Novel insight into the underlying dysregulation mechanisms of immune cell-to-cell communication by analyzing multitissue single-cell atlas of two COVID-19 patients.

How does SARS-CoV-2 cause lung microenvironment disturbance and inflammatory storm is still obscure. We here performed the single-cell transcriptome sequencing from lung, blood, and bone marrow of two dead COVID-19 patients and detected the cellular communication among them. Our results demonstrated that SARS-CoV-2 infection increase the frequency of cellular communication between alveolar type I cells (AT1) or alveolar type II cells (AT2) and myeloid cells triggering immune activation and inflammation microenvironment and then induce the disorder of fibroblasts, club, and ciliated cells, which may cause increased pulmonary fibrosis and mucus accumulation. Further study showed that the increase of T cells in the lungs may be mainly recruited by myeloid cells through ligands/receptors (e.g., ANXA1/FPR1, C5AR1/RPS19, and CCL5/CCR1). Interestingly, we also found that certain ligands/receptors (e.g., ANXA1/FPR1, CD74/COPA, CXCLs/CXCRs, ALOX5/ALOX5AP, CCL5/CCR1) are significantly activated and shared among lungs, blood and bone marrow of COVID-19 patients, implying that the dysregulation of ligands/receptors may lead to immune cell's activation, migration, and the inflammatory storm in different tissues of COVID-19 patients. Collectively, our study revealed a possible mechanism by which the disorder of cell communication caused by SARS-CoV-2 infection results in the lung inflammatory microenvironment and systemic immune responses across tissues in COVID-19 patients.

3044 Cases reveal important prognosis signatures of COVID-19 patients.

Critical patients and intensive care unit (ICU) patients are the main population of COVID-19 deaths. Therefore, establishing a reliable method is necessary for COVID-19 patients to distinguish patients who may have critical symptoms from other patients. In this retrospective study, we firstly evaluated the effects of 54 laboratory indicators on critical illness and death in 3044 COVID-19 patients from the Huoshenshan hospital in Wuhan, China. Secondly, we identify the eight most important prognostic indicators (neutrophil percentage, procalcitonin, neutrophil absolute value, C-reactive protein, albumin, interleukin-6, lymphocyte absolute value and myoglobin) by using the random forest algorithm, and find that dynamic changes of the eight prognostic indicators present significantly distinct within differently clinical severities. Thirdly, our study reveals that a model containing age and these eight prognostic indicators can accurately predict which patients may develop serious illness or death. Fourthly, our results demonstrate that different genders have different critical illness rates compared with different ages, in particular the mortality is more likely to be attributed to some key genes (e.g. ACE2, TMPRSS2 and FURIN) by combining the analysis of public lung single cells and bulk transcriptome data. Taken together, we urge that the prognostic model and first-hand clinical trial data generated in this study have important clinical practical significance for predicting and exploring the disease progression of COVID-19 patients.

Characteristics of lymphocyte subset alterations in COVID-19 patients with different levels of disease severity.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a respiratory disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which had rapidly spread all over the world and caused public health emergencies in the past two years. Although the diagnosis and treatment for COVID-19 have been well defined, the immune cell characteristics and the key lymphocytes subset alterations in COVID-19 patients have not been thoroughly investigated. METHODS: The levels of immune cells including T cells, B cells, and natural killer (NK) cells in 548 hospitalized COVID-19 patients, and 30 types of lymphocyte subsets in 125 hospitalized COVID-19 patients admitted to Wuhan Huoshenshan Hospital of China were measured using flow cytometry. The relationship between lymphocytes subsets with the cytokine interleukin-6 (IL-6) and the characteristics of lymphocyte subsets in single-cell RNA sequencing (scRNA-seq) data obtained from peripheral blood mononuclear cells (PBMCs) were also analysed in COVID-19 patients. RESULTS: In this study, we found that patients with critical COVID-19 infection exhibited an overall decline in lymphocytes including CD4+ T cells, CD8+ T cells, total T cells, B cells, and NK cells compared to mild and severe patients. However, the number of lymphocyte subsets, such as CD21low CD38low B cells, effector T4 cells, and PD1+ depleted T8 cells, was moderately increased in critical COVID-19 patients compared to mild cases. Notably, except for effector memory T4 cells, plasma blasts and Tregs, the number of all lymphocyte subsets was markedly decreased in COVID-19 patients with IL-6 levels over 30-fold higher than those in healthy cases. Moreover, scRNA-seq data showed obvious differences in the distribution and numbers of lymphocyte subsets between COVID-19 patients and healthy persons, and subsets-specific marker genes of lymphocyte subsets including CD4, CD19, CCR7, and IL7R, were markedly decreased in COVID-19 patients compared with those in healthy cases. CONCLUSION: A comprehensive decrease in immune cell and lymphocyte subsets in critical COVID-19 patients, and peripheral lymphocyte subset alterations showed a clear association with clinical characteristics.

