Epidemiological characteristics of seasonal influenza under implementation of zero-COVID-19 strategy in China.

OBJECTIVE: Respiratory viral diseases have posed a persistent threat to public health due to their high transmissibility. Influenza virus and SARS-Cov-2 are both respiratory viruses that have caused global pandemics. A zero-COVID-19 strategy is a public health policy imposed to stop community transmission of COVID-19 as soon as it is detected. In this study, we aim to examine the epidemiological characteristics of seasonal influenza in the past five years before and after the emergence of COVID-19 in China and observe the possible impact of the strategy on influenza. METHODS: Data from two data sources were retrospectively analyzed. A comparison on influenza incidence rate between Hubei and Zhejiang provinces was conducted based on data from the Chinese Center for Disease Control and Prevention (CDC). Then a descriptive and comparative analysis on seasonal influenza based on data from Zhongnan Hospital of Wuhan University and Hangzhou Ninth People`s Hospital before and after the outbreak of SARS-CoV-2 was conducted. RESULTS: From 2010-2017, both provinces experienced relatively low influenza activity until the 1st week of 2018, when they reached peak incidence rates of 78.16/100000PY, 34.05/100000PY respectively. Since then, influenza showed an obvious seasonality in Hubei and Zhejiang until the onset of COVID-19. During 2020 and 2021, there was a dramatic decline in influenza activity compared to 2018 and 2019. However, influenza activity seemed to rebound at the beginning of 2022 and surged in summer, with positive rates of 20.52% and 31.53% in Zhongnan Hospital of Wuhan University and Hangzhou Ninth People`s Hospital respectively as of the time writing this article. CONCLUSIONS: Our results reinforce the hypothesis that zero-COVID-19 strategy may impact the epidemiological pattern of influenza. Under the complex pandemic situation, implementation of NPIs could be a beneficial strategy containing not only COVID-19 but also influenza.

How to correct relative voxel scale factors for calculations of vector-difference Fourier maps in cryo-EM.

The atomic coordinates derived from cryo-electron microscopy (cryo-EM) maps can be inaccurate when the voxel scaling factors are not properly calibrated. Here, we describe a method for correcting relative voxel scaling factors between pairs of cryo-EM maps for the same or similar structures that are expanded or contracted relative to each other. We find that the correction of scaling factors reduces the amplitude differences of Fourier-inverted structure factors from voxel-rescaled maps by up to 20-30%, as shown by two cryo-EM maps of the SARS-CoV-2 spike protein measured at pH 4.0 and pH 8.0. This allows for the calculation of the difference map after properly scaling, revealing differences between the two structures for individual amino acid residues. Unexpectedly, the analysis uncovers two previously overlooked differences of amino acid residues in structures and their local structural changes. Furthermore, we demonstrate the method as applied to two cryo-EM maps of monomeric apo-photosystem II from the cyanobacteria Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. The resulting difference maps reveal many changes in the peripheral transmembrane PsbX subunit between the two species.

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617.

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

Ubiquitous e-Teaching and e-Learning: China’s Massive Adoption of Online Education and Launching MOOCs Internationally during the COVID-19 Outbreak

China had made a remarkable headway in online education provision during the first quarter of 2020 due to the coronavirus disease 2019 (COVID-19) outbreak, a global public health crisis that acted as a catalyst for the uptake in online education as a method for students’ e-learning and teachers’ e-teaching at a vast number of institutions worldwide. China’s launching of XuetangX Global and iCourse International, two massive online open course (MOOC) platforms in April 2020 to provide distant e-learning solutions to global learners at a time they were most needed, proves to be a timely move as the global challenge caused by this pandemic turned out to be an opportunity in disguise for online education internationally. This article centers around China’s opportune development in online education and launching university MOOCs internationally in the height of the worsening COVID-19 pandemic in early 2020 and examines its preparedness, implementation, and impact.

