The furin motif in the SARS-CoV-2 spike (S) protein is important for lung cell entry. It is embedded in an extended loop structure and preceded by a highly conserved QTQTX motif that is required for efficient furin cleavage of the SARS-CoV-2 WA-1 S protein. BA.3.2 is an emerging SARS-CoV-2 saltation variant that is spreading globally in April 2026 and encodes a highly mutated S protein. Here, we analyzed whether the QTQTX motif is also required for spike protein cleavage and lung cell entry of BA.3.2. We report that two patient-derived spike sequences of the BA.3.2 subvariant BA.3.2.2 lack the first QT repeat of the QTQTX motif and show that this motif is largely dispensable for both cleavage and lung cell entry of BA.3.2.2, which we found to depend on TMPRSS2. Our results suggest that the reconfiguration of the BA.3.2 S protein during persistent infection may have significantly altered the determinants of furin cleavage.IMPORTANCEThe furin motif in the SARS-CoV-2 spike (S) protein is unique among sarbecoviruses and constitutes a virulence determinant. A QTQTX motif located immediately upstream of the furin motif is required for furin cleavage of the S protein of the virus that circulated early in the pandemic. Here, we show that the QTQTX motif is largely dispensable for S protein processing and S protein-driven lung cell entry of the emerging saltation variant BA.3.2, which is currently spreading globally. Thus, BA.3.2 evolution within immunocompromised individuals may have relaxed the requirements for furin processing of the spike protein.

High-risk human papillomaviruses (HPVs) cause cervical, anogenital, and head and neck cancers. A key oncogenic mechanism involves viral exploitation of host DNA damage tolerance pathways to support productive replication, a process that also drives genomic instability. Despite evidence that specific cellular polymerases participate in HPV genome replication, the full complement of polymerases deployed across the viral life cycle remains poorly defined. This minireview examines the potential roles of both replicative and translesion synthesis (TLS) polymerases at distinct stages of the HPV life cycle, and new tools that can be used to assess their potential importance in driving cancer, the life cycle, and altering mutagenic potential.

Human adenoviruses (HAdVs) are double-stranded DNA viruses that can cause a wide range of infections, including respiratory, gastrointestinal, and ocular diseases, as well as less common conditions such as hepatitis, hemorrhagic cystitis, and encephalitis. While most infections in immunocompetent individuals remain clinically asymptomatic, immunocompromised patients are at a high risk of developing severe HAdV infections. Due to their exceptional transduction efficiency, HAdVs are also widely used as vaccine vectors, such as in vector-based COVID-19 vaccines. Although HAdVs are present as pathogens and vectors, the interaction between HAdVs and the human immune system remains insufficiently studied. Most adenoviral basic research in this context has focused on HAdV-C5. However, differences in capsid structure and genome organization exist not only between adenovirus species but also among types within the same species. These variations may influence the ability of the virus to evade or counteract the host immune system, ultimately influencing viral infectivity and replication efficiency. Further studies are needed to elucidate these differences. In this review, we summarize the differences in immune responses, with a particular emphasis on triggered IFN responses, across various HAdV species and their influence on infection outcomes, as well as their implications for the use of adenoviral vectors. Future research addressing these differences will be essential to better understand adenovirus pathogenesis and the rational design of adenovirus-based vectors.

