Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes.

SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed and open. The locked conformations feature tightly packed trimers with structural elements incompatible with RBD in "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably due to their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. Intriguingly, locked conformations were the only conformations captured for S proteins of bat and pangolin origin SARS-related coronaviruses. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures to other available spike structures of Sarbecoviruses to identify conserved features and discuss their possible functions.

Safety and superior immunogenicity of heterologous boosting with an RBD-based SARS-CoV-2 mRNA vaccine in Chinese adults

Homologous and heterologous booster with COVID-19 mRNA vaccines represent the most effective strategy to prevent the ongoing Omicron pandemic. The additional protection from these prototype SARS-CoV-2 S-targeting vaccine was attributed to the increased RBD-specific memory B cells with expanded potency and breadth. Herein, we show the safety and immunogenicity of heterologous boosting with the RBD-targeting mRNA vaccine AWcorna (also term ARCoV) in Chinese adults who have received two doses inactivated vaccine. The superiority over inactivated vaccine in neutralization antibodies, as well as the safety profile, support the use of AWcorna as heterologous booster in China.

Combating the SARS-CoV-2 Omicron variant with non-Omicron neutralizing antibodies

The highly mutated and transmissible Omicron variant has provoked serious concerns over its decreased sensitivity to the current coronavirus disease 2019 (COVID-19) vaccines and evasion from most anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs). In this study, we explored the possibility of combatting the Omicron variant by constructing bispecific antibodies based on non-Omicron NAbs. We engineered ten IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, eight out of ten bispecific antibodies showed high binding affinity to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron, as well as authentic Omicron(+R346K) variants. Six bispecific antibodies containing the cross-NAb GW01 neutralized Omicron variant and retained their abilities to neutralize other sarbecoviruses. Bispecific antibodies inhibited Omicron infection by binding to the ACE2 binding site. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody FD01 in complex with the Omicron spike (S) revealed 5 distinct trimers and one unique bi-trimer conformation. The structure and mapping analyses of 34 Omicron S variant single mutants elucidated that two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3-RBD-up conformation, enlarged the interface area, accommodated the S371L mutation, improved the affinity between a single IgG and the Omicron RBD, and hindered ACE2 binding by forming bi-trimer conformation. Our study offers an important foundation for anti-Omicron NAb design. Engineering bispecific antibodies based on non-Omicron NAbs may provide an efficient solution to combat the Omicron variant.

The adenosine analogue prodrug ATV006 is orally bioavailable and has potent preclinical efficacy against SARS-CoV-2 and its variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 pandemic, is rapidly evolving. Due to the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOC), including the currently most prevalent Delta variant, orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously we showed that adenosine analogue 69-0 (also known as GS-441524), possesses potent anti-SARS-CoV-2 activity. Herein, we report that esterification of the 5-hydroxyl moieties of 69-0 markedly improved the antiviral potency. The 5-hydroxyl-isobutyryl prodrug, ATV006, showed excellent oral bioavailability in rats and cynomolgus monkeys and potent antiviral efficacy against different VOCs of SARS-CoV-2 in cell culture and three mouse models. Oral administration of ATV006 significantly reduced viral loads, alleviated lung damage and rescued mice from death in the K18-hACE2 mouse model challenged with the Delta variant. Moreover, ATV006 showed broad antiviral efficacy against different mammal-infecting coronaviruses. These indicate that ATV006 represents a promising oral drug candidate against SARS-CoV-2 VOCs and other coronaviruses.

Plasma cell-free DNA promise disease monitoring and tissue injury assessment of COVID-19

COVID-19 is a huge threat to global health. Due to the lack of definitive etiological therapeutics currently, effective disease monitoring is of high clinical value for better healthcare and management of the large number of COVID-19 patients. In this study, we recruited 37 COVID-19 patients, collected 176 blood samples upon diagnosis and during treatment, and analyzed cell-free DNA (cfDNA) in these samples. We report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA characteristics reflect patient-specific physiological conditions during treatment. Further analysis on tissue origin tracing of cfDNA reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, we demonstrate the translational merit of cfDNA as valuable analyte for effective disease monitoring, as well as tissue injury assessment in COVID-19 patients.

