Dear Editor, Chikungunya virus(CHIKV)is a single-strand positive-sense RNA virus belonging to the family Togaviridae.Since 2004,CHIKV outbreaks have become more frequent and widespread,affecting millions of people and establishing Chikungunya disease as a significant global public health issue.1 In Asia,the Asian lineage(CHIKV-Asian)and the East-Central-South-African lineage(CHIKV-ECSA)have become the most prevalent.In July 2025,a large-scale outbreak occurred in Guangdong Province,China,resulting in more than 9000 confirmed cases.2 To date,no drug has been approved for clinical therapy,highlighting the urgent need to develop effective treatments against CHIKV.

ABSTRACT Inhibiting the catalytic activity of 3CLpro is a mainstream strategy to block coronavirus replication. However, the appearance of SARS-CoV-2 3CLpro resistance to protease inhibitors raises concerns for effective therapies. In this work, we first investigated the resistance profile of simnotrelvir, an approved anti-SARS-CoV-2 drug that targets 3CLpro. We found that the T21I/E166A mutations in 3CLpro equally emerged when SARS-CoV-2 was passaged in the HEK293T-hACE2 cells with increasing concentrations of simnotrelvir. The SARS-CoV-2 isolate carrying 3CLproT21I/E166A (SARS2-T21I/E166A) showed cross-resistance to simnotrelvir, nirmatrelvir, and ensitrelvir, but not significant resistance to bofutrelvir. Biochemical and cellular assays confirmed that 3CLproT21I/E166A was associated with the differential resistance to these protease inhibitors. Crystallographic structural analysis indicated that the alanine substitution disrupted hydrogen bonding interactions surrounding the γ-lactam rings (P1) of the inhibitors, which is similar to the model rebuilding observed with the previously reported E166V mutation. However, in contrast to the valine substitution, the alanine substitution resulted in a more spacious S2 subsite, thereby causing stronger interaction between the P1 and residues F140 and Ser1 of protomer B. Further computational simulations demonstrated that the covalent binding of bofutrelvir preserves strong binding affinity despite modifications in the S2 subsite caused by the E166A mutation, suggesting that inhibitors containing an aldehyde warhead may partially overcome resistance. Notably, both simnotrelvir and bofutrelvir exhibited therapeutic efficacy against the SARS2-T21I/E166A variant in K18-hACE2 mice. These findings advance our understanding of the resistance profiles and mechanistic underpinnings of SARS-CoV-2 3CLpro and underscore the necessity for diversified antiviral therapeutic strategies.IMPORTANCEConsidering that the nirmatrelvir-resistant SARS-CoV-2 has emerged in immunocompromised patients who received long-term Paxlovid therapy, it is essential to investigate the response of resistance 3CLpro mutants to various protease inhibitors. Simnotrelvir, a novel inhibitor targeting SARS-CoV-2 3CLpro, has been authorized for the treatment of mild-to-moderate COVID-19 in China and has treated over 1 million patients. However, the resistance profile of simnotrelvir to SARS-CoV-2 remains unknown. Here, we identified that 3CLpro with T21I/E166A mutations confers resistance to simnotrelvir and showed cross-resistance to nirmatrelvir and ensitrelvir, but not bofutrelvir. More importantly, we further revealed that E166A showed a novel resistance mechanism to both the covalent inhibitors consisting of a γ-lactam ring and non-covalent inhibitors like ensitrelvir, which is different from that of E166V previously reported. In contrast, bofutrelvir maintains high affinity to T21I/E166A, suggesting that inhibitors with aldehyde warhead can partly neutralize the resistance.

