Journal of Virology最新文献

{"title":"Nanoparticle vaccine based on the pre-fusion F glycoprotein of respiratory syncytial virus elicits robust protective immune responses.","authors":"Zhulong Hu, Siyu Tian, Yu Zhou, Yanqun Wang, Yu Li, Senyan Zhang, Peilan Wei, Zhen Zhuang, Luo Ren, Jiao Liu, Na Zang, Rui Yu, Yanbin Ding, Yan Guo, Cai Jing, Hang Chen, Caixia Zhang, Yuanfeng Yao, Chunping Deng, Rui Wei, Peng Zhou, Yongjuan Zou, Dawei Zhao, Shuyun Liu, Meijuan Fu, Xuejun Mo, Guodong Peng, Enmei Liu, Jincun Zhao, Yuanyuan Li, Jing Jin","doi":"10.1128/jvi.00903-25","DOIUrl":"10.1128/jvi.00903-25","url":null,"abstract":"<p><p>Respiratory syncytial virus (RSV) is a global public health concern. Currently, RSV vaccines are approved only for use in older adults, while preventing the disease in infants and children, as well as ensuring vaccine durability, remains a significant challenge. The pre-fusion conformation of the RSV fusion (F) glycoprotein is a primary target for vaccine development, as it elicits significantly higher neutralizing antibody titers than the post-fusion form. Here, we conjugated either the first-generation RSV pre-fusion F antigen, DS-Cav1, or the second-generation antigen, Sc9-10, to a computationally designed nanoparticle platform, NPM, via a Catcher/Tag system. Conjugating RSV pre-fusion F to NPM significantly enhanced immunogenicity, stability, and bioactivity compared to display on the I53-50 nanoparticle platform. In a cotton rat challenge model, Sc9-10-NPM vaccine candidates provided effective protection across a wide dosage range, regardless of the adjuvant used. These results support the continued development of this promising nanoparticle-based RSV vaccine candidate.IMPORTANCERespiratory syncytial virus (RSV) is a major cause of severe respiratory illness in infants and young children worldwide, yet few vaccines are approved for use in these vulnerable groups. In this study, we developed a new vaccine candidate based on a second-generation RSV pre-fusion F protein, engineered for improved stability and immune response. This protein was displayed on a specially designed nanoparticle platform to enhance its effectiveness and durability. The vaccine elicited strong immune responses and provided complete protection in preclinical models, even without the use of potent adjuvants that may cause side effects. Importantly, it did not trigger adverse vaccine-enhanced disease (VED). These findings suggest that this vaccine design could offer a safer and more effective way to protect infants and other at-risk populations from RSV. Additionally, the nanoparticle platform may be applicable to vaccines against other infectious diseases.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0090325"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455995/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144959104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SARS-CoV-2 causes chronic lung inflammation and impaired respiratory capacity in aged Roborovski dwarf hamsters.","authors":"Amirhossein Karimi, Carolin M Lieber, Kaori Sakamoto, Richard K Plemper","doi":"10.1128/jvi.00755-25","DOIUrl":"10.1128/jvi.00755-25","url":null,"abstract":"<p><p>Roborovski dwarf hamsters are permissive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and progress to acute viral pneumonia with profound lung tissue injury, recapitulating hallmarks of severe coronavirus disease 2019 (COVID-19) in vulnerable patient groups such as older adults. In this study, we established dwarf hamster whole-body plethysmography and assessed disease severity and propensity for long-term compromise of lung recovery from severe COVID-19-like disease in young, adult, and aged animals. Aged dwarf hamsters infected intranasally with variant of concern (VOC) omicron BA.4 experienced more severe clinical signs, carried a higher lung virus load, and had a greater risk of succumbing to infection. Resting airway hypersensitivity was transiently increased in aged, but not young, dwarf hamsters 3-4 days post-infection. Pharmacologically induced respiratory distress revealed compromised lung capacity in animals of both age groups at peak disease. Aged animals showed impaired respiratory function for 45 days, mounted a weaker antiviral response, and developed chronic pneumonia with lasting tissue damage. Treatment of acute disease with approved antivirals, paxlovid-like nirmatrelvir + ritonavir or molnupiravir, prevented long-term respiratory sequelae in aged animals. Nirmatrelvir + ritonavir fully suppressed transient respiratory distress and mediated complete survival of aged animals. This study shows a high positive correlation between host age and SARS-CoV-2 disease severity in dwarf hamsters, establishes a model for chronic pneumonia with impaired respiratory capacity