SARS-CoV-2-Triggered Mast Cell Rapid Degranulation Induces Alveolar Epithelial Inflammation and Lung Injury

SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. Here we showed that SARS-CoV-2-triggeed MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation alterred various signaling pathways in alveolar epithelial cells, particularly, led to the production of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/449680v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@899996org.highwire.dtl.DTLVardef@1c26c0eorg.highwire.dtl.DTLVardef@1442cdcorg.highwire.dtl.DTLVardef@dd4204_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefSARS-CoV-2 triggers an immediate mast cell (MC) degranulation, which initiates the alveolar epithelial inflammation and disrupts the tight junction. MC stabilizers that block degranulation reduce virus-induced lung inflammation and injury. HighlightsO_LIThe binding of RBD of Spike protein of SARS-CoV-2-to ACE2 receptor protein triggers an immediate MC degranulation C_LIO_LIMC degranulation induces transcriptomic changes include an upregulated inflammatory signaling and a downregulated cell-junction signaling C_LIO_LIMC degranulation leads to alveolar epithelial inflammation and disruption of tight junctions C_LIO_LIMC stabilizer that inhibits degranulation reduces SARS-CoV-2-induced lung inflammation and injury in vivo C_LI

Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global crisis, urgently necessitating the development of safe, efficacious, convenient-to-store, and low-cost vaccine options. A major challenge is that the receptor-binding domain (RBD)-only vaccine fails to trigger long-lasting protective immunity if used solely for vaccination. To enhance antigen processing and cross-presentation in draining lymph nodes (DLNs), we developed an interferon (IFN)-armed RBD dimerized by immunoglobulin fragment (I-R-F). I-R-F efficiently directs immunity against RBD to DLN. A low dose of I-R-F induces not only high titer long-lasting neutralizing antibodies but also comprehensive T cell responses than RBD, and even provides comprehensive protection in one dose without adjuvant. This study shows that the I-R-F vaccine provides rapid and complete protection throughout upper and lower respiratory tracts against high dose SARS-CoV-2 challenge in rhesus macaques. Due to its potency and safety, this engineered vaccine may become one of the next-generation vaccine candidates in the global race to defeat COVID-19.

Spike mutations decrease SARS-CoV-2 sensitivity to neutralizing antibodies but not ACE2-Ig in vitro

The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to [~]15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.

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

Dysfunctional immune response in the COVID-19 patients is a recurrent theme impacting symptoms and mortality, yet the detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 205 COVID-19 patients and controls to create a comprehensive immune landscape. Lymphopenia and active T and B cell responses were found to coexist and associated with age, sex and their interactions with COVID-19. Diverse epithelial and immune cell types were observed to be virus-positive and showed dramatic transcriptomic changes. Elevation of ANXA1 and S100A9 in virus-positive squamous epithelial cells may enable the initiation of neutrophil and macrophage responses via the ANXA1-FPR1 and S100A8/9-TLR4 axes. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis and designing effective therapeutic strategies for COVID-19. HIGHLIGHTSO_LILarge-scale scRNA-seq analysis depicts the immune landscape of COVID-19 C_LIO_LILymphopenia and active T and B cell responses coexist and are shaped by age and sex C_LIO_LISARS-CoV-2 infects diverse epithelial and immune cells, inducing distinct responses C_LIO_LICytokine storms with systemic S100A8/A9 are associated with COVID-19 severity C_LI

Remdesivir Metabolite GS-441524 Efficiently Inhibits SARS-CoV-2 Infection in Mouse Model