High-Dose Intravenous Immunoglobulin in Severe Coronavirus Disease 2019: A Multicenter Retrospective Study in China.

BACKGROUND: The effective treatment of coronavirus disease 2019 (COVID-19) remains unclear. We reported successful use of high-dose intravenous immunoglobulin (IVIg) in cases of severe COVID-19, but evidence from larger case series is still lacking. METHODS: A multi-center retrospective study was conducted to evaluate the effectiveness of IVIg administered within two weeks of disease onset at a total dose of 2 g/kg body weight, in addition to standard care. The primary endpoint was 28-day mortality. Efficacy of high-dose IVIg was assessed by using the Cox proportional hazards regression model and the Kaplan-Meier curve adjusted by inverse probability of treatment weighting (IPTW) analysis, and IPTW after multiple imputation (MI) analysis. RESULTS: Overall, 26 patients who received high-dose IVIg with standard therapy and 89 patients who received standard therapy only were enrolled in this study. The IVIg group was associated with a lower 28-day mortality rate and less time to normalization of inflammatory markers including IL-6, IL-10, and ferritin compared with the control. The adjusted HR of 28-day mortality in high-dose IVIg group was 0.24 (95% CI 0.06-0.99, p<0.001) in IPTW model, and 0.27 (95% CI 0.10-0.57, p=0.031) in IPTW-MI model. In subgroup analysis, patients with no comorbidities or treated in the first week of disease were associated with more benefit from high-dose IVIg. CONCLUSIONS: High-dose IVIg administered in severe COVID-19 patients within 14 days of onset was linked to reduced 28-day mortality, more prominent with those having no comorbidities or treated at earlier stage.

Clinical Characteristics of Refractory Coronavirus Disease 2019 in Wuhan, China.

BACKGROUND: Since December 2019, coronavirus disease 2019 (COVID-19), caused by severe adult respiratory syndrome coronavirus 2, occurred in Wuhan, and rapidly spread throughout China. This study aimed to clarify the characteristics of patients with refractory COVID-19. METHODS: In this retrospective single-center study, we included 155 consecutive patients with confirmed COVID-19 in Zhongnan Hospital of Wuhan University from 1 January to 5 February. The cases were divided into general and refractory COVID-19 groups according to the clinical efficacy of treatment after hospitalization, and the differences between groups were compared. RESULTS: Compared with patients with general COVID-19 (45.2%), those with refractory disease were older, were more likely to be male, and had more underlying comorbid conditions, a lower incidence of fever, higher maximum temperatures among patients with fever, higher incidences of shortness of breath and anorexia, more severe disease assessment at admission, higher neutrophil, aspartate aminotransferase, lactate dehydrogenase, and C-reactive protein levels, lower platelet counts and albumin levels, and higher incidences of bilateral pneumonia and pleural effusion (P < .05). Patients with refractory COVID-19 were more likely to receive oxygen, mechanical ventilation, expectorant, and adjunctive treatment, including corticosteroids, antiviral drugs, and immune enhancers (P < .05). Considering the factors of disease severity at admission, mechanical ventilation, and intensive care unit transfer, patients with refractory COVID-19 were also more likely to be male, have manifestations of anorexia on admission, and receive oxygen, expectorant, and adjunctive agents (P < .05). CONCLUSION: In nearly 50% of patients with COVID-19 obvious clinical and radiological remission was not achieved within 10 days after hospitalization. Male, anorexia, and no fever at admission was predictive of poor treatment efficacy.

Coronavirus Nsp1: Immune Response Suppression and Protein Expression Inhibition.