Molecular Basis of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted between humans and minks, and some mutations in the spike (S) protein, especially in the receptor-binding domain (RBD), have been identified in mink-derived viruses. Here, we examined binding of the mink angiotensin-converting enzyme 2 (ACE2) receptor to mink-derived and important human-originating variants, and we demonstrated that most of the RBD variants increased the binding affinities to mink ACE2 (mkACE2). Cryo-electron microscopy structures of the mkACE2-RBD Y453F (with a Y-to-F change at position 453) and mkACE2-RBD F486L complexes helped identify the key residues that facilitate changes in mkACE2 binding affinity. Additionally, the data indicated that the Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and human vaccinated sera efficiently prevented infection of human cells by pseudoviruses expressing Y453F, F486L, or N501T RBD. Our findings provide an important molecular mechanism for the rapid adaptation of SARS-CoV-2 in minks and highlight the potential influence of the main mink-originating variants for humans. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a broad range of hosts. Mink-derived SARS-CoV-2 can transmit back to humans. There is an urgent need to understand the binding mechanism of mink-derived SARS-CoV-2 variants to mink receptor. In this study, we identified all mutations in the receptor-binding domain (RBD) of spike (S) protein from mink-derived SARS-CoV-2, and we demonstrated the enhanced binding affinity of mink angiotensin-converting enzyme 2 (ACE2) to most of the mink-derived RBD variants as well as important human-originating RBD variants. Cryo-electron microscopy structures revealed that the Y453F and F486L mutations enhanced the binding forces in the interaction interface. In addition, Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and the SARS-CoV-2 pseudoviruses with Y453F, F486L, or N501T mutations were neutralized by human vaccinated sera. Therefore, our results provide valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Broader-species receptor binding and structural bases of Omicron SARS-CoV-2 to both mouse and palm-civet ACE2s.

The Omicron variant of SARS-CoV-2 carries multiple unusual mutations, particularly in the receptor-binding domain (RBD) of the spike (S) protein. Moreover, host-adapting mutations, such as residues 493, 498, and 501, were also observed in the Omicron RBD, which indicates that it is necessary to evaluate the interspecies transmission risk of the Omicron variant. Herein, we evaluated the interspecies recognition of the Omicron BA.1 and Delta RBDs by 27 ACE2 orthologs, including humans. We found that Omicron BA.1 expanded its receptor binding spectra to palm-civet, rodents, more bats (least horseshoe bat and greater horseshoe bat) and lesser hedgehog tenrec. Additionally, we determined the cryo-electron microscopy (cryo-EM) structure of the Omicron BA.1 S protein complexed with mouse ACE2 (mACE2) and the crystal structure of Omicron RBD complexed with palm-civet ACE2 (cvACE2). Several key residues for the host range have been identified. These results suggest that surveillance should be enhanced on the Omicron variant for its broader-species receptor binding to prevent spillover and expansion of reservoir hosts for a prolonged pandemic.

Structural basis of human ACE2 higher binding affinity to currently circulating Omicron SARS-CoV-2 sub-variants BA.2 and BA.1.1.

The currently circulating Omicron sub-variants are the SARS-CoV-2 strains with the highest number of known mutations. Herein, we found that human angiotensin-converting enzyme 2 (hACE2) binding affinity to the receptor-binding domains (RBDs) of the four early Omicron sub-variants (BA.1, BA.1.1, BA.2, and BA.3) follows the order BA.1.1 > BA.2 > BA.3 ≈ BA.1. The complex structures of hACE2 with RBDs of BA.1.1, BA.2, and BA.3 reveal that the higher hACE2 binding affinity of BA.2 than BA.1 is related to the absence of the G496S mutation in BA.2. The R346K mutation in BA.1.1 majorly affects the interaction network in the BA.1.1 RBD/hACE2 interface through long-range alterations and contributes to the higher hACE2 affinity of the BA.1.1 RBD than the BA.1 RBD. These results reveal the structural basis for the distinct hACE2 binding patterns among BA.1.1, BA.2, and BA.3 RBDs.