The outbreak of coronavirus disease 2019 (COVID-19) rapidly spreads across worldwide and becomes a global pandemic. Remdesivir is the only COVID-19 treatment approved by U.S. Food and Drug Administration (FDA); however, its effectiveness is still under questioning as raised by the results of a large WHO Solidarity Trial. Herein, we report that the parent nucleotide of remdesivir, GS-441524, potently inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero E6 and other cells. It exhibits good plasma distribution and longer half-life (t1/2=4.8h) in rat PK study. GS-441524 is highly efficacious against SARS-CoV-2 in AAV-hACE2 transduced mice and murine hepatitis virus (MHV) in mice, reducing the viral titers in CoV-attacked organs, without noticeable toxicity. Given that GS-441524 was the predominant metabolite of remdesivir in the plasma, the anti-COVID-19 effect of remdesivir may partly come from the effect of GS-441524. Our results also supported that GS-441524 as a promising and inexpensive drug candidate in the treatment of COVID-19 and future emerging CoVs diseases.

Duple extinguishment of COVID-19: single compound synergized inhibition of SARS-CoV-2 replication and direct suppression of inflammatory cytokines in vitro/vivo

We firstly disclose single compound yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from Au22Glutathione18 (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication (EC50: ~0.24 M) and intrinsically down-regulates NF{kappa}B pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The lung viral load and inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir while their lung viral load are decreased to same level. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 golden hamsters. One Sentence SummaryWe found that gold cluster molecule directly inhibits SARS-CoV-2 replication and intrinsically suppresses inflammatory cytokines expression in COVID-19 transgenic mouse and golden hamster model, gold cluster providing a better lung injury protection than Remdesivir in COVID-19 golden hamsters via intranasally dropping administration.

The Strand-biased Transcription of SARS-CoV-2 and Unbalanced Inhibition by Remdesivir

SARS-CoV-2, a positive single-stranded RNA virus, caused the COVID-19 pandemic. During the viral replication and transcription, the RNA dependent RNA polymerase (RdRp) "jumps" along the genome template, resulting in discontinuous negative-stranded transcripts. In other coronaviruses, the negative strand RNA was found functionally relevant to the activation of host innate immune responses. Although the sense-mRNA architectures of SARS-CoV-2 were reported, its negative strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand. During negative strand synthesis, apart from canonical sub-genomic ORFs, numerous non-canonical fusion transcripts are formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor Remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the anti-viral vaccine development and drug design. One Sentence SummaryStrand-biased transcription of SARS-CoV-2.

Characterization of Microbial Co-infections in the Respiratory Tract of hospitalized COVID-19 patients

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of Coronavirus disease 2019 (COVID-19). However, microbial composition of the respiratory tract and other infected tissues, as well as their possible pathogenic contributions to varying degrees of disease severity in COVID-19 patients remain unclear. MethodBetween January 27 and February 26, 2020, serial clinical specimens (sputum, nasal and throat swab, anal swab and feces) were collected from a cohort of hospitalized COVID-19 patients, including 8 mildly and 15 severely ill patients (requiring ICU admission and mechanical ventilation), in the Guangdong province, China. Total RNA was extracted and ultra-deep metatranscriptomic sequencing was performed in combination with laboratory diagnostic assays. Co-infection rates, the prevalence and abundance of microbial communities in these COVID-19 patients were determined. FindingsNotably, respiratory microbial co-infections were exclusively found in 84.6% of severely ill patients (11/13), among which viral and bacterial co-infections were detected by sequencing in 30.8% (4/13) and 69.2% (9/13) of the patients, respectively. In addition, for 23.1% (3/13) of the patients, bacterial co-infections with Burkholderia cepacia complex (BCC) and Staphylococcus epidermidis were also confirmed by bacterial culture. Further, a time-dependent, secondary infection of B. cenocepacia with expressions of multiple virulence genes in one severely ill patient was demonstrated, which might be the primary cause of his disease deterioration and death one month after ICU admission. InterpretationOur findings identified distinct patterns of co-infections with SARS-CoV-2 and various respiratory pathogenic microbes in hospitalized COVID-19 patients in relation to disease severity. Detection and tracking of BCC-associated nosocomial infections are recommended to improve the pre-emptive treatment regimen and reduce fatal outcomes of hospitalized patients infected with SARS-CoV-2. FundingNational Science and Technology Major Project of China, National Major Project for Control and Prevention of Infectious Disease in China, the emergency grants for prevention and control of SARS-CoV-2 of Ministry of Science and Technology and Guangdong province, Guangdong Provincial Key Laboratory of Genome Read and Write, Guangdong Provincial Academician Workstation of BGI Synthetic Genomics, and Shenzhen Engineering Laboratory for Innovative Molecular Diagnostics.

Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Author summaryIn this study, we compared SARS-CoV-2 populations of 13 Chinese COVID-19 patients. Those viral populations contained a considerable proportion of viral sub-genomic messenger RNAs (sgmRNA), reflecting an active viral replication activity in the respiratory tract tissues. The comparison of 66 identified intra-host variants further showed a low viral genetic distance between intra-household patients and a narrow transmission bottleneck size. Despite the co-existence of genetically distinct viruses within the same host, most intra-host minor variants were not shared between transmission pairs, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Furthermore, the narrow bottleneck and active viral activity in the respiratory tract show that the passage of a small number of virions can cause infection. Our data have therefore delivered a key genomic resource for the SARS-CoV-2 transmission research and enhanced our understanding of the evolutionary dynamics of SARS-CoV-2.

Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to many countries around the world. However, where is it origin and which animals are sensitive to cross-species transmission is unclear. The interaction of virus and cell receptor is a key determinant of host range for the novel coronavirus. Angiotensin-converting enzyme 2 (ACE2) is demonstrated as the primary entry receptor for SARS-CoV-2. In this study, we evaluated the SARS-CoV-2 entry mediated by ACE2 of 11 different species of animals, and discovered that ACE2 of Rhinolophus sinicus (Chinese horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (pig), Capra hircus (goat) and especially Manis javanica (Malayan pangolin) were able to render SARS-CoV-2 entry in non-susceptible cells. This is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the presume that SARS-CoV-2 may have a pangolin origin. However, none of the ACE2 proteins from Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (chicken), Notechis scutatus (mainland tiger snake), Mus musculus (house mouse) rendered SARS-CoV-2 entry. Specifically, a natural isoform of Macaca mulatta (Rhesus monkey) ACE2 with a mutation of Y217N was resistance to infection, which rises the possible impact of this type of ACE2 during monkey studies of SARS-CoV-2. Overall, these results clarify that SARS-CoV-2 could engage receptors of multiple species of animals and it is a perplexed work to track SARS-CoV-2 origin and its intermediate hosts. IMPORTANCEIn this study, we illustrated that SARS-CoV-2 is able to engage receptors of multiple species of animals. This indicated that it may be a perplexed work to track SARS-CoV-2 origin and discover its intermediate hosts. This feature of virus is considered to potentiate its diverse cross-species transmissibility. Of note, here is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the possibility that SARS-CoV-2 may have a pangolin origin. And we also demonstrated that not all species of bat were sensitive to SARS-CoV-2 infection. At last, it is also important to detect the expression ratio of the Y217N ACE2 to the prototype in Rhesus monkeys to be recruited for studies on SARS-CoV-2 infection.

