The nucleoside analog 4'-fluorouridine suppresses the replication of multiple enteroviruses by targeting 3D polymerase.

Human enteroviruses are the major pathogens causing hand-foot-and-mouth disease in infants and young children throughout the world, and infection with enterovirus is also associated with severe complications, such as aseptic meningitis and myocarditis. However, there are no antiviral drugs available to treat enteroviruses infection at present. In this study, we found that 4'-fluorouridine (4'-FlU), a nucleoside analog with low cytotoxicity, exhibited broad-spectrum activity against infections of multiple enteroviruses with EC50 values at low micromolar levels, including coxsackievirus A10 (CV-A10), CV-A16, CV-A6, CV-A7, CV-B3, enterovirus A71 (EV-A71), EV-A89, EV-D68, and echovirus 6. With further investigation, the results indicated that 4'-FlU directly interacted with the RNA-dependent RNA polymerase of enterovirus, the 3D pol, and impaired the polymerase activity of 3D pol, hence inhibiting viral RNA synthesis and significantly suppressing viral replication. Our findings suggest that 4'-FlU could be promisingly developed as a broad-spectrum direct-acting antiviral agent for anti-enteroviruses therapy.

Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research.

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications, enabling the development of effective therapeutic and preventive strategies. In this study, our initial attempts to introduce a NanoLuc luciferase (NLuc) reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies. We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus. Therefore, we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71 (EV-A71), coxsackievirus A10 (CVA10), coxsackievirus A7 (CVA7), coxsackievirus A16 (CVA16), namely EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT, and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency. Notably, the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses. A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics. Moreover, these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages. In addition, the HiBiT-tagged viruses were successfully tested in antiviral drug assays, and the sensitivity of the viruses to drugs was not affected by the HiBiT tag. Ultimately, our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal, convenient, and invaluable method for advancing research in the realm of enterovirus virology. Furthermore, HiBiT-tagged enteroviruses exhibit great potential for diverse applications, including the development of antivirals and the elucidation of viral infection mechanisms.

Construction of an infectious clone for enterovirus A89 and mutagenesis analysis of viral infection and cell binding.

Enterovirus A89 (EV-A89) is an unconventional strain belonging to the Enterovirus A species. Limited research has been conducted on EV-A89, leaving its biological and pathogenic properties unclear. Developing reverse genetic tools for EV-A89 would help to unravel its infection mechanisms and aid in the development of vaccines and anti-viral drugs. In this study, an infectious clone for EV-A89 was successfully constructed and recombinant enterovirus A89 (rEV-A89) was generated. The rEV-A89 exhibited similar characteristics such as growth curve, plaque morphology, and dsRNA expression with parental strain. Four amino acid substitutions were identified in the EV-A89 capsid, which were found to enhance viral infection. Mechanistic studies revealed that these substitutions increased the virus's cell-binding ability. Establishing reverse genetic tools for EV-A89 will significantly contribute to understanding viral infection and developing anti-viral strategies.IMPORTANCEEnterovirus A species contain many human pathogens and have been classified into conventional cluster and unconventional cluster. Most of the research focuses on various conventional members, while understanding of the life cycle and infection characteristics of unconventional viruses is still very limited. In our study, we constructed the infectious cDNA clone and single-round infectious particles for the unconventional EV-A89, allowing us to investigate the biological properties of recombinant viruses. Moreover, we identified key amino acids residues that facilitate EV-A89 infection and elucidate their roles in enhancing viral binding to host cells. The establishment of the reverse genetics system will greatly facilitate future study on the life cycle of EV-A89 and contribute to the development of prophylactic vaccines and anti-viral drugs.

A cysteine protease inhibitor GC376 displays potent antiviral activity against coxsackievirus infection.

Infection with coxsackievirus A10 (CV-A10) can cause hand-foot-mouth disease and is also associated with severe complications, including viral pneumonia, aseptic and viral meningitis. Coxsackievirus infection may also play a role in the pathogenesis of acute myocardial infarction and in the increased risk of type 1 diabetes mellitus in adults. However, there are no approved vaccines or direct antiviral agents available to prevention or treatment of coxsackievirus infection. Here, we reported that GC376 potently inhibited CV-A10 infection in different cell lines without cytotoxicity, significantly suppressed production of viral proteins, and strongly reduced the yields of infectious progeny virions. Further study indicated that GC376, as viral 3C protease inhibitor, had the potential to restrain the cleavage of the viral polyprotein into individually functional proteins, thus suppressed the replication of CV-A10. Furthermore, the drug exhibited antiviral activity against coxsackieviruses of various serotypes including CV-A6, CV-A7 and CV-A16, suggesting that GC376 is a broad-spectrum anti-coxsackievirus inhibitor and the 3C protease is a promising target for developing anti-coxsackievirus agents.

