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Background: SARS-CoV-2-specific adaptive immunity more than 1 year after initial infection has not been well characterised. The aim of this study was to investigate the durability and cross-reactivity of immunological memory acquired from natural infection against SARS-CoV-2 in individuals recovered from COVID-19 2 years after infection., Methods: In this longitudinal cohort study, we recruited patients who had recovered from laboratory-confirmed COVID-19 and were discharged from Jinyintan Hospital (Wuhan, China) between Jan 7 and May 29, 2020. We carried out three successive follow-ups between June 16 and Sept 3, 2020 (6 months), Dec 16, 2020, and Feb 7, 2021 (1 year), and Nov 16, 2021, and Jan 10, 2022 (2 years), in which blood samples were taken. We included participants who did not have re-infection or receive a SARS-CoV-2 vaccination (infected-unvaccinated), and participants who received one to three doses of inactivated vaccine 1-2 years after infection (infected-vaccinated). We evaluated the presence of IgG antibodies, neutralising antibodies, and memory B-cell and memory T-cell responses against the prototype strain and delta and omicron variants., Findings: In infected-unvaccinated participants, neutralising antibody titres continually declined from 6-month to 2-year follow-up visits, with a half-life of about 141·2 days. Neutralising antibody responses to omicron sublineages (BA.1, BA.1.1, BA.2, BA.4/5, BF.7, BQ.1, and XBB) were poor. Memory B-cell responses to the prototype strain were retained at 2 years and presented cross-reactivity to the delta and omicron BA.1 variants. The magnitude of interferon γ and T-cell responses to SARS-CoV-2 were not significantly different between 1 year and 2 years after infection. Multifunctional T-cell responses against SARS-CoV-2 spike protein and nucleoprotein were detected in most participants. Recognition of the BA.1 variant by memory T cells was not affected in most individuals. The antibody titres and the frequencies of memory B cells, but not memory T cells, increased in infected-vaccinated participants after they received the inactivated vaccine., Interpretation: This study improves the understanding of the duration of SARS-CoV-2-specific immunity without boosting, which has implications for the design of vaccination regimens and programmes. Our data suggest that memory T-cell responses primed by initial viral infection remain highly cross-reactive after 2 years. With the increasing emergence of variants, effective vaccines should be introduced to boost neutralising antibody and overall T-cell responses to newly emerged SARS-CoV-2 variants., Funding: Chinese Academy of Medical Sciences, National Natural Science Foundation of China, Fundamental Research Funds for the Central Universities for Peking Union Medical College, Beijing Natural Science Foundation, UK Medical Research Council., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Viral RNA-host protein interactions are indispensable during RNA virus transcription and replication, but their detailed structural and dynamical features remain largely elusive. Here, we characterize the binding interface for the SARS-CoV-2 stem-loop 3 (SL3) cis-acting element to human TIA1 protein with a combined theoretical and experimental approaches. The highly structured SARS-CoV-2 SL3 has a high binding affinity to TIA1 protein, in which the aromatic stacking, hydrogen bonds, and hydrophobic interactions collectively direct this specific binding. Further mutagenesis studies validate our proposed 3D binding model and reveal two SL3 variants have enhanced binding affinities to TIA1. And disruptions of the identified RNA-protein interactions with designed antisense oligonucleotides dramatically reduce SARS-CoV-2 infection in cells. Finally, TIA1 protein could interact with conserved SL3 RNA elements within other betacoronavirus lineages. These findings open an avenue to explore the viral RNA-host protein interactions and provide a pioneering structural basis for RNA-targeting antiviral drug design., (© 2023. The Author(s).)