Atlas of currently available human neutralizing antibodies against SARS-CoV-2 and escape by Omicron sub-variants BA.1/BA.1.1/BA.2/BA.3.

SARS-CoV-2 Omicron variant has presented significant challenges to current antibodies and vaccines. Herein, we systematically compared the efficacy of 50 human monoclonal antibodies (mAbs), covering the seven identified epitope classes of the SARS-CoV-2 RBD, against Omicron sub-variants BA.1, BA.1.1, BA.2, and BA.3. Binding and pseudovirus-based neutralizing assays revealed that 37 of the 50 mAbs lost neutralizing activities, whereas the others displayed variably decreased activities against the four Omicron sub-variants. BA.2 was found to be more sensitive to RBD-5 antibodies than the other sub-variants. Furthermore, a quaternary complex structure of BA.1 RBD with three mAbs showing different neutralizing potencies against Omicron provided a basis for understanding the immune evasion of Omicron sub-variants and revealed the lack of G446S mutation accounting for the sensitivity of BA.2 to RBD-5 mAbs. Our results may guide the application of the available mAbs and facilitate the development of universal therapeutic antibodies and vaccines against COVID-19.

Ligand/Receptor-mediated Cell Communication Alteration Causes the Lung Inflammation Microenvironment and the Redistribution of Inter-tissue Immune Cells in COVID-19 Patients (preprint)

How SARS-CoV-2 causes disturbances of the lung microenvironment and systemic immune response remains a mystery. Here, we first analyze detailedly paired single-cell transcriptome data of the lungs, blood and bone marrow of two patients who died of COVID-19. Second, our results demonstrate that SARS-CoV-2 infection significantly increases the cellular communication frequency between AT1/AT2 cells and highly inflammatory myeloid cells, and induces the pulmonary inflammation microenvironment, and drives the disorder of fibroblasts, club and ciliated cells, thereby causing the increase of pulmonary fibrosis and mucus accumulation. Third, our works reveal that the increase of the lung T cell infiltration is mainly recruited by myeloid cells through certain ligands/receptors (ANXA1/FPR1, C5AR1/RPS19 and CCL5/CCR1), rather than AT1/AT2. Fourth, we find that some ligands and receptors such as ANXA1/FPR1, CD74/COPA, CXCLs/CXCRs, ALOX5/ALOX5AP, CCL5/CCR1, are significantly activated and shared among patients’ lungs, blood and bone marrow, implying that dysregulated ligands and receptors may cause the migration, redistribution and the inflammatory storm of immune cells in different tissues. Overall, our study reveals a latent mechanism by which the disorders of ligands and receptors caused by SARS-CoV-2 infection drive cell communication alteration, the pulmonary inflammatory microenvironment and systemic immune responses across tissues in COVID-19 patients.

The Geographical Distribution and Influencing Factors of COVID-19 in China.

The study of the spatial differentiation of COVID-19 in cities and its driving mechanism is helpful to reveal the spatial distribution pattern, transmission mechanism and diffusion model, and evolution mechanism of the epidemic and can lay the foundation for constructing the spatial dynamics model of the epidemic and provide theoretical basis for the policy design, spatial planning and implementation of epidemic prevention and control and social governance. Geodetector (Origin version, Beijing, China) is a great tool for analysis of spatial differentiation and its influencing factors, and it provides decision support for differentiated policy design and its implementation in executing the city-specific policies. Using factor detection and interaction analysis of Geodetector, 15 indicators of economic, social, ecological, and environmental dimensions were integrated, and 143 cities were selected for the empirical research in China. The research shows that, first of all, risks of both infection and death show positive spatial autocorrelation, but the geographical distribution of local spatial autocorrelation differs significantly between the two. Secondly, the inequalities in urban economic, social, and residential environments interact with COVID-19 spatial heterogeneity, with stronger explanatory power especially when multidimensional inequalities are superimposed. Thirdly, the spatial distribution and spread of COVID-19 are highly spatially heterogeneous and correlated due to the complex influence of multiple factors, with factors such as Area of Urban Construction Land, GDP, Industrial Smoke and Dust Emission, and Expenditure having the strongest influence, the factors such as Area of Green, Number of Hospital Beds and Parks, and Industrial NOx Emissions having unignorable influence, while the factors such as Number of Free Parks and Industrial Enterprises, Per-GDP, and Population Density play an indirect role mainly by means of interaction. Fourthly, the factor interaction effect from the infected person's perspective mainly shows a nonlinear enhancement effect, that is, the joint influence of the two factors is greater than the sum of their direct influences; but from the perspective of the dead, it mainly shows a two-factor enhancement effect, that is, the joint influence of the two factors is greater than the maximum of their direct influences but less than their sum. Fifthly, some suggestions are put forward from the perspectives of building a healthy, resilient, safe, and smart city, providing valuable reference and decision basis for city governments to carry out differentiated policy design.