Previous epidemiological studies have shown that E-cigarette and tobacco users are more likely to develop COVID-19 symptoms than non-users. To investigate the underlying mechanisms, we examined the effects of nicotine, the major component of tobacco and E-cigarette, and its major metabolite, cotinine, on the susceptibility of human respiratory epithelial cells to SARS-CoV-2 infection. We found that pre-treatment with nicotine and cotinine significantly and additively enhanced viral infection. While nicotine increased the expression of the viral receptor ACE2 and the serine protease TMPRSS2, cotinine upregulated cysteine protease cathepsin B and promoted viral spike protein cleavage. These findings suggest that nicotine and cotinine enhance SARS-CoV-2 infection at the cell entry stage through distinct mechanisms. Using a SARS-CoV-2 pseudovirus system, we further investigated the effects and mechanisms of nicotine and cotinine on viral entry. We found that both compounds enhanced pseudovirus infection, but with different time courses. Nicotine's effect correlated with the upregulation of ACE2 and TMPRSS2, whereas cotinine's effect corresponded with increased cathepsin B and viral spike protein cleavage. The cathepsin B inhibitor E64d completely abolished cotinine-enhanced viral spike protein cleavage and viral entry. In contrast, ACE2 and TMPRSS2 inhibitors (chloromethylketone and camostat) had limited effects on viral spike protein cleavage and only partially reduced the viral entry enhancement induced by nicotine and cotinine. These results indicate that nicotine promotes virus-receptor binding and cell entry, while cotinine facilitates viral entry through cathepsin B-mediated spike protein cleavage.IMPORTANCEThis study highlights the potential risks of tobacco and E-cigarette use in increasing susceptibility to COVID-19. We show that the major neurostimulant in tobacco and E-cigarette, nicotine, and its metabolite, cotinine, additively enhance SARS-CoV-2 infection in human respiratory epithelial cells by promoting viral entry. Specifically, nicotine and cotinine upregulate the expression of distinct, yet complementary sets of key cellular proteins required for viral entry. These findings suggest that both tobacco smoking and E-cigarette vaping may exacerbate COVID-19 infection rates and severity and provide new insights into how nicotine and cotinine contribute to viral susceptibility. This research underscores the need for public health measures to address the heightened risk posed by tobacco smoking and E-cigarette vaping to encounter the SARS-CoV-2 infection.

Human parainfluenza viruses (PIVs) are a leading cause of respiratory illness, particularly in vulnerable populations, where infection can lead to severe disease. Despite their clinical impact, there are currently no licensed vaccines or effective antiviral treatments available. PIVs have two large surface proteins, the fusion and hemagglutinin-neuraminidase (HN) proteins, both of which are targets of neutralizing antibodies. In this study, we identified and characterized two human monoclonal antibodies (mAbs), 5217-2 and 5217-9, which bind recombinant PIV3 HN protein, recognize PIV3-infected cells, and neutralize in vitro. We determined the antigenic sites of the PIV3 HN-specific mAbs via biolayer interferometry and found mAb 5217-9 targets a previously defined neutralizing antigenic site, while mAb 5217-2 binds a distinct antigenic site, expanding the known antigenic landscape. To further understand the newly defined epitope, we determined a cryo-electron microscopy (cryo-EM) structure of mAb 5217-2 in complex with PIV3 HN, revealing an epitope adjacent to the PIV3 HN protein receptor binding site. We also determined the structure of the previously discovered anti-PIV3 HN mAb PIV3HN-09, previously shown to be partially protective in vivo. In a hamster challenge model of PIV3, mAb 5217-2 significantly reduced lung viral titers, demonstrating its protective capacity. Furthermore, as the mAb PIV3HN-05 was previously shown to cross-neutralize PIV1, we evaluated its protective efficacy in a PIV1 challenge model, demonstrating reduced lung viral titers. Overall, these findings provide new insights into PIV3 HN epitopes to support structure-based vaccine design and demonstrate protective mAbs for both PIV3 and PIV1.IMPORTANCEHuman parainfluenza viruses (PIVs) are a major cause of respiratory illness, yet there are currently no licensed vaccines or targeted antiviral therapies. A better understanding of how antibodies recognize viral surface proteins is critical for developing effective interventions. In this study, we identified human monoclonal antibodies that neutralize PIV3 and mapped the specific regions of the viral hemagglutinin-neuraminidase (HN) protein they recognize. Structural analysis revealed a previously uncharacterized antibody-binding site near the HN active site and expanded the known antigenic landscape of this protein. We also demonstrate protective activity of these antibodies in animal models against PIV3 and PIV1 infection. These findings provide new targets and structural insights that can guide the development of vaccines and antibody-based therapies against PIVs.