The coronavirus disease 2019 (COVID-19) pandemic has devastated global health. Identifying key host factors essential for SARS-CoV-2 RNA replication is expected to unravel cellular targets for the development of broad-spectrum antiviral drugs which have been quested for the preparedness of future viral outbreaks. Here, we have identified host proteins that associate with nonstructural protein 12 (nsp12), the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using a mass spectrometry (MS)-based proteomic approach. Among the candidate factors, CDK2 (Cyclin-dependent kinase 2), a member of cyclin-dependent kinases, interacts with nsp12 and causes its phosphorylation at T20, thus facilitating the assembly of the RdRp complex consisting of nsp12, nsp7 and nsp8 and promoting efficient synthesis of viral RNA. The crucial role of CDK2 in viral RdRp function is further supported by our observation that CDK2 inhibitors potently impair viral RNA synthesis and SARS-CoV-2 infection. Taken together, we have discovered CDK2 as a key host factor of SARS-CoV-2 RdRp complex, thus serving a promising target for the development of SARS-CoV-2 RdRp inhibitors., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.

The emergence of SARS-CoV-2 Omicron and other variants of concern (VOCs) has brought huge challenges to control the COVID-19 pandemic, calling for urgent development of effective vaccines and therapeutic drugs. In this study, we focused on characterizing the impacts of divergent VOCs on the antiviral activity of lipopeptide-based fusion inhibitors that we previously developed. First, we found that pseudoviruses bearing the S proteins of five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) and one variant of interest (Lambda) exhibited greatly decreased infectivity relative to the wild-type (WT) strain or single D614G mutant, especially the Omicron pseudovirus. Differently, the most of variants exhibited an S protein with significantly enhanced cell fusion activity, whereas the S protein of Omicron still mediated decreased cell-cell fusion. Next, we verified that two lipopeptide-based fusion inhibitors, IPB02V3 and IPB24, maintained the highly potent activities in inhibiting various S proteins-driven cell fusion and pseudovirus infection. Surprisingly, both IPB02V3 and IPB24 lipopeptides displayed greatly increased potencies against the infection of authentic Omicron strain relative to the WT virus. The results suggest that Omicron variant evolves with a reduced cell fusion capacity and is more sensitive to the inhibition of fusion-inhibitory lipopeptides; thus, IPB02V3 and IPB24 can be further developed as potent, broad-spectrum antivirals for combating Omicron and the potential future outbreak of other emerging variants.