Enantiomers of Chloroquine and Hydroxychloroquine Exhibit Different Activities Against SARS-CoV-2 in vitro, Evidencing S-Hydroxychloroquine as a Potentially Superior Drug for COVID-19

In all of the clinical trials for COVID-19 conducted thus far and among those ongoing involving chloroquine or hydroxychloroquine, the drug substance used has invariably been chloroquine (CQ) diphosphate or hydroxychloroquine (HCQ) sulfate, i.e., the phosphoric or sulfuric acid salt of a racemic mixture of R- and S-enantiomer (50/50), respectively. As a result, the clinical outcome from previous CQ or HCQ trials were, in fact, the collective manifestation of both R and S- enantiomers with inherent different pharmacodynamic and pharmacokinetic properties, and toxicity liabilities. Our data for the first time demonstrated the stereoselective difference of CQ and HCQ against live SARS-CoV-2 virus in a Biosafety Level 3 laboratory. S-chloroquine (S-CQ) and S-hydroxychloroquine (S-HCQ) significantly more active against SARS-CoV-2, as compared to R-CQ and R-HCQ, respectively. In addition, Mpro, as one of the critical enzymes for viral transcription and replication, also exhibited an enantioselective binding affinity toward the S-enantiomers. The most significant finding from this study is the pronounced difference of the two enantiomers of CQ and HCQ observed in hERG inhibition assay. The IC50 value of S-HCQ was higher than 20 M against hERG channel, which was much less active over all tested CQ and HCQ compounds. Moreover, S-HCQ alone did not prolong QT interval in guinea pigs after 3 days and 6 days of administration, indicating a much lower cardiac toxicity potential. With these and previous findings on the enantio-differentiated metabolism, we recommend that future clinical studies should employ S-HCQ, substantially free of the R-enantiomer, to potentially improve the therapeutic index for the treatment of COVID-19 over the racemic CQ and HCQ.

Intra-host Variation and Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients

As of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports1,2. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.

Single-Cell Analysis Reveals Macrophage-Driven T Cell Dysfunction in Severe COVID-19 Patients

The vastly spreading COVID-19 pneumonia is caused by SARS-CoV-2. Lymphopenia and cytokine levels are tightly associated with disease severity. However, virus-induced immune dysregulation at cellular and molecular levels remains largely undefined. Here, the leukocytes in the pleural effusion, sputum, and peripheral blood biopsies from severe and mild patients were analyzed at single-cell resolution. Drastic T cell hyperactivation accompanying elevated T cell exhaustion was observed, predominantly in pleural effusion. The mechanistic investigation identified a group of CD14+ monocytes and macrophages highly expressing CD163 and MRC1 in the biopsies from severe patients, suggesting M2 macrophage polarization. These M2-like cells exhibited up-regulated IL10, CCL18, APOE, CSF1 (M-CSF), and CCL2 signaling pathways. Further, SARS-CoV-2-specific T cells were observed in pleural effusion earlier than in peripheral blood. Together, our results suggest that severe SARS-CoV-2 infection causes immune dysregulation by inducing M2 polarization and subsequent T cell exhaustion. This study improves our understanding of COVID-19 pathogenesis.

Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections

The World Health Organization has recently declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, as pandemic. There is currently a lack of knowledge in the antibody response elicited from SARS-CoV-2 infection. One major immunological question is concerning the antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by using plasma from patients infected by SARS-CoV-2 or SARS-CoV, and plasma obtained from infected or immunized mice. Our results show that while cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses is rare, indicating the presence of non-neutralizing antibody response to conserved epitopes in the spike. Whether these non-neutralizing antibody responses will lead to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding the antigenicity differences between SARS-CoV-2 and SARS-CoV, but also has important implications in vaccine development.

Multiple approaches for massively parallel sequencing of HCoV-19 genomes directly from clinical samples

COVID-19 has caused a major epidemic worldwide, however, much is yet to be known about the epidemiology and evolution of the virus. One reason is that the challenges underneath sequencing HCoV-19 directly from clinical samples have not been completely tackled. Here we illustrate the application of amplicon and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of HCoV-19 from clinical samples covering a range of sample types and viral load. We also examine and compare the bias, sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. This is, to date, the first work systematically implements amplicon and capture approaches in sequencing HCoV-19, as well as the first comparative study across methods. Our work offers practical solutions for genome sequencing and analyses of HCoV-19 and other emerging viruses.