Induced regulatory T cells modified by knocking down T-bet in combination with ectopic expression of inhibitory cytokines effectively protect graft-versus-host disease.

Induced regulatory T (iTreg) cells play a vital role in immune tolerance and in controlling chronic inflammation. Generated in the periphery, iTreg cells are suitable for responding to alloantigens and preventing transplant rejection. Nevertheless, their clinical application has been impeded by the plasticity and instability attributed to the loss of forkhead box protein 3 expression, raising concerns that iTreg may be converted to effector T cells and even exert a pathogenic effect. Herein, second-generation short hairpin RNAs loaded with 3 pairs of small interfering RNAs were utilized to target the T-box transcription factor TBX21. In addition, 2 immunosuppressive cytokines, namely, transforming growth factor beta and interleukin 10, were constitutively expressed. This novel engineering strategy allowed the generation of stably induced regulatory T (SI Treg) cells, which maintained the expression of forkhead box protein 3 even in an unfavorable environment and exerted potent immunosuppressive functions in vitro. Furthermore, SI Treg cells demonstrated an effector transcriptional profile. Finally, SI Treg cells showed a significant protective effect against graft-versus-host disease-related deaths in a xenotransplantation model. Collectively, these results signify that SI Treg cells hold great promise for future clinical application and offer a rational therapeutic approach for transplant rejection.

Complement C3 Facilitates Stratification of Stages of Chronic Hepatitis B and Signifies Development of Acute-on-Chronic Liver Failure in Acute Decompensated Cirrhosis.

INTRODUCTION: Patients with chronic hepatitis B (CHB) have a dynamic disease process and risk of end-stage liver disease. It is critical to unambiguously differentiate the stages of the disease and focus on therapy prior to onset of an irreversible clinical endpoint. METHODS: We retrospectively analyzed a wide range of CHB patients at different stages. The predictive power of serum complement component 3 (C3) levels for the development of acute-on-chronic liver failure (ACLF) in patients with decompensated cirrhosis was established and validated. RESULTS: The decrease in serum C3 levels paralleled the severity of diseases related to hepatitis B virus. Patients with decompensated cirrhosis who developed ACLF had significantly lower serum C3 levels than others on admission (0.50 vs. 0.80 g/L, P < 0.001). Data analysis also revealed that low serum C3 was a significant risk factor for developing ACLF (hazard ratio = 0.32, P < 0.01). The area under the receiver operating characteristic curve (auROC) for serum C3 levels that predicted the development of ACLF in patients with decompensated cirrhosis was 0.90, which had sensitivity and specificity of 88.2% and 88.7%, respectively. A similar result was observed in the validation set (auROC = 0.86 for predicting development of ACLF in patients with decompensated cirrhosis). CONCLUSIONS: Serum C3 levels are valuable in assessing the severity of CHB-related stages. Low C3 levels signifies the development of ACLF in patients with decompensated cirrhosis.

Generally, acute-on-chronic liver failure is a rapidly worsening liver failure syndrome. This disease is intractable and with high mortality. Acute decompensation of the liver is defined as the occurrence of complications of liver disease (i.e., ascites, hepatic encephalopathy, gastrointestinal bleeding, and bacterial infection). Clinically, acute decompensation in hepatitis B virus-related cirrhosis (a result of chronic liver injury by virus) often develops into acute-on-chronic liver failure. In addition, the complement component 3 is a serum protein, which participates in the immune response against virus infection and has been reported to be associated with liver failure. We tried to explore the feature of serum complement component 3 to differentiate the stages of the disease and assess its predictive value for acute-on-chronic liver failure. So, we analyzed the complement component 3 data from a broad range of hepatitis B virus-cirrhosis patients. Through analysis, we found that complement component 3 levels are valuable in assessing the severity of chronic hepatitis B-related stages. Low complement component 3 levels can also signify the development of acute-on-chronic liver failure in patients with decompensated cirrhosis.