Coronavirus nonstructural protein 15 (Nsp15) is a conserved uridine-preferring endoribonuclease (EndoU). Studies using mouse hepatitis virus (MHV), SARS-CoV-2, and other coronaviruses have shown that Nsp15 associates with replication-transcription complexes (RTCs) and contributes to viral immune evasion. Structural studies of alpha- and beta-coronavirus Nsp15 proteins reveal a hexameric enzyme that engages viral RNA substrates and cleaves at unpaired uridines through an RNase A-like, largely metal-independent mechanism stimulated by divalent cations. The Nsp15 hexamer functions as a dynamic, cooperative platform capable of accommodating extended double-stranded and structured RNA substrates. Genetic studies in several coronaviruses indicate that EndoU activity is dispensable for viral RNA synthesis in cell culture, but critical for suppressing host antiviral responses. Loss of EndoU activity promotes accumulation of immunostimulatory RNA species and activation of dsRNA-sensing pathways, including MDA5-dependent interferon signaling, PKR-mediated translational arrest, and the OAS/RNase L system. Mechanistically, Nsp15 is proposed to suppress these responses by selectively processing uridine-rich and structurally accessible regions in viral RNA, including poly(U)-containing negative-strand RNAs, and elements within untranslated regions and transcription regulatory sequences. Beyond catalysis, Nsp15 may contribute to RTC organization and regulate viral RNA recombination or defective viral genome formation, although these roles remain less well-defined and may vary among coronavirus species. Together, these findings support a model in which Nsp15 functions as a regulator of viral RNA composition and immunogenicity rather than solely as a degradative nuclease. This review summarizes recent advances in Nsp15 structure, RNA processing, immune evasion, and antiviral targeting, and highlights key unresolved questions.

Koch's postulates remain unachievable for most crustacean viruses due to uncultivable pathogens and chronic infection dynamics. We propose viromic Koch's postulates, a culture-independent framework, to address this limitation. The modified postulates uncover comprehensive information about viruses throughout the chain from clinical samples to viral preparations to challenge trials by combining the virome from metatranscriptomic, proteomic, or molecular analyses with diagnoses from clinical or pathological observations. Applying this approach to iron prawn syndrome (IPS), a chronic disease that causes stunted growth in Macrobrachium rosenbergii, we identified infectious precocity virus (IPV) as the exclusive viral correlate through metatranscriptomic analysis. IPV showed 100% prevalence in symptomatic prawns, but was absent in controls. Viral preparations from diseased tissue reproduced IPS pathology in healthy prawns upon inoculation, with IPV replication kinetics correlating to growth retardation. Histopathology and in situ hybridization localized IPV to neuroendocrine tissues, confirming tropism and replication sites. Our findings definitively establish IPV as the causative agent of IPS. Furthermore, this study demonstrates how systematizing existing culture-independent verification steps-referred to here as viromic Koch's postulates-can serve as a practical, standardized operational roadmap for pathogen verification in uncultivable systems, facilitating the investigation of viral diseases in aquaculture.

A non-replicating Tiantan strain-based vaccinia virus (NTV) holds significant application prospects for vaccination and gene therapy and has recently entered clinical trials as a novel and safer vaccine candidate against monkeypox. However, optimization is still required, particularly regarding its production capacity and immunogenicity. In this study, a recombinant virus was constructed by modifying the F1L and C7L genes in a non-replicating viral backbone using CRISPR/Cas9-mediated gene editing and homologous recombination. The resulting construct, designated NTV-ΔF1L-C7L, exhibited significantly enhanced replication in vaccine production cell lines, with viral yields increasing by more than 680-fold in MRC-5 cells compared to those of the parental NTV. Its pathogenicity in mice was significantly reduced, showing more than a 10-fold decrease compared to the pathogenicity of the vaccinia virus Tiantan strain (VTT). Following two intramuscular doses, NTV-ΔF1L-C7L elicited high titers of orthopoxvirus-specific IgG and neutralizing antibodies against vaccinia and monkeypox viruses, along with a robust cellular immune response exhibiting a Th1 bias, which was significantly stronger than that induced by either parental NTV or VTT vaccination. Complete protection (100%) against a lethal challenge with the vaccinia virus Western Reserve strain was achieved in mice immunized with either a low dose (10[3] PFU) or a single dose (10[5] PFU) of NTV-ΔF1L-C7L, comparable to that conferred by VTT. These findings demonstrate that NTV-ΔF1L-C7L combines high safety with enhanced replication and immunogenicity, supporting its value as a novel vaccine vector and its potential application in controlling the current global monkeypox outbreak.IMPORTANCEA highly attenuated NTV exhibits an improved safety profile; however, its production capacity and immunogenicity require optimization for clinical application. In this study, a novel recombinant virus, NTV-ΔF1L-C7L, was developed by targeting the deletion of F1L and the insertion of C7L into the NTV backbone. This attenuated live vaccine, NTV-ΔF1L-C7L, demonstrates several significant advantages. Its robust in vitro replication supports scalability for large-scale vaccine production. It has demonstrated strong immunoprotective efficacy in mice while maintaining a high safety margin, exhibiting reduced pathogenicity in vivo , and inducing robust humoral and cellular immune responses against vaccinia and monkeypox virus. These immune responses confer complete protection against lethal VACV challenge at low-dose or single-dose immunization. These findings establish a solid experimental foundation for the further development of NTV-ΔF1L-C7L as a next-generation VACV-based vector or a candidate vaccine against mpox.