Coronaviruses have brought severe challenges to public health all over the world in the past 20years. SARS-CoV-2, the causative agent of the COVID-19 pandemic that has led to millions of deaths, belongs to the genus beta-coronavirus. Alpha- and beta-coronaviruses encode a unique protein, nonstructural protein 1 (Nsp1) that both suppresses host immune responses and reduces global gene expression levels in the host cells. As a key pathogenicity factor of coronaviruses, Nsp1 redirects the host translation machinery to increase synthesis of viral proteins. Through multiple mechanisms, coronaviruses impede host protein expression through Nsp1, while escaping inhibition to allow the translation of viral RNA. In this review, we discuss current data about suppression of the immune responses and inhibition of protein synthesis induced by coronavirus Nsp1, as well as the prospect of live-attenuated vaccine development with virulence-attenuated viruses with mutations in Nsp1.

Clinical and immunological characteristics in COVID-19 convalescent patients.

The humoral and cellular immunity of convalescent COVID-19 patients is involved in pathogenesis and vaccine immunity. In this study, through CoV-psV neutralization assay and IFN-γ ELISpot testing in 30 cases of COVID-19 patients after 9 months post-SARS-CoV-2 infection, it found that the ratio of memory/naive CD4+ T lymphocytes cells and levels of anti-SARS-CoV-2-IgM and RBD-IgM were slightly but significantly higher in COVID-19 severe convalescent patients than that in non-severe patients. The specific cellular and humoral immunity against SARS-CoV-2 were detectable, regardless of the severity of the disease in the acute phase. This information may help understanding the immune status after SARS-CoV-2 infection.

Computational insights into the membrane fusion mechanism of SARS-CoV-2 at the cellular level.

The membrane fusion mechanism of SARS-CoV-2 offers an attractive target for the development of small molecule antiviral inhibitors. Fusion involves an initial binding of the crown-like trimeric spike glycoproteins of SARS-CoV-2 to the receptor angiotensin II-converting enzyme 2 (ACE2) on the permissive host cellular membrane and a prefusion to post-fusion conversion of the spike trimer. During this conversion, the fusion peptides of the spike trimer are inserted into the host membrane to bring together the host and viral membranes for membrane fusion in highly choreographic events. However, geometric constraints due to interactions with the membranes remain poorly understood. In this study, we build structural models of super-complexes of spike trimer/ACE2 dimers based on the molecular structures of the ACE2/neutral amino acid transporter B(0)AT heterodimer. We determine the conformational constraints due to the membrane geometry on the enzymatic activity of ACE2 and on the viral fusion process. Furthermore, we find that binding three ACE2 dimers per spike trimer is essential to open the central pore as necessary for triggering productive membrane fusion through an elongation of the central stalk. The reported findings thus provide valuable insights for targeting the membrane fusion mechanism for drug design at the molecular level.

Prevalence and Characteristics of Rheumatoid-Associated Autoantibodies in Patients with COVID-19.

OBJECTIVE: Patients with rheumatic immune diseases were more likely to develop severe or critical COVID-19. We aimed to determine whether rheumatoid factor antibodies were present in COVID patients and the level and type of rheumatoid factor antibodies produced in COVID-19 patients were related to the degree of the patient's condition. The study also aimed to determine the prevalence and characteristics of rheumatoid factor antibodies in patients with COVID-19. METHODS: Sera collected from 129 patients with COVID-19 were tested for rheumatoid factor antibodies by ELISA. Five patients were tracked for several months to monitor dynamic changes of these antibodies. RESULTS: Rheumatoid-associated autoantibodies were detected in 20.16% of patients (26/129) following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, IgM-RF was primarily present in critically ill patients, while IgA-RF was mainly present in mild patients. Five patients were able to track for several months to monitor dynamic changes of these antibodies. Rheumatoid factor antibodies peaks in the later phase of the disease and last for longer time. Anti-Jo-1 antibody was found in one of the five patients. CONCLUSION: This was the case series report that rheumatoid-associated autoantibodies are present in patients with COVID-19. The clinical significance of these antibodies was not fully understood and needed further characterization. These autoantibodies are related to the severity of the patient's disease and exist for a long time in the patient's body, while their impact on the patient's health is unknown.

Rapid, reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2.