Chikungunya virus (CHIKV) belongs to the genus Alphavirus of the family Togaviridae. CHIKV infection generally causes severe clinical symptoms, including debilitating arthralgia, fever, hemorrhage and cutaneous rashes. In recent years, the transmission range of CHIKV has continued to expand, resulting in recurrent local outbreaks in densely populated and economically developed regions and posing a severe threat to public health. Accordingly, safe and effective anti-CHIKV therapeutics are urgently needed for the clinical treatment of infected patients. Nevertheless, no specific anti-CHIKV drugs have been approved for clinical use or advanced into clinical trials, and relevant research and development remain confined to the preclinical stage. This review systematically illustrates the key steps of the CHIKV life cycle, core viral components and vital functional domains, and summarizes the current research progress of anti-CHIKV agents. It comprehensively outlines the discovery strategies, structural optimization directions and activity evaluation approaches of anti-CHIKV small-molecule compounds, and further investigates their action targets. Notably, nsP2 and nsP4 represent promising targets for broad-spectrum anti-CHIKV even anti-alphavirus drug development due to their structural conservation across alphavirus species and druggable features. This work provides a solid theoretical basis and valuable reference for the future research and development of novel anti-CHIKV drugs.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus causing severe systemic infection with high mortality rates. Previously, SFTSV RNA was reported in the semen of infected patients, but whether SFTSV infects the male reproductive tract remains unclear. Here we demonstrate that SFTSV exhibits broad tropism for male reproductive organs in mice, resulting in low sperm count and local inflammation. SFTSV infects Leydig cells in the testis, triggering apoptosis, pyroptosis and inflammation, thereby disrupting testosterone production. Single-cell RNA sequencing identified infiltration of CCR2+ and SPP1+ macrophages expressing S100A4, a key driver of epididymal hyperinflammation and fibrosis. An S100A4 inhibitor reduced pathology and mortality in infected mice. Potential male-to-female sexual transmission risk was observed in mice. In infected patients, viral shedding in semen correlated with disease severity and spermatogenic dysfunction, with viral persistence lasting nearly 3 months after symptom onset. These findings suggest a potential risk of sexual transmission and adverse effects on male reproductive health.

Respiratory syncytial virus (RSV) and influenza virus are the principal contributors to respiratory tract infections worldwide. Clinical treatment of RSV or influenza A virus (IAV) is limited and may be postponed because of misdiagnosis or coinfection with multiple pathogens. Here, we described VV251, the prodrug of 4'-fluorouridine, a novel oral nucleoside analog that exhibits potent inhibitory activity against RSV laboratory strains and clinical isolates, as well as IAV, in vitro. The EC50 values of VV251 against these viruses ranged from nanomolar to low micromolar. Moreover, VV251 significantly reduced the viral titer of RSV A2 or IAV PR8 in a BALB/c infection model. Importantly, we further established a simultaneous RSV/IAV coinfection model to investigate the antiviral activity of VV251. Oral administration of VV251 once daily significantly inhibited viral replication and decreased the mortality of RSV/IAV-coinfected mice. Our findings establish VV251 as a broad-spectrum drug candidate for the treatment of RSV, IAV, and RSV/IAV coinfection.IMPORTANCERespiratory syncytial virus (RSV) and influenza virus are the two predominant causative agents of acute respiratory tract infections, leading to a substantial number of hospitalizations and thousands of deaths annually. The cost of treating disease caused by infection with RSV and influenza viruses is a huge financial burden on the world. We have identified a nucleotide analog with favorable pharmacokinetic properties and antiviral activity against RSV and influenza A virus (IAV) during mono- or coinfection in a mouse model. This compound has the potential to be used for the treatment of RSV, IAV, and RAV/IAV coinfection.

Enteroviruses, which belong to the family Picornaviridae, cause hand, foot, and mouth disease (HFMD), respiratory symptoms, and severe neurological complications in children. Since vaccines cannot provide cross-protection against different serotypes of enteroviruses, the development of broad-spectrum anti-enteroviral drugs is imperative. The viral 3C protease (3Cpro), which is essential for polyprotein processing represents a validated target for therapeutic intervention. Importantly, enterovirus 3Cpro shares conserved structural and catalytic features with coronavirus main protease (Mpro, also known as 3C-like protease, 3CLpro), providing a rationale for cross-target inhibitor repurposing. Through targeted screening of peptidomimetic protease inhibitors, a clinical-stage SARS-CoV-2 Mpro inhibitor was identified as a potent inhibitor of enterovirus A71 (EV71) 3Cpro. Bofutrelvir displayed nanomolar antiviral activity in multiple cell lines and demonstrated broad-spectrum efficacy against several enteroviruses including coxsackievirus B5, coxsackievirus A16 (CA16) and echovirus 11. In EV71 infected neonatal mice, intraperitoneal administration of bofutrelvir markedly reduced viral loads in brain, spinal cord, and muscle, alleviated clinical symptoms, and suppressed tissue inflammation. Oral administration of bofutrelvir also provided therapeutic benefits in neonatal mice models of both EV71 and CA16. Crystallographic analysis revealed that bofutrelvir binds in the conserved substrate-binding cleft of EV71 3Cpro, elucidating its molecular mechanism of inhibition. These findings identify bofutrelvir as a broad-spectrum peptidomimetic 3Cpro inhibitor with strong antiviral efficacy against enteroviruses and highlight its potential for repurposing as a promising antiviral candidate for the treatment of enteroviral infections.