Abstract: The pandemic of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to major public health challenges globally. The increasing viral lineages identified indicate that the SARS-CoV-2 genome is evolving at a rapid rate. Viral genomic mutations may cause antigenic drift or shift, which are important ways by which SARS-CoV-2 escapes the human immune system and changes its transmissibility and virulence. Herein, we summarize the functional mutations in SARS-CoV-2 genomes to characterize its adaptive evolution to inform the development of vaccination, treatment as well as control and intervention measures., (Copyright © 2022 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNA RBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNA RBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2., Competing Interests: Declaration of interests Patents related to the data presented have been filed. W.W. is the founder of Therorna, Inc., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The memory immune response is crucial for preventing reinfection or reducing disease severity. However, the robustness and functionality of the humoral and T-cell response to SARS-CoV-2 remains unknown 12 months after initial infection. The aim of this study is to investigate the durability and functionality of the humoral and T-cell response to the original SARS-CoV-2 strain and variants in recovered patients 12 months after infection., Methods: In this longitudinal cohort study, we recruited participants who had recovered from COVID-19 and who were discharged from the Wuhan Research Center for Communicable Disease Diagnosis and Treatment at the Chinese Academy of Medical Sciences, Wuhan, China, between Jan 7 and May 29, 2020. Patients received a follow-up visit between Dec 16, 2020, and Jan 27, 2021. We evaluated the presence of IgM, IgA, and IgG antibodies against the SARS-CoV-2 nucleoprotein, Spike protein, and the receptor-binding domain 12 months after initial infection, using ELISA. Neutralising antibodies against the original SARS-CoV-2 strain, and the D614G, beta (B.1.351), and delta (B.1.617.2) variants were analysed using a microneutralisation assay in a subset of plasma samples. We analysed the magnitude and breadth of the SARS-CoV-2-specific memory T-cell responses using the interferon γ (IFNγ) enzyme-linked immune absorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) assay. The antibody response and T-cell response (ie, IFN-γ, interleukin-2 [IL-2], and tumour necrosis factor α [TNFα]) were analysed by age and disease severity. Antibody titres were also analysed according to sequelae symptoms., Findings: We enrolled 1096 patients, including 289 (26·4%) patients with moderate initial disease, 734 (67·0%) with severe initial disease, and 73 (6·7%) with critical initial disease. Paired plasma samples were collected from 141 patients during the follow-up visits for the microneutralisation assay. PBMCs were collected from 92 of 141 individuals at the 12-month follow-up visit, of which 80 were analysed by ELISpot and 92 by ICS assay to detect the SARS-CoV-2-specific memory T-cell responses. N-IgG (899 [82·0%]), S-IgG (1043 [95·2%]), RBD-IgG (1032 [94·2%]), and neutralising (115 [81·6%] of 141) antibodies were detectable 12 months after initial infection in most individuals. Neutralising antibodies remained stable 6 and 12 months after initial infection in most individuals younger than 60 years. Multifunctional T-cell responses were detected for all SARS-CoV-2 viral proteins tested. There was no difference in the magnitude of T-cell responses or cytokine profiles in individuals with different symptom severity. Moreover, we evaluated both antibody and T-cell responses to the D614G, beta, and delta viral strains. The degree of reduced in-vitro neutralising antibody responses to the D614G and delta variants, but not to the beta variant, was associated with the neutralising antibody titres after SARS-CoV-2 infection. We also found poor neutralising antibody responses to the beta variant; 83 (72·2%) of 115 patients showed no response at all. Moreover, the neutralising antibody titre reduction of the recovered patient plasma against the delta variant was similar to that of the D614G variant and lower than that of the beta variant. By contrast, T-cell responses were cross-reactive to the beta variant in most individuals. Importantly, T-cell responses could be detected in all individuals who had lost the neutralising antibody response to SARS-CoV-2 12 months after the initial infection., Interpretation: SARS-CoV-2-specific neutralising antibody and T-cell responses were retained 12 months after initial infection. Neutralising antibodies to the D614G, beta, and delta viral strains were reduced compared with those for the original strain, and were diminished in general. Memory T-cell responses to the original strain were not disrupted by new variants. This study suggests that cross-reactive SARS-CoV-2-specific T-cell responses could be particularly important in the protection against severe disease caused by variants of concern whereas neutralising antibody responses seem to reduce over time., Funding: Chinese Academy of Medical Sciences, National Natural Science Foundation, and UK Medical Research Council., Competing Interests: We declare no competing interests., (© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license.)

The outbreak of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has created a global health crisis. SARS-CoV-2 infects varieties of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative viral entry pathways. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. Beyond known host proteins, i.e., ACE2, TMPRSS2, and NRP1, we identified multiple host components, among which LDLRAD3, TMEM30A, and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike's N-terminal domain (NTD). Their essential and physiological roles have been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in an ACE2-independent fashion. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of COVID-19 countermeasures., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10 -12  M) and strong neutralizing activity (IC 50  = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications., (© 2022. The Author(s).)