The SARS-CoV-2 (SARS2) and SARS-CoV (SARS1) viruses pose significant threats due to their high mutation rates and potential for cross-species transmission, which has rendered many antibodies ineffective. To counter potential future emergences of novel SARS-like viruses, it is imperative to develop nanobodies with broad-spectrum neutralizing capacity. Here, we employed an alternating immunization strategy in alpacas using SARS1 and SARS2 RBDs to generate cross-reactive nanobodies. Four crossing-binding nanobodies (aSR29, aSR196, aSR347, and aSR348) were identified, exhibiting high affinities for SARS1/SARS2 RBDs and Omicron subvariants. Structural analysis revealed that aSR29 targets a cryptic, highly conserved epitope at the inner base of the RBD, a region shielded from conventional antibodies by steric hindrance, efficiently neutralizing SARS1, SARS2, and multiple Variants of Concern (VOCs), including XBB.1.5 and BQ.1.1. Building on this unique binding mode, we engineered a bispecific fusion protein, aSR29-ACE2-Fc. This construct synergistically combines high-affinity RBD binding with the steric blockade of viral attachment, resulting in a 33-fold increase in neutralization potency against SARS2 and enhanced resilience against immune escape. Our findings validate an efficient strategy for eliciting broad-spectrum nanobodies and demonstrate the therapeutic potential of VHH-ACE2 fusion proteins against current and future SARS-like viruses.IMPORTANCEThe continued evolution of SARS-CoV-2 has rendered most existing antibodies ineffective, highlighting the urgent need for broad-spectrum countermeasures against current and future SARS-like viruses. Here, we developed an alternating immunization strategy to generate nanobodies that target a hidden, highly conserved region on the viral spike protein. One such nanobody, aSR29, potently neutralizes diverse variants, including XBB.1.5 and BQ.1.1. By fusing aSR29 with the viral receptor ACE2, we created a bispecific molecule that blocks viral entry through two independent mechanisms, achieving a 33-fold increase in neutralization potency. This work provides a practical and scalable strategy for pandemic preparedness, demonstrating that engineered nanobody-ACE2 fusions can serve as effective broad-spectrum therapeutics against emerging SARS-like viruses.

Nucleoside and nucleotide analogs are an impactful class of antivirals, yet their efficacy is often limited by inefficient intracellular accumulation and enzymatic activation. Many analogs are administered as nucleoside prodrugs, dependent on host or viral kinases for sequential phosphorylation, with the first monophosphate-forming step widely considered rate-limiting. Medicinal chemistry strategies such as utilizing masked-phosphate prodrugs may improve uptake and activation, but require alternative metabolic processing to generate the active triphosphate. Nanoparticle delivery provides an alternative or complementary approach by directly encapsulating phosphorylated nucleotide analogs. Nanoparticles made from materials including lipids, polymers, dendrimers, or polysaccharides can protect phosphorylated drugs from degradation, improve cellular uptake, and enable controlled or tissue-specific release. This review provides a brief context and overview of recent advances in nanoparticle-mediated delivery of phosphorylated antiviral nucleotides, drawing on parallels from oncology, and evaluates the technological advances and limitations influencing their continued development as antiviral carriers.

