Coronaviruses pose a global pandemic threat, making development of a pan-coronavirus inhibitor crucial for preparedness and containment in the event of a new coronavirus outbreak. The 3C-like protease (3CLpro) is a key target for antiviral development, as it is essential for viral replication and conserved across human coronaviruses. We previously developed an assay to monitor SARS-CoV-2 3CLpro activity in cells. This assay uses a single vector that coexpresses the 3CLpro enzyme and the reporter, which consists of two luciferase fragments linked by a 3CLpro cleavage site. Cleavage of this site by 3CLpro decreases luciferase activity, whereas inhibition of 3CLpro increases the luciferase activity. Here, we adapted this assay to examine 3CLpro activity from six other human coronaviruses: SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. We further determined the effects of different cleavage sites to improve the signal-to-background ratio. The Nsp4-Nsp5 site and super-active substrate (SAS) resulted in the largest dynamic range for most coronaviruses in our assay. Using the broad-spectrum 3CLpro inhibitor GC376, we observed increased reporter activity, indicating the assay’s efficacy for identifying inhibitors across multiple coronaviruses. The adaptation and improvement of the assay can facilitate the development of inhibitors against 3CLpro from multiple or novel coronaviruses.

Detection and clinical analysis of human coronavirus infection in pediatric patients with acute respiratory tract infection in the Xiamen Area, China, 2022-2024.

Abstract Background Antibody kinetics following SARS-CoV-2 infection differs from the classical trajectory, where infection initially elicits IgM responses before subsequent class-switching to IgG and IgA, complicating the use of IgM as a marker of recent SARS-CoV-2 infection. This atypical pattern may reflect cross-reactive memory responses against highly-related human coronaviruses (HCoV). Here, we investigated the interplay between early IgG, IgM, and IgA responses to SARS-CoV-2 infections and HCoV antibody responses. Methods Serum IgM, IgA, and IgG responses against the spike (S) of four HCoVs; and the S, nucleocapsid (N), and receptor binding domain (RBD) of SARS-CoV-2 were quantified by a multiplex immunoassay in acute SARS-CoV-2 cases (N = 51) 0–14 days post onset and pre-pandemic controls (N = 30). Results SARS-CoV-2 IgG levels and percent seropositivity were higher at 0–7 days compared to controls, increasing further at 8–14 days. Beta-HCoV IgA, IgG, and IgM were higher in cases than controls, increasing between 0 and 7 and 8–14 days. Among early (0–7 days) SARS-CoV-2 S IgG responders, IgG levels correlated positively with beta-HCoV S IgG (HCoV-HKU1: R = 0.62, P = 0.023 and HCoV-OC43: R = 0.81, P < 0.001) for IgG. High SARS-CoV-2 S IgM among infected individuals correlated positively with HCoV S IgM levels. Significance SARS-CoV-2 infection elicits cross-reactivity across IgM, IgG, and IgA responses to HCoVs, suggesting limitations in the use of IgM as a serological marker of recent infection. These findings highlight the complexity of immune responses to SARS-CoV-2 and the importance of accounting for potential cross-reactivity to related viruses when investigating serological markers of infection and assessing correlates of protection.

Epidemiological characteristics of human coronaviruses among populations with acute respiratory infections: surveillance data from jing'an district, Shanghai, 2024–2025

Coronaviruses remain a challenge due to the limited or incomplete protection provided by existing vaccines, highlighting the need for improved antigen-based designs that can reduce mortality, block transmission, and provide long-lasting, broad-spectrum protection. In this study, we adapted artificial antibody strategies to display receptor-binding domains (RBDs) from representative human coronaviruses, utilizing an engineered human IgG1 framework modified at the Fab and Fc domains to support diverse antigen presentation and enhanced immunopotentiation. The results indicate that bivalent, tetravalent, and multivalent RBD constructs developed within this framework confer broad-spectrum immune protection against severe acute respiratory syndrome coronavirus 2 and other pathogenic coronaviruses. Moreover, Fc-mediated antigen delivery, primarily engaging the neonatal Fcγ receptor, enhances mucosal, cellular, and sustained immune responses. This underscores the versatility and practical utility of the modified IgG1 framework, based on artificial antibody strategies, for developing broad-spectrum mucosal vaccine antigens, representing promising vaccine candidates targeting human coronaviruses.