The global epidemic caused by the coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in the infection of over 200 million people. To extend the knowledge of interactions between SARS-CoV-2 and humans, we systematically investigate the interactome of 29 viral proteins in human cells by using an antibody-based TurboID assay. In total, 1,388 high-confidence human proximal proteins with biotinylated sites are identified. Notably, we find that SARS-CoV-2 manipulates the antiviral and immune responses. We validate that the membrane protein ITGB1 associates angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 entry. Moreover, we reveal that SARS-CoV-2 proteins inhibit activation of the interferon pathway through the mitochondrial protein mitochondrial antiviral-signaling protein (MAVS) and the methyltransferase SET domain containing 2, histone lysine methyltransferase (SETD2). We propose 111 potential drugs for the clinical treatment of coronavirus disease 2019 (COVID-19) and identify three compounds that significantly inhibit the replication of SARS-CoV-2. The proximity labeling map of SARS-CoV-2 and humans provides a resource for elucidating the mechanisms of viral infection and developing drugs for COVID-19 treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Seasonal human coronaviruses (HCoVs) including HCoV-229E, -OC43, -NL63, and -HKU1 widely spread in global human populations. However, the relevance of humoral response against seasonal HCoVs to COVID-19 pathogenesis is elusive. In this study, we profiled the temporal changes of IgG antibody against spike proteins (S-IgG) of SARS-CoV-2 and seasonal HCoVs in 838 plasma samples collected from 344 COVID-19 patients. We tested the antigenic cross-reactivities of S protein between SARS-CoV-2 and seasonal HCoVs and evaluated the correlations between the levels of HCoV-OC43 S-IgG and the disease severity in COVID-19 patients. We found that SARS-CoV-2 S-IgG titres mounted until days 22-28, whereas HCoV-OC43 antibody titres increased until days 15-21 and then plateaued until day 46. However, IgG titres against HCoV-NL63, -229E, and -HKU1 showed no significant increase. A two-way cross-reactivity was identified between SARS-CoV-2 and HCoV-OC43. Neutralizing antibodies against SARS-CoV-2 were not detectable in healthy controls who were positive for HCoV-OC43 S-IgG. HCoV-OC43 S-IgG titres were significantly higher in patients with severe disease than those in mild patients at days 1-21 post symptom onset (PSO). Higher levels of HCoV-OC43 S-IgG were also observed in patients requiring mechanical ventilation. At days 1-10 PSO, HCoV-OC43 S-IgG titres correlated to disease severity in the age group over 60. Our data indicate that there is a correlation between cross-reactive antibody against HCoV-OC43 spike protein and disease severity in COVID-19 patients.

The ongoing pandemic of COVID-19, caused by SARS-CoV-2, has severely impacted the global public health and socio-economic stability, calling for effective vaccines and therapeutics. In this study, we continued our efforts to develop more efficient SARS-CoV-2 fusion inhibitors and achieved significant findings. First, we found that the membrane-proximal external region (MPER) sequence of SARS-CoV-2 spike fusion protein plays a critical role in viral infectivity and can serve as an ideal template for design of fusion-inhibitory peptides. Second, a panel of novel lipopeptides was generated with greatly improved activity in inhibiting SARS-CoV-2 fusion and infection. Third, we showed that the new inhibitors maintained the potent inhibitory activity against emerging SARS-CoV-2 variants, including those with the major mutations of the B.1.1.7 and B.1.351 strains circulating in the United Kingdom and South Africa, respectively. Fourth, the new inhibitors also cross-inhibited other human CoVs, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Fifth, the structural properties of the new inhibitors were characterized by circular dichroism (CD) spectroscopy and crystallographic approach, which revealed the mechanisms underlying the high binding and inhibition. Combined, our studies provide important information for understanding the mechanism of SARS-CoV-2 fusion and a framework for the development of peptide therapeutics for the treatment of SARS-CoV-2 and other CoVs.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system., (© 2021. The Author(s).)

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S 470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S 450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic., (© 2021. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating pandemic worldwide. Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic, and scientists all over the world have made great efforts to this end. However, manipulation of the SARS-CoV-2 should be performed in the biosafety level 3 laboratory. This makes experiments complicated and time-consuming. Therefore, a safer system for working with this virus is urgently needed. Here, we report the construction of plasmid-based, non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae. Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes. Using SARS-CoV-2 replicons, the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed, and the half-maximal effective concentration (EC 50 ) value of remdesivir and E64-D was estimated by different quantification methods respectively, indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation., (© 2021. Wuhan Institute of Virology, CAS.)

