patients with coronavirus disease 2019

Objective: To understand the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) specific IgM, IgC and IgA responses in corona Virus disease 2019(COVID-19) patients. Method: The SARS-CoV-2-specific IgM and IgG levels were determined by the enzyme-linked immunosorbent assay while IgA 1evels were detected by the chemiluminescence immune detection systems.

The spatial-temporal distribution and transmission route of COVID-19 in Shenzhen in the early stage

Objective: To provide reference for future epidemiological investigation, prevention and control of infectious diseases by analyzing the spatial-temporal distribution and transmission characteristics of corona virus diseases 2019 (COVID- 19) in the early stage in Shenzhen.

Influencing factors of virus clearance of COVID-19 patients

Objective: To analysis the related factors that influenced the elimination of virus in patients with coronavirus disease 2019 (COVID-19), and help with its prevention and control.

A Retinol Derivative Inhibits SARS-CoV-2 Infection by Interrupting Spike-Mediated Cellular Entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the global pandemic and life-threatening coronavirus disease 2019 (COVID-19). Although vaccines and therapeutic antibodies are available, their efficacy is continuously undermined by rapidly emerging SARS-CoV-2 variants. Here, we found that all-trans retinoic acid (ATRA), a vitamin A (retinol) derivative, showed potent antiviral activity against all SARS-CoV-2 variants in both human cell lines and human organoids of the lower respiratory tract. Mechanistically, ATRA directly binds in a deep hydrophobic pocket of the receptor binding domain (RBD) located on the top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation. In summary, our results reveal the pharmacological biotargets and structural mechanism of ATRA and other retinoids in SARS-CoV-2 infection and suggest that ATRA and its derivatives could be potential hit compounds against a broad spectrum of coronaviruses. IMPORTANCE Retinoids, a group of compounds including vitamin A and its active metabolite all-trans retinoic acid (ATRA), regulate serial physiological activity in multiple organ systems, such as cell growth, differentiation, and apoptosis. The ATRA analogues reported to date include more than 4,000 natural and synthetic molecules that are structurally and/or functionally related to ATRA. Here, we found that ATRA showed potent antiviral activity against all SARS-CoV-2 variants by directly binding in a deep hydrophobic pocket of the receptor binding domain (RBD) located on top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation, suggesting the pharmacological feasibility of using ATRA or its derivatives as a remedy for and prevention of COVID-19 disease.

A glucose-like metabolite deficient in diabetes inhibits cellular entry of SARS-CoV-2.

The severity and mortality of COVID-19 are associated with pre-existing medical comorbidities such as diabetes mellitus. However, the underlying causes for increased susceptibility to viral infection in patients with diabetes is not fully understood. Here we identify several small-molecule metabolites from human blood with effective antiviral activity against SARS-CoV-2, one of which, 1,5-anhydro-D-glucitol (1,5-AG), is associated with diabetes mellitus. The serum 1,5-AG level is significantly lower in patients with diabetes. In vitro, the level of SARS-CoV-2 replication is higher in the presence of serum from patients with diabetes than from healthy individuals and this is counteracted by supplementation of 1,5-AG to the serum from patients. Diabetic (db/db) mice undergo SARS-CoV-2 infection accompanied by much higher viral loads and more severe respiratory tissue damage when compared to wild-type mice. Sustained supplementation of 1,5-AG in diabetic mice reduces SARS-CoV-2 loads and disease severity to similar levels in nondiabetic mice. Mechanistically, 1,5-AG directly binds the S2 subunit of the SARS-CoV-2 spike protein, thereby interrupting spike-mediated virus-host membrane fusion. Our results reveal a mechanism that contributes to COVID-19 pathogenesis in the diabetic population and suggest that 1,5-AG supplementation may be beneficial to diabetic patients against severe COVID-19.

Possible recombination between two variants of concern in a COVID-19 patient.

We identified an individual who was coinfected with two SARS-CoV-2 variants of concern, the Beta and Delta variants. The ratio of the relative abundance between the two variants was maintained at 1:9 (Beta:Delta) in 14 days. Furthermore, possible evidence of recombinations in the Orf1ab and Spike genes was found.

Soaring Asymptomatic Infected Individuals Bring About Barriers and Difficulties for Interruption of COVID-19 Prevalence in China.

Background: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic, which has caused unprecedented damage to human health and life. The present study aimed to carry out and discover asymptomatic infected individuals in Shenzhen, China. The data will provide the control measures to stop COVID-19 prevalence. Methods: The study was a retrospective review of medical records from 462 confirmed patients with COVID-19 and 45 asymptomatic infected individuals in Shenzhen from January 19 to April 30, 2020; this is a retrospective, observational multicenter study. Results: A total of 462 confirmed cases were diagnosed in Shenzhen from January 19 to April 30, 2020. The cohort included 423 domestic cases (91.56%, 95% confidence interval [CI]: 88.67-93.76) and 39 (8.44%, 95% CI: 6.24-11.33) imported cases from other countries. Moreover, a total of 45 asymptomatic infections were found, encompassing 31 (68.89%, 95% CI: 54.34-80.47) local infections and 14 (31.11%, 95% CI: 19.53-45.66) individuals imported from other countries. The proportion of asymptomatic infected persons in Shenzhen is continuously increasing (Z = 13.19, p < 0.0001). The total number of local asymptomatic infections was more than that in other provinces (χ2 = 118.83, p < 0.0001). The proportion of asymptomatic infected individuals among cases imported from other countries was higher than the domestic cases (χ2 = 22.51, p < 0.0001, odds ratio = 4.90, 95% CI: 2.40-9.98). Conclusions: The proportion of asymptomatic infection is increasing. Hence, development and application of the diagnosis method with high sensitivity and specificity play a critical role in reducing COVID-19 global epidemics.