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.

Risk factors for the delayed viral clearance in COVID-19 patients.

Comorbidities are important for the disease outcome of COVID-19, however, which underlying diseases that contribute the most to aggravate the conditions of COVID-19 patients are still unclear. Viral clearance is the most important laboratory test for defining the recovery of COVID-19 infections. To better understand which underlying diseases that are risk factors for delaying the viral clearance, we retrospectively analyzed 161 COVID-19 clinical cases in the Zhongnan Hospital of Wuhan University, Wuhan, China between January 5 and March 13, 2020. The demographic, clinical and laboratory data, as well as patient treatment records were collected. Univariable and multivariable analysis were performed to explore the association between delayed viral clearance and other factors by using logistic regression. Survival analyses by Kaplan-Meier and Cox regression modeling were employed to identify factors negatively influencing the viral clearance negatively. We found that hypertension and intravenous immunoglobulin adversely affected the time of viral RNA shedding. Hypertension was the most important risk factor to delay the SARS-CoV-2 virus clearance, however, the use of Angiotensin-Converting Enzyme Inhibitors(ACEI)/Angiotensin Receptor Blockers(ARB) did not shorten the time for virus clearance in these hypertensive patients' virus clearance. We conclude that patients having hypertension and intravenous immunoglobulin may delay the viral clearance in COVID-19 patients.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Corrigendum: High-Dose Intravenous Immunoglobulin in Severe Coronavirus Disease 2019: A Multicenter Retrospective Study in China.

[This corrects the article DOI: 10.3389/fimmu.2021.627844/full.].

Nonstructural Protein 1 of SARS-CoV-2 Is a Potent Pathogenicity Factor Redirecting Host Protein Synthesis Machinery toward Viral RNA.

The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.

Risk factors for delayed negative conversion of SARS-CoV-2 in patients with COVID-19 pneumonia: a retrospective cohort study.

The epidemic of coronavirus disease 2019 (COVID-19) began in China and had spread rapidly to many other countries. This study aimed to identify risk factors associated with delayed negative conversion of SARS-CoV-2 in COVID-19 patients. In this retrospective single-centre study, we included 169 consecutive patients with confirmed COVID-19 in Zhongnan Hospital of Wuhan University from 15th January to 2nd March. The cases were divided into two groups according to the median time of SARS-CoV-2 negative conversion. The differences between groups were compared. In total, 169 patients had a median virus negative conversion time of 18 days (interquartile range: 11-25) from symptom onset. Compared with the patients with short-term negative conversion, those with long-term conversion had an older age, higher incidence of comorbidities, chief complaints of cough and chest distress/breath shortness and severer illness on admission, higher level of leucocytes, neutrophils, aspartate aminotransferase, creatine kinase and erythrocyte sedimentation rate (ESR), lower level of CD3+CD4+ lymphocytes and albumin and more likely to receive mechanical ventilation. In multivariate analysis, cough, leucocytes, neutrophils and ESR were positively correlated with delayed virus negative conversion, and CD3+CD4+ lymphocytes were negatively correlated. The integrated indicator of leucocytes, neutrophils and CD3+CD4+ lymphocytes showed a good performance in predicting the negative conversion within 2 weeks (area under ROC curve (AUC) = 0.815), 3 weeks (AUC = 0.804), 4 weeks (AUC = 0.812) and 5 weeks (AUC = 0.786). In conclusion, longer quarantine periods might be more justified for COVID-19 patients with cough, higher levels of leucocytes, neutrophils and ESR and lower levels of CD3+CD4+ lymphocytes.

The indicative role of markers for liver injury on the severity and prognosis of coronavirus disease 2019 patients.