The immune mechanisms of long coronavirus disease 2019 (COVID) are not yet fully understood. We aimed to investigate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific memory immune responses in discharged COVID-19 patients with and without long COVID symptoms. In this cross-sectional study, we included 1041 hospitalized COVID-19 patients with the original virus strain in Wuhan (China) 12 months after initial infection. We simultaneously conducted a questionnaire survey and collected peripheral blood samples from the participants. Based on the presence or absence of long COVID symptoms during the follow-up period, we divided the patients into 2 groups: a long COVID group comprising 480 individuals and a convalescent group comprising 561 individuals. Both groups underwent virus-specific immunological analyses, including enzyme-linked immunosorbent assay, interferon-γ-enzyme-linked immune absorbent spot, and intracellular cytokine staining. At 12 months after infection, 98.5% (1026/1041) of the patients were found to be seropositive and 93.3% (70/75) had detectable SARS-CoV-2-specific memory T cells. The long COVID group had significantly higher levels of receptor binding domain (RBD)–immunoglobulin G (IgG) levels, presented as OD450 values, than the convalescent controls (0.40 ± 0.22 vs 0.37 ± 0.20; P =.022). The magnitude of SARS-CoV-2-specific T-cell responses did not differ significantly between groups, nor did the secretion function of the memory T cells. We did not observe a significant correlation between SARS-CoV-2-IgG and magnitude of memory T cells. This study revealed that long COVID patients had significantly higher levels of RBD-IgG antibodies when compared with convalescent controls. Nevertheless, we did not observe coordinated SARS-CoV-2-specific cellular immunity. As there may be multiple potential causes of long COVID, it is imperative to avoid adopting a "one-size-fits-all" approach to future treatment modalities. [ABSTRACT FROM AUTHOR]

The profile of antibodies against antigenic epitopes of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) during neutralizing antibody (NAb) decay has not been clarified. Using a SARS‐CoV‐2 proteome microarray that contained viral antigenic peptides, we analyzed the characteristics of the humoral response in patients with coronavirus disease 19 (COVID‐19) in a longitudinal study. A total of 89 patients were recruited, and 226 plasma samples were serially collected in 2020. In the antigenic peptide microarray, the level of immunoglobulin G (IgG) antibodies against peptides within the S2 subunit (S‐82) and a conserved gene region in variants of interest, open reading frame protein 10 (ORF10‐3), were closely associated with the presence of SARS‐CoV‐2 NAbs. In an independent evaluation cohort of 232 plasma samples collected from 116 COVID‐19 cases in 2020, S82‐IgG titers were higher in NAbs‐positive samples (p = 0.002) than in NAbs‐negative samples using enzyme‐linked immunosorbent assay. We further collected 66 plasma samples from another cohort infected by Omicron BA.1 virus in 2022. Compared with the samples with lower S82‐IgG titers, NAb titers were significantly higher in the samples with higher S82‐IgG titers (p = 0.04). Our findings provide insights into the understanding of the decay‐associated signatures of SARS‐CoV‐2 NAbs. [ABSTRACT FROM AUTHOR]

The bat coronaviruses (CoV) BANAL-20-52 and BANAL-20-236 are two newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) closely related coronaviruses (SC2r-CoV) and the genome of BANAL-20-52 shares the highest homology with SARS-CoV-2. However, the risk of their potential zoonotic transmission has not been fully evaluated. Here, we determined their potential host susceptibility among 13 different bat species and 26 different animal species, and found that both might have extensive host ranges, indicating high zoonotic transmission potential. We also determined the cryo-EM structures of BANAL-20-52 and BANAL-20-236 S proteins at pH 5.5 and the complex of BANAL-20-236 S1 and Rhinolophus affinis ACE2, and found that both trimeric S proteins adopt all three receptor binding domains (RBDs) in "closed" conformation and are more compact than SARS-CoV-2. Strikingly, the unique sugar moiety at N370 of bat SC2r-CoVs acts like a "bolt" and crosses over two neighboring subunits, facilitating the S proteins in the locked conformation and underpinning the architecture stability. Removal of the glycosylation at N370 by a T372A substitution substantially enhances virus infectivity but becomes highly sensitive to trypsin digestion at pH 5.5, a condition roughly mimicking the insectivorous bat's stomach digestion. In contrast, WT S proteins of SC2r-CoVs showed considerable resistance to trypsin digestion at pH 5.5, indicating that the highly conserved T372 in bat CoVs might result from the selective advantages in stability during the fecal-oral transmission over A372. Moreover, the results of cross-immunogenicity among S proteins of SARS-CoV-2, BANAL-20-52, and BANAL-20-236 showed that A372 pseudoviruses are more sensitive to anti-S sera than T372, indicating that immune evasion might also play a role in the natural selection of T372 over A372 during evolution. Finally, residues 493 and 498 of the S protein affect host susceptibility, and residue 498 also influences the immunogenicity of the S protein. Together, our findings aid a better understanding of the molecular basis of CoV entry, selective evolution, and immunogenicity and highlight the importance of surveillance of susceptible hosts of these viruses to prevent potential outbreaks. [ABSTRACT FROM AUTHOR]