Coronavirus, a large family of positive-sense RNA viruses, are responsible for both mild and severe respiratory illnesses, ranging from the common cold to life-threatening conditions. Despite significant advances in vaccine and antiviral development, the high mutability of human coronaviruses (HCoVs), such as SARS-CoV-2, presents a major challenge in treating these infections. Effective, broad-spectrum antiviral drugs are urgently needed to address both current and future HCoV outbreaks. Here, we conducted high-throughput screening of a natural product library containing 3407 compounds to identify potential antiviral agents against HCoV-OC43 and HCoV-229E. We identified several natural products with inhibitory effects on HCoV-229E, HCoV-OC43, and the SARS-CoV-2 variants Delta (B.1.617.2) and Omicron (BA.5) in vitro without evident cytotoxicity. Among these, dibenzoylmethane (DBM) not only demonstrated broad-spectrum anticoronavirus activity in vitro but also effectively inhibited HCoV-OC43 replication in a BALB/c mouse model. Pharmacokinetic analysis revealed that DBM, when administered orally, maintained effective concentrations in the blood over an extended period, suggesting its suitability for oral administration. Mechanistically, DBM was found to regulate caspase-6, a host factor that suppresses interferon signalling and promotes HCoV replication. These findings highlight DBM as a promising candidate for the development of therapeutics targeting HCoVs, offering potential for treating infections by both established and emerging HCoVs.

4'-Fluorouridine (4'-FU), despite demonstrating potent anti-SFTSV efficacy in vitro and in vivo, faces hindrances in its further development as a promising drug due to its weak chemical stability. Here, we report the discovery and development of VV261, a novel 4'-FU double prodrug with three isobutyryl groups on the ribose moiety and a nicotinoyloxymethyl group linked to the imide-nitrogen on the base moiety, exhibiting notable chemical stability and favorable pharmacokinetic properties. In SFTSV-infected mice, VV261 at 5 mg/kg/d for 7 days demonstrated complete protection against lethal SFTSV infection, prevented weight loss, and even a 2 day treatment significantly reduced both viral RNA copies and infectious virus titers in multiple organs, and notably alleviated splenic tissue lesions. After further preclinical evaluations, VV261, identified as a promising candidate drug for the treatment of SFTS, has entered Phase I clinical trials in China, the first such candidate to reach this stage for SFTS.

The 3CL protease (3CLpro) of SARS-CoV-2 is a key enzyme that plays an essential role in mediating viral replication and transcription. In this study, we synthesized and evaluated a series of peptidomimetic compounds containing a tetrahydropyrrole spirodihydroindolone moiety. Among the target compounds, 13c and 17d exhibited obvious 3CLpro inhibitory activities with IC50 = 3.71 and 6.21 nM, respectively. In metabolic stability testing of liver microsomes, compound 13c showed improved stability in human liver microsomes. In addition, 13c displayed significant anti-SARS-CoV-2 activity and high safety in Vero E6 cells (EC50 = 19.26 nM, SI > 400). Further investigations indicated that 13c showed potent activity against HCoV-OC43 and favorable safety in Huh7 cells (EC50 = 61 nM, SI > 100). These findings suggest that compound 13c is a promising lead compound in the development of novel 3CLpro inhibitors.