The ongoing emergence of antiviral drug resistance underscores the critical need for new broad-spectrum antiviral agents. Sulfonamides and their derivatives have emerged as promising candidates for the development of new antiviral therapeutics. In this study, a series of camphor-10-sulfonamide derivatives was synthesized through a feasible and sustainable synthetic approach starting from naturally available precursors and evaluated for antiviral properties. Their activity was examined against three structurally distinct viruses-herpes simplex virus type 1 (HSV-1), human coronavirus (HCoV-OC43), and feline calicivirus (FCV)-representing both DNA and RNA, enveloped and non-enveloped types. The compounds were examined for their effects on viral replication, the stage of viral adsorption to the cell, and extracellular virions. The weakest cytotoxicity and the most pronounced activity of all the tested substances was demonstrated by the tryptophan derivative 7a. A time-dependent inhibition of the stage of adsorption of HCoV-OC43 (Δlg = 2.0 at 120 min) and FCV (Δlg = 1.75 at 60 min) to susceptible cells was established, as well as virucidal activity on the three types of virions tested, with the most pronounced effect at 120 min-for HSV-1 (Δlg = 2.75) and Δlg = 2.0 for HCoV-OC43 and FCV. Molecular docking studies performed using Glide (Schrödinger) provided insights into the active conformations of the most effective ligands and predicted possible interactions with relevant viral targets, supporting their potential as lead structures for further therapeutic development.

Accurate and timely identification of upper respiratory tract pathogens can improve patient management by decreasing unnecessary additional testing, reducing the cost of care, and informing antimicrobial stewardship. We evaluated the FDA-cleared Diasorin LIAISON PLEX® Respiratory Flex (RSP Flex), a highly multiplexed, rapid nucleic acid detection assay that can identify 14 viral and 5 bacterial respiratory pathogens. In total, 215 residual patient upper respiratory tract specimens were tested using the RSP Flex assay and results compared to standard-of-care (SOC) molecular testing. Positive percentage agreement (PPA) was 100% for the following targets: adenovirus, human coronavirus, Bordetella pertussis, Bordetella parapertussis, Mycoplasma pneumoniae, and Chlamydia pneumoniae. Performance for the remaining panel targets were: human enterovirus/rhinovirus PPA = 97.5% (39/40), influenza A virus PPA = 92.3% (12/13), human metapneumovirus PPA = 87.5% (14/16), influenza B virus PPA = 92.9% (13/14), parainfluenza 1 virus PPA = 85.7% (6/7), parainfluenza 2 virus PPA = 80% (4/5), parainfluenza 3 virus PPA = 85.7% (12/14), parainfluenza 4 virus PPA = 87.5% (7/8), respiratory syncytial virus (RSV) PPA = 92.9% (13/14), and SARS-CoV-2 PPA = 95.0% (19/20). The negative percentage agreement (NPA) for all targets was 100% except adenovirus NPA = 98.4% (180/183), enterovirus/rhinovirus NPA = 96.9% (155/160), and M. pneumoniae NPA = 91.9% (34/37). For all targets, the RSP Flex assay had an overall PPA of 93.8%, NPA of 99.5% and complete concordance of 99.1% compared to SOC testing. The LIAISON PLEX RSP Flex assay is a fully automated, sample-to-customizable answer system that offers a wide range of bacterial and viral target testing with high accuracy.

The effect of human coronaviruses (HCoVs) on airway epithelial cells is known and very well described. However, their influence on the human lung endothelium remains poorly understood. In this study, we assess the effect of low pathogenic HCoV-229E on the inflammatory and antiviral response of Human Lung Microvascular Endothelial Cells (HMVEC-L). Human Lung Microvascular Endothelial Cells were infected with HCoV-229E and then mRNA expression, protein release, cell viability, apoptosis rate, and presence of AP-N in flow cytometry were assessed. The infection of HMVEC-L with HCoV-229E led to the massive replication picked at 96 h post infection (hpi); however, the virus efficiently replicated at 48 hpi. Despite the inflammatory (IL-8, IL-6) and antiviral (IFN-β, OAS-1, PKR, MX-1) response, as well as the pattern recognition receptors activation (TLR3, TLR7/8, RIG-I, MDA5) occurred only at hour 72. Additionally, the cytopathic effect and the increase in apoptosis were observed. AP-N blockade inhibited inflammatory and antiviral induction, as well as decreased HCoV-229E genome copy numbers in HMVEC-L. We proved that the lung microvascular endothelium may be infected by human coronavirus 229E and that this infection may lead to a robust, but delayed inflammatory and antiviral response. Secondly, HMVEC-L may play an important role during Coronaviridae upper and lower airway infections.