Nonmuscle myosin IIA promotes the internalization of influenza A virus and regulates viral polymerase activity through interacting with nucleoprotein in human pulmonary cells.

Influenza A virus (IAV), responsible for seasonal epidemics and recurring pandemics, represents a global threat to public health. Given the risk of a potential IAV pandemic, it is increasingly important to better understand virus-host interactions and develop new anti-viral strategies. Here, we reported nonmuscle myosin IIA (MYH9)-mediated regulation of IAV infection. MYH9 depletion caused a profound inhibition of IAV infection by reducing viral attachment and internalization in human lung epithelial cells. Surprisingly, overexpression of MYH9 also led to a significant reduction in viral productive infection. Interestingly, overexpression of MYH9 retained viral attachment, internalization, or uncoating, but suppressed the viral ribonucleoprotein (vRNP) activity in a minigenome system. Further analyses found that excess MYH9 might interrupt the formation of vRNP by interacting with the viral nucleoprotein (NP) and result in the reduction of the completed vRNP in the nucleus, thereby inhibiting subsequent viral RNA transcription and replication. Together, we discovered that MYH9 can interact with IAV NP protein and engage in the regulation of vRNP complexes, thereby involving viral replication. These findings enlighten new mechanistic insights into the complicated interface of host-IAV interactions, ultimately making it an attractive target for the generation of antiviral drugs.

Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway.

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.

Single-cell analysis of the adaptive immune response to SARS-CoV-2 infection and vaccination.

Amid the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, vaccination and early therapeutic interventions are the most effective means to combat and control the severity of the disease. Host immune responses to SARS-CoV-2 and its variants, particularly adaptive immune responses, should be fully understood to develop improved strategies to implement these measures. Single-cell multi-omic technologies, including flow cytometry, single-cell transcriptomics, and single-cell T-cell receptor (TCR) and B-cell receptor (BCR) profiling, offer a better solution to examine the protective or pathological immune responses and molecular mechanisms associated with SARS-CoV-2 infection, thus providing crucial support for the development of vaccines and therapeutics for COVID-19. Recent reviews have revealed the overall immune landscape of natural SARS-CoV-2 infection, and this review will focus on adaptive immune responses (including T cells and B cells) to SARS-CoV-2 revealed by single-cell multi-omics technologies. In addition, we explore how the single-cell analyses disclose the critical components of immune protection and pathogenesis during SARS-CoV-2 infection through the comparison between the adaptive immune responses induced by natural infection and by vaccination.

Enterovirus A Shows Unique Patterns of Codon Usage Bias in Conventional Versus Unconventional Clade.

Enterovirus A (EV-A) species cause hand, foot and mouth disease (HFMD), threatening the health of young children. Understanding the mutual codon usage pattern of the virus and its host(s) has fundamental and applied values. Here, through examining multiple codon usage parameters, we found that the codon usage bias among EV-A strains varies and is clade-specific. EVA76, EVA89, EVA90, EVA91 and EVA92, the unconventional clade of EV-A strains, show unique codon usage pattern relative to the two conventional clades, including EVA71, CVA16, CVA6 and CVA10, etc. Analyses of Effective Number of Codon (ENC), Correspondence Analysis (COA) and Parity Rule 2 (PR2), etc., revealed that the codon usage patterns of EV-A strains are shaped by mutation pressure and natural selection. Based on the neutrality analysis, we determined the dominant role of natural selection in the formation of the codon usage bias of EV-A. In addition, we have determined the codon usage compatibility of potential hosts for EV-A strains using codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses, and found that EV-A showed host-specific codon adaptation patterns in different clades. Finally, we confirmed that the unique codon usage pattern of the unconventional clade affected protein expression level in human cell lines. In conclusion, we identified novel characteristics of codon usage bias in distinct EV-A clades associated with their host range, transmission and pathogenicity.

Review of clinical characteristics, immune responses and regulatory mechanisms of hepatitis E-associated liver failure.