A series of novel Mpro inhibitors was designed and synthesized to combat the coronavirus, such as HCoV-OC43 and SARS-CoV-2, and several compounds showed comparable antiviral activity to nirmatrelvir. Among them, an octahydroindole-based peptidomimetic covalent inhibitor 28f showed strong inhibitory activity against Mpros and exhibited broad-spectrum anticoronavirus activity with EC50 values ranging from 0.027 to 4.41 μM. Besides, this compound displayed potent antiviral activity against EV71. Compared to FB2001, 28f displayed better pharmacokinetic properties, and the value of oral bioavailability in CD-1 mice and Beagle dogs was improved to 10.4 and 10.2%, respectively. In addition, oral treatment with 28f could significantly reduce the viral loads of HCoV-OC43 in mice, and compound 28f could also effectively reduce lung viral loads in a K18-hACE2 transgenic mouse model without ritonavir. Taken together, compound 28f is a promising orally bioavailable broad-spectrum antiviral drug candidate that deserves further research.

Monkeypox virus (MPXV), an orthopoxvirus that has long been endemic in Africa, has posed a significant global health threat since 2022. The I7L protease, a highly conserved cysteine proteinase essential for orthopoxvirus replication, represents a promising target for broad-spectrum antiviral drug development. Here, the first crystal structure of MPXV I7L protease is reported, revealing its unique dimeric form and different conformations of a cap region nearby the active site. Molecular dynamics simulations and AlphaFold3 prediction of protease-substrate structures both suggest that this highly flexible cap acts as a conformational switch, regulating the substrate access to the active site. Additionally, the structural basis of substrate recognition and the catalytic mechanism of the protease are elucidated, mapping determinants of substrate specificity. These insights enable us to design covalent inhibitors to mimic the natural substrates and develop a fluorescence resonance energy transfer (FRET)-based protease assay to effectively assess the inhibitory activity, leading to the discovery of first-in-class inhibitors of MPXV I7L protease with nanomolar potency. Therefore, this work provides a comprehensive understanding of the MPXV I7L protease's structure, dynamics, and function, and presents an example of successful rational design of covalent peptidomimetic inhibitors, serving as a good starting point for drug development against MPXV.

Human adenovirus (HAdV) is a significant viral pathogen that causes severe acute respiratory infections (SARIs) in children and immunocompromised patients. Currently, no specific treatment options are available for HAdV infections. This study used a green fluorescence protein-based, high-throughput screening (HTS) assay on a botanical drug library containing 3697 botanical compounds to identify agents that could inhibit HAdV. Four compounds with anti-HAdV-C5 activity in the low-micromolar range were identified and inhibited other wild-type HAdVs known to cause SARIs. Among these compounds, 13-methylberberine chloride presented the highest select index values. Berberine is a commercially available natural product-derived isoquinoline alkaloid with multiple pharmacological effects and is widely used in Asian countries. We systematically evaluated the anti-HAdV activity of six berberine-derived compounds in vitro and performed a time-of-drug-addition assay to explore their antiviral modes of action. Mechanistic studies revealed that berberine and its analogs inhibit HAdV replication by downregulating the MAPK signaling pathway, particularly ERK activation, which is crucial for viral replication and progeny production. Our findings suggest that berberine is a promising candidate for the development of anti-HAdV therapies.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever disease caused by the SFTS virus (SFTSV). Despite pandemic concerns arising from repeated instances of human-to-human transmission and a high fatality rate, effective anti-SFTSV interventions remain unavailable. Here, utilizing single-cell RNA sequencing (scRNA-seq) and flow cytometry data, we revealed that the deficiency and dysfunction states of T cells, particularly the impaired cytotoxicity and exhausted state of CD4+ T cells, were significantly associated with lethal consequences in SFTS patients. Using an infectious mouse model, we further observed that depletion of CD4+ T and CD8+ T cells was related to elevated viremia and increased fatality rates in SFTSV-infected mice. Accordingly, we designed virus envelope glycoprotein-targeting bispecific T cell engager (BiTE) antibodies to redirect T cells to eliminate SFTSV-infected cells, effectively rescuing mice from lethal SFTSV infection. Collectively, Gn-targeted BiTEs hold potential as a therapeutic option for treating SFTS.