Changes in Serum Neutralizing Antibodies Levels During Convalescence of COVID-19 Patients.

Detection of serum-specific SARS-CoV-2 antibody has become a complementary means for the identification of coronavirus disease 2019 (COVID-19). As we already know, the neutralizing antibody titers in patients with COVID-19 decrease during the course of time after convalescence, whereas the duration of antibody responses in the convalescent patients has not been defined clearly. In the current study, we collected 148 serum samples from 37 confirmed COVID-19 cases with different disease severities. The neutralizing antibodies (Nabs), IgM and IgG against COVID-19 were determined by CLIA Microparticle and microneutralization assay, respectively. The time duration of serum titers of SARS-CoV-2 antibodies were recorded. Our results indicate that IgG (94.44%) and Nabs (89.19%) can be detected at low levels within 190-266 days of disease onset. The findings can advance knowledge regarding the antibody detection results for COVID-19 patients and provide a method for evaluating the immune response after vaccination.

Dynamics and Decoupling Analysis of Carbon Emissions from Construction Industry in China

The construction industry is the backbone of most countries, but its carbon emissions are huge and growing rapidly, constraining the achievement of global carbon-peaking and carbon-neutrality goals. China’s carbon emissions are the highest in the world, and the construction industry is the largest contributor. Due to significant differences between provinces in pressure, potential, and motivation to reduce emissions, the “one-size-fits-all”emission reduction policy has failed to achieve the desired results. This paper empirically investigates the spatial and temporal evolution of carbon emissions in China’s construction industry and their decoupling relationship with economic growth relying on GIS tools and decoupling model in an attempt to provide a basis for the formulation of differentiated construction emission reduction policies and plans in China. The study shows that, firstly, the changes in carbon emissions and carbon intensity in the provincial construction industry are becoming increasingly complex, with a variety of types emerging, such as declining, “inverted U-shaped”, growing, “U-shaped”, and smooth fluctuating patterns. Secondly, the coefficient of variation is higher than 0.65 for a long time, indicating high spatial heterogeneity. However, spatial agglomeration and correlation are low, with only a few cluster-like agglomerations formed in the Pearl River Delta, Yangtze River Delta, Bohai Bay, Northeast China, and Loess and Yunnan–Guizhou Plateau regions. Thirdly, most provinces have not reached peak carbon emissions from the construction industry, with 25% having reached peak and being in the plateau stage, respectively. Fourthly, the decoupling relationship between carbon emissions from the construction industry and economic growth, as well as their changes, is increasingly diversified, and most provinces are in a strong and weak decoupling state. Moreover, a growing number of provinces that have achieved decoupling are moving backward to re-coupling, due to the impact of economic transformation and the outbreaks of COVID-19, with the degraded regions increasingly concentrated in the northeast and northwest. Fifthly, we classify China’s 30 provinces into Leader, Intermediate, and Laggard policy zones and further propose differentiated response strategies. In conclusion, studying the trends and patterns of carbon-emission changes in the construction industry in different regions, revealing their spatial differentiation and correlation, and developing a classification management strategy for low carbonized development of the construction industry help significantly improve the reliability, efficiency, and self-adaptability of policy design and implementation.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.

Emergence of a young case infected with avian influenza A (H5N6) in Anhui Province, East China during the COVID-19 pandemic.

In the context of the coronavirus disease 2019 pandemic, we investigated the epidemiological and clinical characteristics of a young patient infected by avian influenza A (H5N6) virus in Anhui Province, East China, and analyzed genomic features of the pathogen in 2020. Through the cross-sectional investigation of external environment monitoring (December 29-31, 2020), 1909 samples were collected from Fuyang City. It was found that the positive rate of H5N6 was higher than other areas obviously in Tianma poultry market, where the case appeared. In addition, dual coinfections were detected with a 0.057% polymerase chain reaction positive rate the surveillance years. The virus was the clade 2.3.4.4, which was most likely formed by genetic reassortment between H5N6 and H9N2 viruses. This study found that the evolution rates of the hemagglutinin and neuraminidase genes of the virus were higher than those of common seasonal influenza viruses. The virus was still highly pathogenic to poultry and had a preference for avian receptor binding.