Coronaviruses (CoVs) replicate unusually large RNA genomes that necessitate proofreading by the 3'-to-5' exoribonuclease (ExoN) formed by nonstructural proteins 14 (nsp14) and 10 (nsp10). Previous studies suggested that inactivation of the ExoN catalytic site in severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is lethal, leaving unresolved whether the virus can tolerate impaired proofreading activity. Here, we investigated the functional requirement for ExoN in SARS-CoV-2 replication by combining a continuous fluorescence-based biochemical assay with an optimized single-bacmid reverse genetics system. Mutational analysis of residues involved in RNA binding or catalysis revealed graded effects on ExoN activity in vitro. Alanine substitution of Lys9, a residue positioned near the RNA-binding interface, did not reduce ExoN activity, whereas charge reversal at this position (K9E) impaired activity more strongly than alanine substitutions of the catalytic motif I residues D90 and E92 (D90A/E92A). Correspondingly, recombinant SARS-CoV-2 carrying K9A was readily recovered, whereas the D90A/E92A mutant was recovered only after an extended delay, and K9E could not be rescued despite repeated attempts. The D90A/E92A mutant exhibited reduced replication while maintaining the engineered ExoN substitutions during serial passage. Deep sequencing of viral populations revealed a marked increase in genome-wide sequence variation in the D90A/E92A mutant, demonstrating a stable mutator phenotype. Together, these findings indicate that SARS-CoV-2 can tolerate substantial impairment of ExoN activity but depends on a minimal activity threshold for viability. This system provides a platform for defining how SARS-CoV-2 proofreading controls genome stability, viral fitness, and sensitivity to antiviral strategies that exploit reduced replication fidelity.IMPORTANCECoronaviruses have unusually large RNA genomes because they encode a proofreading enzyme that removes copying errors during replication. It has been unclear whether SARS-CoV-2 can survive when this proofreading function is strongly weakened because earlier studies suggested that loss of the enzyme's catalytic activity is lethal. We show that SARS-CoV-2 can tolerate substantial impairment of proofreading but only when residual exonuclease activity remains above a minimal threshold. A virus with impaired proofreading replicates less efficiently and accumulates mutations across its genome, whereas a more severe defect prevents virus recovery. These findings clarify how coronavirus proofreading balances genome stability with viral fitness and provide a useful system for studying how reduced replication fidelity affects viral evolution, antiviral sensitivity, and attenuation. Defining this activity threshold may also help guide antiviral strategies that target coronavirus proofreading.

Human immunodeficiency virus 1 (HIV-1) remains a global health burden affecting over 39 million people worldwide, with approximately one million new infections occurring each year. Sexual transmission remains the predominant route of HIV-1 acquisition, during which the virus crosses mucosal barriers and interacts with host immune cells to facilitate infection. Dendritic cells (DCs) contribute to systemic viral spread and seeding of reservoirs by efficiently capturing HIV-1, leading to infection and subsequent transmission of the virus to CD4[+] T cells. Siglec-1 (CD169) is a key receptor involved in HIV-1 capture, and its blockade may help prevent viral transmission. Here, we used newly developed single-domain antibodies, also known as nanobodies, against Siglec-1 as a candidate inhibitor to limit HIV-1 transmission. Using a Siglec-1-overexpressing cell line, we demonstrated that these nanobodies specifically blocked Siglec-1-mediated HIV-1 binding and transmission. Extending these findings to a more physiologically relevant context, the anti-Siglec-1 nanobodies neither induced immune nor cellular activation in DCs, indicating a favorable safety profile for functional applications. Most notably, the anti-Siglec-1 nanobody 2C2 effectively blocked HIV-1 binding as well as infection of DCs. Moreover, both replication-dependent and replication-independent HIV-1 transmission by DCs was also abrogated by the nanobody. Our findings not only underscore the relevance of Siglec-1 in HIV-1 capture but also highlight the therapeutic potential of utilizing host-targeting strategies against infectious diseases.