Bat coronavirus RaTG13 shares about 96.2% nucleotide sequence identity with that of SARS-CoV-2 and uses human and Rhinolophus affinis (Ra) angiotensin-converting enzyme 2 (ACE2) as entry receptors. Whether there are bat species other than R. affinis susceptible to RaTG13 infection remains elusive. Here, we show that, among 18 different bat ACE2s tested, only RaACE2 is highly susceptible to transduction by RaTG13 S pseudovirions, indicating that the bat species harboring RaTG13 might be very limited. RaACE2 has seven polymorphic variants, RA-01 to RA-07, and they show different susceptibilities to RaTG13 S transduction. Sequence and mutagenesis analyses reveal that residues 34, 38, and 83 in RaACE2 might play critical roles in interaction with the RaTG13 S protein. Of note, RaACE2 polymorphisms have minimal effect on S proteins of SARS-CoV-2 and several SARS-CoV-2 related CoVs (SC2r-CoV) including BANAL-20-52 and BANAL-20-236 in terms of binding, membrane fusion, and pseudovirus entry. Further mutagenesis analyses identify residues 501 and 505 in S proteins critical for the recognition of different RaACE2 variants and pangolin ACE2 (pACE2), indicating that RaTG13 might have not been well adapted to R. affinis bats. While single D501N and H505Y changes in RaTG13 S protein significantly enhance the infectivity and minimize the difference in susceptibility among different RaACE2 variants, an N501D substitution in SARS-CoV-2 S protein displays marked disparity in transduction efficiencies among RaACE2 variants with a significant reduction in infectivity on several RaACE2 variants. Finally, a T372A substitution in RaTG13 S protein not only significantly increases infectivity on all RaACE2 variants, but it also markedly enhances entry on several bat ACE2s including R. sinicus YN, R. pearsonii, and R. ferrumeiqunum. However, the T372A mutant is about 4-fold more sensitive to neutralizing sera from mice immunized with BANAL-20-52 S, suggesting that the better immune evasion ability of T372 over A372 might contribute to the natural selective advantage of T372 over A372 among bat CoVs. Together, our study aids a better understanding of coronavirus entry, vaccine design, and evolution. Author summary: Bat CoV RaTG13 has zoonotic transmission potential, and interactions between S protein and ACE2 play essential roles. RaTG13 uses RaACE2 as an entry receptor, and there are at least seven polymorphic RaACE2 variants. The effect of RaACE2 polymorphism on entry of RaTG13 and other SARS-CoV-2 related bat CoV (SC2r-CoV) has not been determined. In this study, we find that RaTG13 S pseudovirions only highly efficiently transduce cells expressing RaACE2 among available 18 bat species commonly habituating in Southwestern China and Southeastern Asia, suggesting that there might be limited bat species including R. affinis susceptible to infection of RaTG13 virus. The RaACE2 polymorphism has a marked effect on the entry of RaTG13, whereas it shows a minimal effect on the entry of SARS-CoV-2 and SC2r-CoVs like BANAL-20-52, BANAL-20-236, and pangolin-CoV GD. Further sequence and mutagenesis analyses identify residues 34, 38, and 83 in RaACE2 and residues 501 and 505 in the S proteins critical for S protein and receptor interactions, indicating that RaTG13 might have not been well adapted to R. affinis bats. We also find that T372A substitution in RaTG13 S significantly enhances entry on all RaACE2 variants and several bat ACE2s, and immune evasion might contribute to the natural selection of T372 over A372 in bat S proteins. Altogether, our work provides detailed analyses of the effect of RaACE2 polymorphism on the entry of RaTG13 and other SC2r-CoVs and a better understanding of the entry and evolution of CoVs. [ABSTRACT FROM AUTHOR]