Recurrence of coronavirus outbreaks and zoonotic origins of human coronaviruses underscore the importance of developing pan‐coronavirus antivirals. The highly conserved 3C‐like protease (3CL pro ) in coronaviruses, together with the well‐established druggability, makes it an ideal target for broad‐spectrum antiviral therapeutics. Here, the inhibitory activity of approved 3CL pro inhibitors, including nirmatrelvir, ensitrelvir, and simnotrelvir, against fifteen 3CL pro s is first reported by enzymatic assays. Despite their potent inhibition toward 3CL pro s of β‐CoVs, these inhibitors show reduced potency against 3CL pro s 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 3CL pro s across all subgenera (IC 50 s: 19–146 n m ), with an IC 50 value of 61 and 81 n m against α‐CCoV‐HuPn‐2018 and δ‐PDCoV 3CL pro s, respectively. Moreover, it effectively inhibits nirmatrelvir‐resistant 3CL pro 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 3CL pro inhibitors, and provide a proof‐of‐concept guide for developing broad‐spectrum antivirals as potential pan‐CoV therapeutics.

Poxviruses cause severe diseases, including smallpox and mpox, that pose major threats to human health. The poxvirus core protease (CorePro) is essential for viral maturation and is highly conserved in poxviruses, making it an attractive antiviral target1. However, the structure of CorePro remains unknown, hampering antiviral development. Here we determined the apo structure of monkeypox virus (MPXV) CorePro and the structure of CorePro in a complex with the inhibitor aloxistatin, a drug candidate for muscular dystrophy2. These structures show that CorePro forms a homodimer that features a unique 'dancing couple' fold. The catalytic intermediate state of CorePro was characterized by an aldehyde derivative from a natural substrate (I-G18). This derivative binds covalently to the catalytic Cys328, shifting the active site of the viral protease from a closed conformation in the apo form to a favourable open conformation upon substrate binding. On the basis of the CorePro-I-G18 complex, we designed a series of peptidomimetic inhibitors with a nitrile warhead, which could covalently anchor with the catalytic Cys328. These compounds inhibit CorePro with half-maximal inhibitory concentrations of 44.9-100.3 nM, and exhibit potent and broad anti-poxvirus activity. Our studies provide a basis for designing wide-spectrum inhibitors against poxvirus infections.

Bunyaviruses, a subset of segmented negative-sense RNA viruses, include pathogenic species capable of zoonotic transmission to humans via arthropod vectors and rodent hosts. Pathogenic bunyavirus infections can cause severe hemorrhagic fevers and other life-threatening diseases, posing threats to human health and social stability; however, therapeutic strategies for treating bunyavirus infections remain limited. Here, we report that VV251 hydrochloride salt (VV251), an optimized oral prodrug derivative of 4'-fluorouridine (EIDD-2794), exhibits potent efficacy against severe fever with thrombocytopenia syndrome virus (SFTSV) and lymphocytic choriomeningitis virus (LCMV) both in vitro and in vivo. In various cell lines, VV251 inhibits SFTSV and LCMV with EC50 values in the nanomolar to micromolar range. In lethal rodent models, once-daily oral administration of VV251 at low doses (10 mg/kg for SFTSV; 1 mg/kg for LCMV) achieves complete protection (100% survival), matching the efficacy of T-705 at 300 mg/kg. Additional pharmacokinetic analysis indicates that VV251 has favorable absorption and exposure profiles in both Sprague-Dawley rat and cynomolgus monkey models. This study evaluates the antiviral profile of VV251 and supports its further development as a promising therapeutic candidate.IMPORTANCEBunyaviruses encompass numerous highly pathogenic agents that pose significant threats to human health, including the causative agents of Crimean-Congo hemorrhagic fever, Lassa fever, and Rift Valley fever. The World Health Organization has identified Lassa fever as a priority pathogen requiring urgent research and development efforts in emergency contexts, underscoring the critical need for effective oral antiviral therapies to enhance pandemic preparedness. Here, we report that VV251 hydrochloride salt (VV251), an optimized oral prodrug derivative of 4'-fluorouridine (4'-FlU, EIDD-2794), shows significant efficacy against severe fever with thrombocytopenia syndrome virus and lymphocytic choriomeningitis virus infections, with inhibitory activity in cell culture and protective effects in lethal animal models. Building on the established broad-spectrum antiviral activity of 4'-FlU against multiple high-consequence pathogens (including severe acute respiratory syndrome coronavirus 2, respiratory syncytial virus, Lassa virus, and Junin virus), VV251 emerges as a promising next-generation oral antiviral candidate, offering an orally available therapeutic option to combat these formidable pathogens.