Therapeutic potential of a polyherbal extract against COVID-19 and associated inflammation and mucormycosis: In vitro characterization and in vivo validation in a human coronavirus 229E-infected mouse model.

Optimizing Accuracy Rate of Genomic Image Representation of Human Coronavirus Sequences for COVID-19 Detection

Coronaviruses pose a serious threat to public health, driving the need for antiviral therapeutics and vaccines. Therefore, it is paramount to understand how this family of viruses evades cellular antiviral responses and establishes productive infection. The conserved coronavirus non-structural protein (nsp)1 has been shown to inhibit host protein synthesis and promote host mRNA degradation while viral mRNAs are protected. We showed previously that SARS-CoV-2 induces activation of host integrated stress response (ISR) kinases PKR and PERK, which promote phosphorylation of eIF2α and consequent inhibition of host protein synthesis. In contrast, eIF2α remains unphosphorylated during MERS-CoV infection. To investigate the interactions of nsp1 and the ISR kinases, we utilized recombinant SARS-CoV-2 and MERS-CoV expressing nsp1 with mutations in each of two conserved domains. Upon infection with SARS-CoV-2 nsp1 mutants, translation was shut down in wildtype (WT) and PKR knockout (KO) cells but rescued in PERK KO cells, likely due to reduced p-eIF2α. In contrast, translation was rescued during infection with the analogous MERS-CoV nsp1 mutants even in WT cells. Moreover, SARS-CoV-2 WT suppressed expression of GADD34, a negative regulator of eIF2α phosphorylation, while SARS-CoV-2 nsp1 mutants induced GADD34. In contrast MERS-CoV WT induced GADD34. Utilizing single-molecule fluorescencein situhybridization, we found that SARS-CoV-2 and MERS-CoV nsp1 promote host mRNA degradation during WT, but not nsp1 mutant, infection. Finally, while SARS-CoV-2 WT suppressed stress granule formation, nsp1 mutants induced stress granules containing host RNA. Thus, SARS-CoV-2 and MERS-CoV differ in interactions with the ISR and nsp1 control of host protein synthesis.SignificanceCoronaviruses cause disease across a wide range of animal species, and the human coronaviruses SARS-CoV-2 and MERS-CoV have caused epidemics of severe respiratory illness. Thus, it is imperative to understand how these viruses antagonize host responses and cause lethal disease. We show here that the betacoronavirus non-structural protein (nsp)1 promotes shutdown of host protein synthesis while preserving viral protein synthesis and, in addition, promotes degradation of host mRNAs. However, SARS-CoV-2 and MERS-CoV differ in their ability to manipulate the host integrated stress response, indicating that it is important to understand detailed coronavirus-host interactions and how they differ even between lethal coronaviruses. Such insights will inform the development of antiviral therapeutics to treat and prevent current and future coronavirus outbreaks.