Hepatitis E virus (HEV) is the most common cause of acute liver failure (LF) and one of the most common factors causing acute injury in acute-on-chronic LF (ACLF). When HEV-related LF occurs, a series of changes take place in both the intrahepatic environment and extrahepatic microenvironment. The changed types and distribution of immune cells (infiltrating macrophages and increased lymphocytes) in liver tissue, as well the increased proinflammatory cytokines and chemokines in the blood, indicate that the occurrence and progression of HEV-related LF are closely related to immune imbalance. The clinical features and immune reaction in the body during HEV-related acute LF (ALF) and ACLF are complicated. This review highlights recent progress in elucidating the clinical manifestations of HEV-associated ALF and ACLF and discusses the corresponding systemic immune changes and possible regulatory mechanisms.

Angiotensin-Converting Enzyme 2 Potentiates SARS-CoV-2 Infection by Antagonizing Type I Interferon Induction and Its Down-Stream Signaling Pathway.

The innate interferon (IFN) response constitutes the first line of host defense against viral infections. It has been shown that IFN-I/III treatment could effectively contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication in vitro. However, how SARS-CoV-2 survives through the innate antiviral mechanism remains to be explored. Our study uncovered that human angiotensin-converting enzyme 2 (ACE2), identified as a primary receptor for SARS-CoV-2 entry, can disturb the IFN-I signaling pathway during SARS-CoV-2 infection in human lung cells. We identified that ACE2 was significantly upregulated by SARS-CoV-2 and Sendai virus (SeV) infection, and exogenous expression of ACE2 suppressed IFN-I production in a dose-dependent manner. Mechanistically, ACE2 disrupted poly (I:C)-mediated inhibition of SARS-CoV2 replication by antagonizing IFN-I production by blocking IRF3 phosphorylation and nuclear translocation. Moreover, ACE2 quenched the IFN-mediated antiviral immune response by degrading endogenous STAT2 protein, inhibiting STAT2 phosphorylation and nuclear translocation. Interestingly, IFN-inducible short ACE2 (dACE2 or MIRb-ACE2) can also be induced by virus infection and inhibits the IFN signaling. Thus, our findings provide mechanistic insight into the distinctive role of ACE2 in promoting SARS-CoV-2 infection and enlighten us that the development of interventional strategies might be further optimized to interrupt ACE2-mediated suppression of IFN-I and its signaling pathway. IMPORTANCE Efficient antiviral immune responses against SARS-CoV-2 infection play a key role in controlling the coronavirus diseases 2019 (COVID-19) caused by this virus. Although SARS-CoV-2 has developed strategies to counteract the IFN-I signaling through the virus-derived proteins, our knowledge of how SARS-CoV-2 survives through the innate antiviral mechanism remains poor. We herein discovered the distinctive role of ACE2 as a restraining factor of the IFN-I signaling in facilitating SARS-CoV-2 infection in human lung cells. Both full-length ACE2 and truncated dACE2 can antagonize IFN-mediated antiviral response. These findings are key to understanding the counteraction between SARS-CoV-2 pathogenicity and the host antiviral defenses.

Modern Beijing sublineage of Mycobacterium tuberculosis shift macrophage into a hyperinflammatory status.

The high prevalence of the modern Beijing sublineage of Mycobacterium tuberculosis may be related to increased virulence, although the responsible mechanisms remain poorly understood. We previously described enhanced triacylglycerol accumulation in modern Beijing strains. Here we show that modern Beijing strains grow faster in vitro and trigger a vigorous immune response and pronounced macrophage infiltration. Transcriptomic analysis of bone marrow derived macrophages infected with modern Beijing lineage strains revealed a significant enrichment of infection, cholesterol homeostasis and amino acid metabolic pathways. The upregulation of proinflammatory / bactericidal cytokines was confirmed by RT-PCR analysis, which is also in consistent with the reduced bacterial burden in modern strains infected macrophages. These results suggest that modern Beijing strains elicit a hyperinflammatory response which might indicate a stronger virulence and contribute to their extensive global prevalence.

Artificial intelligence-assisted enumeration of ultra-small viruses with dual dark-field plasmon resonance probes.