BACKGROUND:Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that causes severe hemorrhagic fever in humans, but no FDA-approved specific antivirals or vaccines are available to treat or prevent SFTS. METHODS:The plasmids construction and transfection were performed to generate the recombinant SFTSV harboring the nanoluciferase gene (SFTSV-Nluc). Immunostaining plaque assay was performed to measure viral titers, and DNA electrophoresis and Sanger sequencing were performed to evaluate the genetic stability. Luciferase assay and quantitative RT-PCR were performed to evaluate the efficacy of antivirals in vitro. Bioluminescence imaging, titration of virus from excised organs, hematology, and histopathology and immunohistochemistry were performed to evaluate the efficacy of antivirals in vivo. FINDINGS:SFTSV-Nluc exhibited high genetic stability and replication kinetics similar to those of wild-type virus (SFTSVwt), then a rapid high-throughput screening system for identifying inhibitors to treat SFTS was developed, and a nucleoside analog, 4-FlU, was identified to effectively inhibit SFTSV in vitro. SFTSV-Nluc mimicked the replication characteristics and localization of SFTSVwt in counterpart model mice. Bioluminescence imaging of SFTSV-Nluc allowed real-time visualization and quantification of SFTSV replication in the mice. 4-FlU was demonstrated to inhibit the replication of SFTSV with more efficiency than T-705 and without obvious adverse effect in vivo. INTERPRETATION:The high-throughput screening system based on SFTSV-Nluc for use in vitro and in vivo revealed that a safe and effective antiviral nucleoside analog, 4-FlU, may be a basis for the strategic treatment of SFTSV and other bunyavirus infections, paving the way for the discovery of antivirals. FUNDING:This work was supported by grants from the National Key Research and Development Plan of China (2021YFC2300700 to L. Zhang, 2022YFC2303300 to L. Zhang), Strategic Priority Research Program of Chinese Academy of Sciences (XDB0490000 to L. Zhang), National Natural Science Foundation of China (31970165 to L. Zhang, U22A20379 to G. Xiao), the Science and Technology Commission of Shanghai Municipality (21S11903100 to Y. Xie), Hubei Natural Science Foundation for Distinguished Young Scholars (2022CFA099 to L. Zhang).

The increasing emergence and re-emergence of RNA virus outbreaks underlines the urgent need to develop effective antivirals. RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), which exhibits significant promise for antiviral therapy. AGO2-dependent shRNA (agshRNA) generates a single-stranded guide RNA and presents significant advantages over traditional siRNA and shRNA. In this study, we applied a logistic regression algorithm to a previously published chemically siRNA efficacy dataset and built a machine learning-based model with high predictive power. Using this model, we designed siRNA sequences targeting diverse RNA viruses, including human enterovirus A71 (EV71), Zika virus (ZIKV), dengue virus 2 (DENV2), mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and transformed them into agshRNAs. We validated the performance of our agshRNA design by evaluating antiviral efficacies of agshRNAs in cells infected with different viruses. Using the agshRNA targeting EV71 as an example, we showed that the anti-EV71 effect of agshRNA was more potent compared with the corresponding siRNA and shRNA. Moreover, the antiviral effect of agshRNA is dependent on AGO2-processed guide RNA, which can load into the RNA-induced silencing complex (RISC). We also confirmed the antiviral effect of agshRNA in vivo. Together, this work develops a novel antiviral strategy that combines machine learning-based algorithm with agshRNA design to custom design antiviral agshRNAs with high efficiency.