The Coronaviridae family represents a broad group of RNA-containing viruses that infect humans and animals. This family belongs to the order Nidovirales and is divided into four main genera: α-CoV, β-CoV, γ-CoV and δ-CoV. It is particularly noteworthy that representatives of β-CoV have caused serious epidemics in humans, such as the outbreaks of SARS-CoV, MERS-CoV, and COVID-19 caused by SARS-CoV-2. Although the clinical manifestations of CoVs can range from mild cold-like symptoms to severe respiratory diseases, they share common features in their structure, modes of transmission, and natural reservoirs. Identifying natural reservoirs, as well as establishing intermediate hosts, is crucial for understanding the mechanisms of interspecies transmission of CoVs. These processes are often mediated by molecular interactions between viral spike (S) proteins and cellular receptors of different species, which contribute to zoonotic outbreaks. Thus, the interaction of various species and the study of these processes of viral spread, cross-species transmission, and pathogen evolution play a key role in ensuring global biological safety. Therefore, we conducted this review to summarize the data from existing studies focused on the taxonomy of CoVs, their main types, natural reservoirs, intermediate hosts, pathways of interspecies transmission, and the significance of the One Health concept as an interdisciplinary approach to monitoring, prevention and control of CoV infections at the intersection of human, animal, and environmental health. We examined databases such as PubMed, Science Direct, Web of Science, and Google Scholar to identify relevant scientific articles in English available for such a review. The aim of this work is to study the taxonomy and classification of coronaviruses, as well as to identify their natural reservoirs, intermediate hosts, and applicable control measures. A review of human and animal coronaviruses has revealed their evolutionary diversity, their main natural reservoirs, their intermediate hosts, and their interactions with cellular receptors. This information allows for a better understanding of the mechanisms by which the viruses are transmitted from animals to humans. The concept of One Health demonstrated the interconnections between human, animal and environmental factors.

Coronaviruses are the largest, enveloped, single-stranded positive-sense RNA viruses. There are seven coronaviruses known to be associated with human illness. Four human coronaviruses, HCoV 229E, NL63, OC43, and HKU1, have caused upper respiratory tract infections in adults and are prevalent worldwide. The most serious strains, which can cause lower respiratory tract infections and acute respiratory distress syndrome, include SARS-CoV, MERS-CoV, and SARS-CoV-2. At the end of 2019, the leading pandemic, COVID-19, was initially discovered and reported in Wuhan City, China's Hubei region. The disease, also named Wuhan pneumonia, is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The disease spread quickly to other provinces of China and overseas. On February 26, 2020, two instances of the virus were reported in Pakistan, one was a student who had recently arrived from Iran, and the other included a person in the Islamabad Capital Territory. The purpose of this study is to investigate the COVID-19 prevalence in District Haripur. Our study included the analysis of COVID-19 cases based on disease status, age, gender, and area. The secondary data about COVID-19 cases were collected from the DHO (District Health Office) Haripur. Three months of data (April 2021-June 2021) of 986 patients; 603 (61.2%) males and 383(38.8%) females were statistically analyzed. COVID-19 was found to be more prevalent among the 41-60 years age group (38.0%) as compared to the below 20 age group (5.0%), 21-40 age group (36.4%), 61-80 age group (19.4%), and above 80 age group (1.2%). COVID-19 was found to be more prevalent in rural areas (51.4%) as compared to urban areas (48.6%). From the above discussion, we conclude that COVID-19 is more prevalent among people in the age group 41-60 due to a weak immune system.

The frequency of new and re-emerging viral infections is rising, driven by shifts in environmental conditions and altered interactions between hosts, vectors, and pathogens. New treatments may be developed through a better understanding of the molecular processes underlying viral replication. The low-density lipoprotein receptor (LDLR) serves as a critical entry portal for a diverse range of infectious agents, facilitating the initiation and perpetuation of their infection cycles. Furthermore, numerous viruses, such as the dengue virus and the hepatitis C virus (HCV), depend on host cholesterol (CHO) for replication and spread. Consequently, targeting the LDLR with pharmacological agents presents a promising therapeutic strategy for a broad spectrum of viral infections, including those caused by human immunodeficiency virus (HIV), HCV, and severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). In addition, human metapneumovirus (HMPV) is a leading cause of respiratory infections in young children, underscoring the need for effective treatments. This research investigates the role of the LDLR in HMPV and other emerging viruses. A key finding is that HMPV infection depends on LDLR-mediated pathways, as evidenced by the rapid recovery observed after exogenous CHO administration. Therefore, understanding the mechanism of LDLR in viral entry and replication is crucial and presents a promising avenue for developing novel antiviral therapies.