Direct visual enumeration of viruses under dark-field microscope (DFM) using plasmon resonance probes (PRPs) is fast and convenient; however, it is greatly limited in the assay of real samples because of its inability to accurately identify false positives owing to non-specific adsorption. In this study, we propose an artificial intelligence (AI)-assisted DFM enumeration strategy for the accurate assay of Enterovirus A71 (an ultra-small human virus) using two PRPs; a 40 nm silver nanoparticle probe (SNP) that appears bright blue under DFM, and a 120 nm gold nanorod probe (GNP) that appears red under DFM. The capture chip was prepared by immobilizing the SNPs with antibodies on the glass to capture the target virus and to form dichromatic sandwich structures with the GNPs, followed by imaging under a dark field (DF). Subsequently, the DF images of the capture chip were subjected to a two-step screening: first, using image processing, and thereafter using the AI algorithm screening to eliminate false positive results and background noise. The results revealed that the data from the AI-assisted dual PRPs assay were highly consistent with those of quantitative PCR (qPCR), and that the sensitivity with a minimum detectable concentration of 3 copies/µL was 5 times higher than that of qPCR. The entire analysis was completed within 45 min. Therefore, our AI-assisted virus enumeration strategy with two DF PRPs holds great potential for ultra-sensitive and accurate quantification of viruses in real samples.

The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene.

Coxsackievirus A16 (CA16) belongs to the Human Enterovirus A species, which is a common pathogen causing hand, foot, and mouth disease in children. Currently, specific vaccines and drugs against CA16 are unavailable, and there is an unmet need to further understand the virus and invent effective treatment. Constructing a CA16 infectious clone with a reporter gene will greatly facilitate its virological studies. Here, we first reported the construction of a CA16 infectious clone (rCA16) whose progeny is highly replicative and virulent in suckling mice. On the basis of rCA16, we further inserted a NanoLuc (Nluc) reporter gene and made the rCA16-Nluc clone. We found that the Nluc gene in rCA16-Nluc is stable during continuous growing in Vero cells and thus allowed detection of a steady luciferase signal in rCA16-Nluc-infected Vero cells over 10 passages. Its application in antivirals characterization and high-throughput screening is exemplified by measuring IC50, CC50, and selection index of guanidine hydrochloride, ribavirin, chloroquine, and ammonium chloride against CA16. Finally, we showed that rCA16-Nluc based assay greatly simplified the CA16 neutralizing antibody tests. Thus, these two CA16 infectious clones will be robust tools for future enterovirus studies and antivirals development.

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Dynamics of TCR repertoire and T cell function in COVID-19 convalescent individuals.

SARS-CoV-2 outbreak has been declared by World Health Organization as a worldwide pandemic. However, there are many unknowns about the antigen-specific T-cell-mediated immune responses to SARS-CoV-2 infection. Here, we present both single-cell TCR-seq and RNA-seq to analyze the dynamics of TCR repertoire and immune metabolic functions of blood T cells collected from recently discharged COVID-19 patients. We found that while the diversity of TCR repertoire was increased in discharged patients, it returned to basal level ~1 week after becoming virus-free. The dynamics of T cell repertoire correlated with a profound shift of gene signatures from antiviral response to metabolism adaptation. We also demonstrated that the top expanded T cell clones (~10% of total T cells) display the key anti-viral features in CD8+ T cells, confirming a critical role of antigen-specific T cells in fighting against SARS-CoV-2. Our work provides a basis for further analysis of adaptive immunity in COVID-19 patients, and also has implications in developing a T-cell-based vaccine for SARS-CoV-2.

SCIGA: Software for large-scale, single-cell immunoglobulin repertoire analysis.

BACKGROUND: B-cell immunoglobulin repertoires with paired heavy and light chain can be determined by means of 10X single-cell V(D)J sequencing. Precise and quick analysis of 10X single-cell immunoglobulin repertoires remains a challenge owing to the high diversity of immunoglobulin repertoires and a lack of specialized software that can analyze such diverse data. FINDINGS: In this study, specialized software for 10X single-cell immunoglobulin repertoire analysis was developed. SCIGA (Single-Cell Immunoglobulin Repertoire Analysis) is an easy-to-use pipeline that performs read trimming, immunoglobulin sequence assembly and annotation, heavy and light chain pairing, statistical analysis, visualization, and multiple sample integration analysis, which is all achieved by using a 1-line command. Then SCIGA was used to profile the single-cell immunoglobulin repertoires of 9 patients with coronavirus disease 2019 (COVID-19). Four neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified from these repertoires. CONCLUSIONS: SCIGA provides a complete and quick analysis for 10X single-cell V(D)J sequencing datasets. It can help researchers to interpret B-cell immunoglobulin repertoires with paired heavy and light chain.