SARS-CoV-2 suppresses mRNA expression of selenoproteins associated with ferroptosis, endoplasmic reticulum stress and DNA synthesis.

Higher selenium status has been shown to improve the clinical outcome of infections caused by a range of evolutionally diverse viruses, including SARS-CoV-2. However, the impact of SARS-CoV-2 on host-cell selenoproteins remains elusive. The present study investigated the influence of SARS-CoV-2 on expression of selenoprotein mRNAs in Vero cells. SARS-CoV-2 triggered an inflammatory response as evidenced by increased IL-6 expression. Of the 25 selenoproteins, SARS-CoV-2 significantly suppressed mRNA expression of ferroptosis-associated GPX4, DNA synthesis-related TXNRD3 and endoplasmic reticulum-resident SELENOF, SELENOK, SELENOM and SELENOS. Computational analysis has predicted an antisense interaction between SARS-CoV-2 and TXNRD3 mRNA, which is translated with high efficiency in the lung. Here, we confirmed the predicted SARS-CoV-2/TXNRD3 antisense interaction in vitro using DNA oligonucleotides, providing a plausible mechanism for the observed mRNA knockdown. Inhibition of TXNRD decreases DNA synthesis which is thereby likely to increase the ribonucleotide pool for RNA synthesis and, accordingly, RNA virus production. The present findings provide evidence for a direct inhibitory effect of SARS-CoV-2 replication on the expression of a specific set of selenoprotein mRNAs, which merits further investigation in the light of established evidence for correlations between dietary selenium status and the outcome of SARS-CoV-2 infection.

Efficacy and Safety of Tocilizumab Treatment COVID-19 Patients: A Case-Control Study and Meta-Analysis.

INTRODUCTION: As the pandemic progresses, the pathophysiology of COVID-19 is becoming more apparent, and the potential for tocilizumab is increasing. However, the clinical efficacy and safety of tocilizumab in the treatment of COVID-19 patients remain unclear. METHODS: To assess the efficacy and safety of tocilizumab treatment in COVID-19 patients, we performed a retrospective case-control study. The study was conducted, including 95 patients treated with tocilizumab plus standard treatment and matched controls with 95 patients treated with standard treatment therapy by propensity score from February to April 2020. We searched some databases using the search terms for studies published from January 1, 2020, to June 1, 2021. RESULTS: Our case-control study found a lower mortality rate in the tocilizumab treatment group than in the standard treatment group (9.47% versus 16.84%, P = 0.134), but the results were not statistically significant. We also found that the mortality rate in tocilizumab treatment groups was significantly lower than in the standard treatment group in the stratified ICU analysis (OR 0.52, 95% CI 0.44-0.61, P = 0.048 and OR 0.31, 95% CI 0.10-0.99, P = 0.044). We selected 49 studies (including 6568 cases and 11,660 controls) that met the inclusion criteria in the meta-analysis. In the overall analysis, we performed a meta-analysis that showed significantly decreased mortality after patients received tocilizumab (OR 0.81, 95% CI 0.69-0.95, P = 0.008). We also revealed significant associations within some subgroups. The sequential trial analysis showed a true-positive result. No significant associations were observed between tocilizumab and elevated secondary infection risk, discharge, adverse events, and mechanical ventilation in the overall analysis. CONCLUSION: Tocilizumab significantly decreased mortality in COVID-19 patients with no increased discharge, secondary infection risk, adverse events, and mechanical ventilation in a meta-analysis. Our data suggest that clinicians should pay attention to tocilizumab therapy as an effective and safe treatment for COVID-19 patients.

SARS-CoV-2 spillover into hospital outdoor environments.

Facing the ongoing coronavirus infectious disease-2019 (COVID-19) pandemic, many studies focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in indoor environment, on solid surface or in wastewater. It remains unclear whether SARS-CoV-2 can spill over into outdoor environments and impose transmission risks to surrounding people and communities. In this study, we investigated the presence of SARS-CoV-2 by measuring viral RNA in 118 samples from outdoor environment of three hospitals in Wuhan. We detected SARS-CoV-2 in soils (205-550 copies/g), aerosols (285-1,130 copies/m3) and wastewaters (255-18,744 copies/L) in locations close to hospital departments receiving COVID-19 patients or in wastewater treatment sectors. These findings revealed a significant viral spillover in hospital outdoor environments that was possibly caused by respiratory droplets from patients or aerosolized particles from wastewater containing SARS-CoV-2. In contrast, SARS-CoV-2 was not detected in other areas or on surfaces with regular implemented disinfection. Soils may behave as viral warehouse through deposition and serve as a secondary source spreading SARS-CoV-2 for a prolonged time. For the first time, our findings demonstrate that there are high-risk areas out of expectation in hospital outdoor environments to spread SARS-CoV-2, calling for sealing of wastewater treatment unit and complete sanitation to prevent COVID-19 transmission risks.