Vectors based on the adeno-associated virus are widely used as delivery methods in gene therapy applications, yet understanding of the mechanisms governing its intracellular trafficking remains incomplete. Traditional models suggest that AAV escapes from endosomes via membrane disruption, but direct evidence for this process is lacking. Here, we show that AAVR, the essential AAV cell entry receptor, functions as a bona fide retromer cargo. Using in vitro reconstitution assays, we demonstrate that AAVR's cytosolic tail is sufficient to engage the retromer-SNX3 complex and drive membrane tubulation, a hallmark of retrograde trafficking. In AAVR-knockout HuH-7 cells, AAV2 particles are internalized, but accumulate in early endosomal compartments, and fail to support transgene expression. Galectin-8 recruitment assays reveal no evidence of endosomal membrane rupture during productive transduction, distinguishing AAV2 from lytic vectors such as lipid nanoparticles. Moreover, VP1u-deficient and PLA2-mutant AAV2 capsids accumulate at the TGN, indicating that VP1u is dispensable for early trafficking but required for post-TGN progression toward productive transduction. These findings argue against the widely held model of direct endosomal escape and provide a mechanistic basis for the previously described essential role of the TGN in productive AAV transduction, positioning AAV entry as a vesicle-guided, receptor-mediated process.IMPORTANCEThe study demonstrates that AAV2 does not escape endosomes via membrane rupture but instead exploits the cellular receptor AAVR as a retromer cargo to traffic through vesicle-mediated retrograde transport to the trans-Golgi network, a critical checkpoint for productive transduction. Building on prior observations that VP1u-deficient particles are still internalized (R. Popa-Wagner, M. Porwal, M. Kann, M. Reuss, et al., J Virol 86:9163-9174, 2012, https://doi.org/10.1128/jvi.00282-12), we show that these particles traffic to the TGN but accumulate at this compartment, indicating that VP1u and its PLA2 activity become essential specifically at a post-TGN step in the transduction pathway.

White spot syndrome virus (WSSV), a major pathogen causing severe losses in global crustacean aquaculture, ranks among the largest known animal enveloped DNA viruses. While some features of WSSV-host interactions are known, the mechanisms by which the virus exploits host mitochondrial functions to promote its replication remain unclear. Here, using the red claw crayfish (Cherax quadricarinatus) model, we demonstrate that WSSV infection activates host mitophagy. This process is orchestrated by the mitochondria-targeting viral protein wsv156, which acts as a key effector that triggers the formation of mitochondrial aggregates (mito-aggresomes) and initiates mitophagy. Mechanistically, wsv156 recruits and activates dynamin-related protein 1 (CqDrp1) to drive mitochondrial fission and aggregation. Concurrently, wsv156 facilitates the recruitment of the E3 ubiquitin ligase Parkin (CqParkin) to mitochondria, engaging the canonical PTEN-induced putative kinase 1 (CqPINK1)-Parkin pathway to initiate mitophagy. Intriguingly, the viral envelope protein VP26 blocks the final step of autophagic degradation, arresting the process in an incomplete state. We further show that this incomplete mitophagy benefits viral replication through a dual mechanism: it enhances glycolysis to meet the heightened energy and biosynthetic demands of infection while suppressing cell apoptosis to preserve cellular viability. Collectively, our findings uncover how a large DNA virus exploits and precisely modulates host mitophagy, specifically by initiating and then arresting it to craft a supportive intracellular niche for replication. This study not only advances the understanding of WSSV pathogenesis but also provides evolutionary insights into conserved viral strategies for manipulating mitochondrial quality control across diverse hosts, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) poses the most significant viral threat to global crustacean aquaculture. A major barrier to developing effective countermeasures is the limited understanding of its molecular pathogenesis. This study demonstrates that WSSV actively hijacks host mitochondrial quality control by triggering a stalled form of mitophagy. We identify wsv156 as a key orchestrator of this process, driving mito-aggresome formation and activating Parkin-dependent mitophagy while arresting its completion via VP26, then creating a stalled, incomplete state. Importantly, this incomplete mitophagic response is repurposed to benefit viral replication through a dual mechanism: it enhances glycolysis to fuel viral biosynthesis and concurrently suppresses host cell apoptosis to maintain cell viability. Our findings uncover a novel strategy, whereby an enveloped large DNA virus manipulates mitochondrial turnover to create a favorable replicative niche. These results deepen the mechanistic understanding of WSSV infection and highlight wsv156 as a potential target for future antiviral strategies in aquaculture.