Recurrence of coronavirus outbreaks and zoonotic origins of human coronaviruses underscore the importance of developing pan-coronavirus antivirals. The highly conserved 3C-like protease (3CLpro) in coronaviruses, together with the well-established druggability, makes it an ideal target for broad-spectrum antiviral therapeutics. Here, the inhibitory activity of approved 3CLpro inhibitors, including nirmatrelvir, ensitrelvir, and simnotrelvir, against fifteen 3CLpros is first reported by enzymatic assays. Despite their potent inhibition toward 3CLpros of β-CoVs, these inhibitors show reduced potency against 3CLpros from the other three genera, particularly against two newly identified human coronaviruses (α-CCoV-HuPn-2018 and δ-PDCoV). In this context, continued efforts in structure-based optimization of nirmatrelvir lead to the identification of compound 8 that potently inhibits a panel of 32 3CLpros across all subgenera (IC50s: 19-146nm), with an IC50 value of 61 and 81nm against α-CCoV-HuPn-2018 and δ-PDCoV 3CLpros, respectively. Moreover, it effectively inhibits nirmatrelvir-resistant 3CLpro mutants and demonstrates broad-spectrum antiviral efficacy in cells. These findings suggest an important rule that a small, non-cyclic P2 segment and a P4 segment with a suitable size are preferred by the design of ultra-broad-spectrum 3CLpro inhibitors, and provide a proof-of-concept guide for developing broad-spectrum antivirals as potential pan-CoV therapeutics.

Four seasonal endemic human coronaviruses (HCoVs), HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63, are culprits of mild upper respiratory and periodic severe diseases in vulnerable populations. Despite their prevalence, understanding HCoVs’ antigenic and immune signatures remains elusive. SARS-CoV-2 has evolved as the fifth HCoV, requiring seasonal vaccination, and currently, no other HCoV vaccines are available. SARS-CoV-2 co-infection with HCoVs increases disease severity; thus, combined vaccination may provide increased protection against seasonal HCoVs overall. Here, we explored spike (S) receptor binding domain (RBD) vs. N-terminal domain (NTD) B-cell immunodominance in HCoV-positive convalescent donors and immunogenicity in mice. We found that while antibody and B-cell isotypes were relatively dominant to S NTD, mice immunized with S RBD elicited significantly higher binding and neutralizing antibody (nAb) responses. With that knowledge, we used computational methods to infer that HCoV S sequences evolve into two main clades and designed chimeric immunodominant domains (IDDs) from both clades for each HCoV. IDDs were scaffolded onto two-component nanoparticles (NPs) displaying each IDD separately (monovalent IDD NP); three ß-HCoV IDDs (Mosaic-3 IDD NP); or five HCoVs IDDs (Mosaic-5 IDD NP). Mice immunized with mosaic IDD NPs, but not soluble IDD antigens nor monovalent IDD NPs, elicited potent, broadly cross-reactive binding and neutralizing antibody (Ab) responses against SARS-CoV-2 variants, other HCoVs, and Sarbecoviruses. System serology revealed that all four IDD immunogens elicited distinct Ab subclasses and Fc-effector functions, with mosaic-5 IDD NPs eliciting the most de novo Ab subclasses, distributions, and broader Fc-mediated immune mechanisms. Dissection of vaccine-immune sera revealed polyclonal Ab responses against multiple non-overlapping cross-reactive S epitopes. Due to elicitation of broad Ab responses with combinatory functionality, IDD NPs open new horizons for developing first-in-class supraseasonal HCoV vaccine candidates, with potential to decrease frequent SARS-CoV-2 sequence updates and protect against other HCoVs. Moreover, elicitation of Ab breadth that spans pandemic-threat Sarbecoviruses gives mosaic IDD NPs promise towards pandemic preparedness.

Human coronaviruses are pathogenic viruses that have the ability to cause serious diseases or even death in humans. The receptor binding domain (RBD) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the region of the spike protein, through which the virus directly interacts with the human Angiotensin-converting enzyme 2 receptor (ACE-2). Understanding the mutations that occur in the RBD and determining their influence on host cell binding plays an essential role in learning about the transmission mechanism of the disease and developing potential treatment. We chose to apply the error-prone Polymerase Chain Reaction (ep-PCR), usually used in directed evolution and targeted mutagenesis applications, and to optimize it for the SARS-CoV-2 RBD sequence. This was followed by sequencing to analyze the mutation pattern. In this study, a novel ep-PCR protocol was developed specifically for RBD, optimized for dNTP ratios, magnesium ions, manganese ions, and cycle numbers. Distribution of mutations in the RBD sequence was determined. The effect of each parameter on the generation of mutations showed that MnCl2 concentration had the strongest effect, followed by the number of cycles, the dNTP ratio, and MgCl₂ concentration. ep-PCR analysis revealed that 12 best conditions for generating mutations in the 13-17bp range. Our results highlight the virulence-enhancing potential of mutations and enable researchers to evaluate potential vaccine candidates in the future. Furthermore, compared to earlier reports, the method described here requires only Sanger sequencing and a standard PCR cycler, therefore it is much more accessible. Most importantly, it yields combinatorial mutation diversity in one round, along the entire length of the sequence of interest.