BACKGROUND: Liver injury in coronavirus disease 2019 (COVID-19) patients was poorly understood. METHODS: The markers of liver injury, severity of disease and prognosis among 495 COVID-19 patients in Zhongnan Hospital of Wuhan University from 1st January 2019 to 11th March 2019 were retrospectively analyzed. RESULTS: The levels of aspartate aminotransferase (AST) (50.1 ± 38.4 vs. 31.4 ± 39.1, P < 0.001), gamma-glutamyl transpeptidase (GGT) (70.3 ± 70.2 vs. 34.1 ± 34.7, P < 0.001) and fibrinogen-to-albumin-ratio (FAR) (13.4 ± 4.0 vs. 10.4 ± 3.4, P < 0.001) were greater than mild COVID-19 patients, whereas the levels of albumin(35.0 ± 6.2 vs. 39.9 ± 3.7, P < 0.001) and albumin/globulin (A/G) ratio (1.21 ± 0.24 vs. 1.50 ± 0.31, P < 0.001) were lower in severe COVID-19 patients. By comparing the changes of liver injury markers 7-10 days after hospitalization, the level of albumin deteriorated from 35.0 ± 6.2 to 30.20 ± 5.5 (P < 0.001), A/G ratio from 1.21 ± 0.24 to 1.06 ± 0.25 (P < 0.001), and FAR from 13.4 ± 4.0 to 15.4 ± 2.9(P < 0.001) in severe COVID-19 patients, while the changes of albumin, A/G ratio and FAR showed opposite patterns in mild COVID-19 patients. FAR > 12 [2.566 (1.410-4.670), P = 0.012) on admission and changes of albumin >5g/l [22.489 (6.422-78.757), P = 0.001] were two risk factors for death, and the sensitivity and specificity for the poor prognosis were 80.8% and 64.0%, 82.6% and 76.3%, respectively. CONCLUSION: The levels of AST, GGT, albumin and FAR are correlated with disease severity after severe acute respiratory syndrome coronavirus-2 infection. FAR > 12 on admission and changes of albumin > 5 g/l were good predictors for the prognosis of COVID-19 patients.

Clinical progression and changes of chest CT findings among asymptomatic and pre-symptomatic patients with SARS-CoV-2 infection in Wuhan, China.

OBJECTIVES: To analyze characteristics of asymptomatic/pres-ymptomatic patients with SARS-CoV-2 infection. METHODS: Chest computed tomography(CT), indicators for organ and coagulation function, inflammation cytokines, of asymptomatic/pre-symptomatic patients with SARS-CoV-2 infection were retrospectively analyzed in Zhongnan Hospital of Wuhan University from 20 December 2019, to 8 March 2020. RESULTS: The proportion of normal chest CT in asymptomatic and pre-symptomatic patients with SARS-CoV-2 infection were 35.4% (17/48) and 3.3%(2/61), respectively (P< 0.001). In 17 asymptomatic patients, their images of chest CT maintained normal during the whole course of diseases, while the normal images of chest CT in 2 pre-symptomatic patients progressed to abnormal later (P< 0.001). All the six asymptomatic patients with SARS-CoV-2 infection maintained unilateral lesion, while the proportion was 29.4%(5/17) in pre-symptomatic patients(P= 0.003). Compared with asymptomatic patients, pre-symptomatic COVID-19 patients had worse levels of Lymphocyte count (P= 0.001), Albumin (P= 0.045), Aspartate aminotransferase (P= 0.044), γ-glutamyl transpeptadase (P= 0.016), Globulin (P= 0.036), Creatinine (P= 0.021), Lactate dehydrogenase (P= 0.008), C-reactive protein (P< 0.001), Serum amyloid A (P< 0.001), and Erythrocyte sedimentation rate (P< 0.001). Except for above indicators, Alkaline phosphatase (P= 0.009), Procalcitonin (P= 0.010), and D-dimer(P< 0.001) increased further during periods of symptoms compared with those levels in pre-symptomatic period. CONCLUSION: In early stage after SARS-CoV-2 infection, images of chest CT and blood tests of asymptomatic patients were different from pre-symptomatic patients.