Lysosome activation in peripheral blood mononuclear cells and prognostic significance of circulating LC3B in COVID-19.

Coronavirus disease 2019 (COVID-19) has spread rapidly worldwide, causing significant mortality. There is a mechanistic relationship between intracellular coronavirus replication and deregulated autophagosome-lysosome system. We performed transcriptome analysis of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients and identified the aberrant upregulation of genes in the lysosome pathway. We further determined the capability of two circulating markers, namely microtubule-associated proteins 1A/1B light chain 3B (LC3B) and (p62/SQSTM1) p62, both of which depend on lysosome for degradation, in predicting the emergence of moderate-to-severe disease in COVID-19 patients requiring hospitalization for supplemental oxygen therapy. Logistic regression analyses showed that LC3B was associated with moderate-to-severe COVID-19, independent of age, sex and clinical risk score. A decrease in LC3B concentration <5.5 ng/ml increased the risk of oxygen and ventilatory requirement (adjusted odds ratio: 4.6; 95% CI: 1.1-22.0; P = 0.04). Serum concentrations of p62 in the moderate-to-severe group were significantly lower in patients aged 50 or below. In conclusion, lysosome function is deregulated in PBMCs isolated from COVID-19 patients, and the related biomarker LC3B may serve as a novel tool for stratifying patients with moderate-to-severe COVID-19 from those with asymptomatic or mild disease. COVID-19 patients with a decrease in LC3B concentration <5.5 ng/ml will require early hospital admission for supplemental oxygen therapy and other respiratory support.

SOD1 is a Possible Predictor of COVID-19 Progression as Revealed by Plasma Proteomics.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide health emergency. Patients infected with SARS-CoV-2 present with diverse symptoms related to the severity of the disease. Determining the proteomic changes associated with these diverse symptoms and in different stages of infection is beneficial for clinical diagnosis and management. Here, we performed a tandem mass tag-labeling proteomic study on the plasma of healthy controls and COVID-19 patients, including those with asymptomatic infection (NS), mild syndrome, and severe syndrome in the early phase and the later phase. Although the number of patients included in each group is low, our comparative proteomic analysis revealed that complement and coagulation cascades, cholesterol metabolism, and glycolysis-related proteins were affected after infection with SARS-CoV-2. Compared to healthy controls, ELISA analysis confirmed that SOD1, PRDX2, and LDHA levels were increased in the patients with severe symptoms. Both gene set enrichment analysis and receiver operator characteristic analysis indicated that SOD1 could be a pivotal indicator for the severity of COVID-19. Our results indicated that plasma proteome changes differed based on the symptoms and disease stages and SOD1 could be a predictor protein for indicating COVID-19 progression. These results may also provide a new understanding for COVID-19 diagnosis and treatment.

Comparative evaluation of six nucleic acid amplification kits for SARS-CoV-2 RNA detection.

BACKGROUND: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B-F) to evaluate their sensitivity, specificity, predictive values and accuracy. METHODS: Fifty-two clinical samples were used including throat swabs (n = 30), nasal swabs (n = 7), nasopharyngeal swabs (n = 7) and sputum specimens (n = 8), comprising confirmed (n = 26) and negative cases (n = 26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits' evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. RESULTS: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa ≥ 0.75); Kits D and E were less congruent (0.4 ≤ Kappa < 0.75). Differences between all kits were statistically significant (P < 0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. CONCLUSIONS: This is the first comparative study to evaluate CPA and RT-qPCR kits' specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.

The Characterization of Disease Severity Associated IgG Subclasses Response in COVID-19 Patients.

Increasing evidence suggests that dysregulated immune responses are associated with the clinical outcome of coronavirus disease 2019 (COVID-19). Nucleocapsid protein (NP)-, spike (S)-, receptor binding domain (RBD)- specific immunoglobulin (Ig) isotypes, IgG subclasses and neutralizing antibody (NAb) were analyzed in 123 serum from 63 hospitalized patients with severe, moderate, mild or asymptomatic COVID-19. Mild to modest correlations were found between disease severity and antigen specific IgG subclasses in serum, of which IgG1 and IgG3 were negatively associated with viral load in nasopharyngeal swab. Multiple cytokines were significantly related with antigen-specific Ig isotypes and IgG subclasses, and IL-1ß was positively correlated with most antibodies. Furthermore, the old patients (≥ 60 years old) had higher levels of chemokines, increased NAb activities and SARS-CoV-2 specific IgG1, and IgG3 responses and compromised T cell responses compared to the young patients (≤ 18 years old), which are related with more severe cases. Higher IgG1 and IgG3 were found in COVID-19 patients with comorbidities while biological sex had no effect on IgG subclasses. Overall, we have identified diseases severity was related to higher antibodies, of which IgG subclasses had weakly negative correlation with viral load, and cytokines were significantly associated with antibody response. Further, advancing age and comorbidities had obvious effect on IgG1 and IgG3.