These authors contributed equally: Xiuyuan Ou, Ge Xu, Pei Li, Yan Liu Correction to: I Cell Discovery i (2023) 9:78 https://doi.org/10.1038/s41421-023-00581-9 published online 28 July 2023 During the final submission of this manuscript, we inadvertently misplaced the Supplementary Fig. Graph Supplementary information Graph: new supplementary file Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41421-023-00597-1. [Extracted from the article]

COVID-19 is a globally infectious viral epidemic of great public health concern caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Angiotensin-converting enzyme 2 (ACE2) plays its role as the receptor for SARS-CoV-2 through binding with S protein and the binding results in ACE2 expression decrease. The change of ACE2 is supposed to elicit a series of cellular and molecular events. Other than as the receptor, ACE2's roles on infection by regulating other molecules need to be further studied during SARS-CoV-2 infection. In the present study, we established the ACE2 knockdown model using Vero E6 cells to study how ACE2 influenced the downstream signaling molecules. Analysis of transcriptome sequencing discovered that ACE2 alteration per se caused the alteration of immune factors, including some related to the viral infection-related signaling pathways. We found that ACE2 silencing induced the reduced interferon-induced transmembrane protein 3 (IFITM3) expression. Overexpression of IFITM3 promoted the SARS-CoV-2 pseudovirus infection of Vero E6 cells lacking the ACE2. It indicates that ACE2 can affect IFITM3 expression and function to affect the SARS-CoV-2 infection. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and contribute to explaining the rapid spread and pathogenesis especially in the case of ACE2 low expression. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3′ UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development. [Display omitted] • RIC-seq maps SARS-CoV-2-to-host RNA-RNA interactions in infected cells • SARS-CoV-2 stabilizes host mRNAs via base pairings at 3′ UTRs • SARS-CoV-2 stabilizes host mRNAs by recruiting RNA-binding protein YBX3 • Stabilized NFKBIZ contributes to SARS-CoV-2-elicited immunopathogenesis Zhao et al. use RIC-seq technology for global profiling of SARS-CoV-2-to-host RNA-RNA interactions in infected cells. The authors find that SARS-CoV-2 RNA stabilizes host mRNAs through their 3′ UTRs via base pairings by recruiting RNA-binding protein YBX3. The stabilized host mRNA NFKBIZ may contribute to SARS-CoV-2-elicited immunopathogenesis. [ABSTRACT FROM AUTHOR]

[Display omitted] The pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has caused immense global disruption. With the rapid accumulation of SARS-CoV-2 genome sequences, however, thousands of genomic variants of SARS-CoV-2 are now publicly available. To improve the tracing of the viral genomes' evolution during the development of the pandemic, we analyzed single nucleotide variants (SNVs) in 121,618 high-quality SARS-CoV-2 genomes. We divided these viral genomes into two major lineages (L and S) based on variants at sites 8782 and 28144, and further divided the L lineage into two major sublineages (L1 and L2) using SNVs at sites 3037, 14408, and 23403. Subsequently, we categorized them into 130 sublineages (37 in S, 35 in L1, and 58 in L2) based on marker SNVs at 201 additional genomic sites. This lineage/sublineage designation system has a hierarchical structure and reflects the relatedness among the subclades of the major lineages. We also provide a companion website (www.covid19evolution.net) that allows users to visualize sublineage information and upload their own SARS-CoV-2 genomes for sublineage classification. Finally, we discussed the possible roles of compensatory mutations and natural selection during SARS-CoV-2's evolution. These efforts will improve our understanding of the temporal and spatial dynamics of SARS-CoV-2's genome evolution. [ABSTRACT FROM AUTHOR]