The coronavirus proofreading exoribonuclease (ExoN) is essential for genome fidelity and immune evasion of the viruses. Despite its critical roles in the viral life cycle, it is unclear how ExoNs across different coronaviruses diverge in their structures and catalytic properties, which may lead to differences in viral genome mutation rates and, consequently, viral fitness, immune evasion, and resistance to antiviral drugs. Here, we present comparative structural and biochemical analyses of ExoNs between two most representative human coronaviruses, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the merbecovirus subgenus and SARS-CoV-2 from the sarbecovirus subgenus. Our results reveal a markedly lower catalytic activity of ExoN from MERS-CoV than that from SARS-CoV-2. The molecular basis of such a divergence across the two coronaviruses is unveiled by the cryo-EM structures of MERS-CoV ExoN in complex with RNA substrates bearing different 3'-end base pairs or mismatch, which represent the first set of ExoN structures from a coronavirus outside the sarbecovirus subgenus. Our findings also identify two highly conserved structural determinants that dictate efficient excision of different nucleotides at the 3' terminus of RNA substrates by coronavirus ExoNs, a property that is pivotal for their roles in both viral RNA proofreading and immune evasion.

Human common cold coronaviruses (CCCoVs, e.g., 229E, NL63, OC43, HKU1) hold critical yet underexplored significance in understanding coronavirus evolutionary dynamics and immune cross-protection, offering insights for predicting emerging pathogens and developing pan-coronavirus vaccines. However, research is hindered by the lack of animal models due to strict human-specific tropism and the confounding effects of frequent co-infections from clinical samples, which obscure virus-specific pathogenesis. Although lung-humanized mice have been used in SARS-CoV-2 studies, their application to CCCoVs remains unvalidated and relies on logistically challenging fresh human tissues. This study optimizes a transplantation strategy using cryopreserved human fetal lung tissue, achieving enhanced engraftment efficiency. And the refined model supports robust infection by all four major CCCoVs and demonstrates the therapeutic efficacy of Paxlovid against HKU1. Furthermore, comparative analysis reveals phenotypic distinctions in human immune cells between native mouse lungs and human lung implants in lung-immune dual-humanized mice. The model also enables validation of virus-specific T cell responses and assessment of SARS-CoV-2 cross-reactivity post-HKU1 infection. Overall, this study establishes a scalable platform using cryopreserved tissues for respiratory virus research, overcoming prior limitations in modeling human-specific tropism and dissecting immune-pathogen interactions.

Objective: To investigate the genotype and epidemiological characteristics of Human Coronavirus NL63 (HCoV-NL63) epidemic strains in Shanghai, China. Methods: Whole genome sequencing of HCoV-NL63 was performed with the nucleic acid-positive specimens collected from the acute respiratory infection surveillance system in Shanghai from 2019 to 2024. Phylogenetic, homology, and key mutation site analysis of the S gene were conducted in combination with international reference sequences. Results: Seven whole genome sequences of HCoV-NL63 were obtained, including five (2020-2022) of subtype B2 and two (2024) of subtype C3. The B2 subtype sequences were highly homologous to the Guangzhou 2018 strain, while the C3 subtype sequences were highly homologous to the Japanese 2023 strain. There were 44 amino acid mutation sites located on the S gene of five B2 strains, with E572A in the receptor binding domain (RBD) region. There were 15 amino acid mutation sites in the S gene of C3 subtype, with I507L and E572A in the RBD region, and I507L as the signature mutation of C3 subtype, closely related to viral virulence. Conclusion: The B2 and C3 subtypes coexist among the prevalent strains in Shanghai, and the related strains are highly homologous to the domestic and international strains causing severe infections, indicating the need to strengthen continuous monitoring and functional research on HCoV-NL63 variants in Shanghai.