Upregulated IL-6 Indicates a Poor COVID-19 Prognosis: A Call for Tocilizumab and Convalescent Plasma Treatment.

A comprehensive understanding of the dynamic changes in interleukin-6 (IL-6) levels is essential for monitoring and treating patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). By analyzing the correlations between IL-6 levels and health conditions, underlying diseases, several key laboratory detection indices, and the prognosis of 1,473 patients with the coronavirus disease 2019 (COVID-19), the role of IL-6 during SARS-CoV-2 infection was demonstrated. Our results indicated that IL-6 levels were closely related to age, sex, body temperature, oxygen saturation (SpO2) of blood, and underlying diseases. As a stable indicator, the changes in IL-6 levels could indicate the inflammatory conditions during a viral infection. Two specific treatments, namely, tocilizumab and convalescent plasma therapy (CPT), decreased the level of IL-6 and relieved inflammation. CPT has an important role in the therapy for patients with critical COVID-19. We also found that patients with IL-6 levels, which were 30-fold higher than the normal level, had a poor prognosis compared to patients with lower levels of IL-6.

Potential False-Positive and False-Negative Results for COVID-19 IgG/IgM Antibody Testing After Heat-Inactivation.

Objectives: With the worldwide spread of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), various antibody detection kits have been developed to test for SARS-CoV-2- specific IgG, IgM, and total antibody. However, the use of different testing methods under various heat-inactivation conditions might affect the COVID-19 detection results. Methods: Seven different antibody detection kits produced by four manufacturers for detection of SARS-CoV-2 IgG, IgM, and total antibody were tested at Wuhan Huoshenshan Hospital, China. Most of the kits used the indirect immunity, capture, and double-antigen sandwich methods. The effects of various heat-inactivation conditions on SARS-CoV-2-specific IgG, IgM, and total antibody detection were analyzed for the different test methods. Results: Using the indirect immunity method, values for SARS-CoV-2 IgG antibody significantly increased and those for IgM antibody decreased with increasing temperature of heat-inactivation using indirect immunity method. However, values for SARS-CoV-2 IgM and total antibody showed no change when the capture and double-antigen sandwich methods were used. The changes in IgG and IgM antibody values with the indirect immunity method indicated that heat-inactivation could affect COVID-19 detection results obtained using this method. In particular, 18 (22.2%) SARS-CoV-2 IgM positive samples were detected as negative with heat-inactivation at 65°C for 30 min, and one (25%) IgG negative sample was detected as positive after heat-inactivation at 56°C for 60 min and 60°C for 30 min. Conclusions: Heat-inactivation could increase SARS-CoV-2 IgG antibody values, and decrease IgM antibody values, causing potential false-positive or false-negative results for COVID-19 antibody detection using the indirect immunity method. Thus, before conducting antibody testing, the testing platforms should be evaluated in accordance with the relevant requirements to ensure accurate COVID-19 detection results.

The paradoxical problem with COVID-19 ocular infection: Moderate clinical manifestation and potential infection risk.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which induced mainly the respiratory damage also caused ocular surface symptoms. However, the detailed description of ocular manifestations, severity fluctuations in confirmed COVID-19 adult patients still lacked. We analyzed onset clinical symptoms and duration, ocular symptoms, needs for medication, outcomes in 28 conjunctivitis patients who were extracted from 3198 COVID-19 patients hospitalized in Huoshenshan Hospital and Taikangtongji Hospital, Wuhan, China. The expression levels of ACE2, TMPRSS2, ANPEP, DPP4, NRP1 on fetal and adult ocular surface and mouse lacrimal glands were assessed by single cell seq analysis. Our results indicated that conjunctivitis was a rare and self-limited complication in adults with COVID-19 while the existence of coronavirus receptors on human ocular surface and mouse lacrimal glands indicated the risk of SARS-CoV-2 infection. Our research firstly examined SARS-CoV-2 receptors, including the new discovered one, NRP1, on the fetal ocular surface and in the mouse lacrimal glands.

CRISPR-Assisted DNA Detection: A Novel dCas9-Based DNA Detection Technique.

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.