in at-risk hosts, and demonstrates the benefit of antiviral therapy of acute disease for long-term respiratory health.IMPORTANCEIn the COVID-19 pandemic, the frequency of chronic respiratory insufficiency after acute SARS-CoV-2 infection was positively linked to patient age. Roborovski dwarf hamsters recapitulate hallmarks of life-threatening COVID-19 in at-risk patients. In this study, we monitored disease progression and lung function in young and aged dwarf hamsters infected with a VOC omicron isolate and assessed the effect of antiviral treatment on long-term lung function. We established a strong correlation between host age and SARS-CoV-2 disease severity in dwarf hamsters, identified a high propensity of aged animals to develop chronic lung inflammation, and demonstrated a long-term loss of respiratory capacity in the subset of aged animals that survived the acute infection. Antiviral treatment suppressed the development of late sequelae and preserved lung function. These results have important implications for effective SARS-CoV-2 management in aged hosts at high risk of developing severe viral pneumonia with long-term impaired lung function.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0075525"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12456015/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144817040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing the structural boundaries of broadly reactive antibody interactions with diverse H3 influenza hemagglutinin proteins.","authors":"John V Dzimianski, Kaito A Nagashima, Joseph M Cruz, Giuseppe A Sautto, Sara M O'Rourke, Vitor H B Serrão, Ted M Ross, Jarrod J Mousa, Rebecca M DuBois","doi":"10.1128/jvi.00453-25","DOIUrl":"10.1128/jvi.00453-25","url":null,"abstract":"<p><p>Influenza virus infections are an ongoing seasonal disease burden and a persistent pandemic threat. Formulating successful vaccines remains a challenge due to accumulating mutations in circulating strains, necessitating the development of innovative strategies to combat present and future viruses. One promising strategy for attaining greater vaccine effectiveness and longer-lasting protection is the use of computationally optimized broadly reactive antigens (COBRAs). The COBRA approach involves <i>in silico</i> antigen design by generating iterative, layered consensus sequences based on current and historic viruses. Antigens designed by this process show a greater breadth of antibody-mediated protection compared to wild-type antigens, with effectiveness that often extends beyond the sequence design space of the COBRA. In particular, the use of COBRA hemagglutinin (HA) proteins has led to the discovery of broadly reactive antibodies that are suggestive of their therapeutic potential. Understanding the extent to which these antibodies are effective is key to assessing the resilience of vaccine-induced immunity to diverging influenza strains. To investigate this, we tested the binding of broadly reactive antibodies with a diverse panel of H3 HA proteins. Using cryo-electron microscopy, we defined the molecular characteristics of binding for these antibodies at the paratope-epitope interface. Through sequence and structural comparisons, we observed the correlative patterns between antibody affinity and antigen structure. These data shed light on the breadth and limitations of broadly reactive antibody responses in the context of an ever-changing landscape of influenza virus strains, yielding insights into strategies for universal vaccine design.IMPORTANCEFormulating effective influenza vaccines remains a challenge due to a constantly changing landscape of circulating viruses. This is particularly true for H3N2 viruses that undergo a high degree of antigenic drift. Several new vaccine designs can elicit broadly neutralizing antibodies that are effective against a range of influenza strains. More insight is needed, however, into how resilient these antibodies will be to future strains that evolve in the context of this selective pressure. Here, we measured the precise binding characteristics of three broadly neutralizing antibodies to 18 different hemagglutinin (HA) proteins representing almost 50 years of virus evolution. Using single-particle cryo-electron microscopy and X-ray crystallography, we determined the structural characteristics of the epitopes bound by these antibodies and identified specific amino acids that greatly impact the effectiveness of these antibodies. This provides important insights into the longevity of antibody efficacy that can help guide design choices in next-generation vaccines.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0045325"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455949/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Prediction of COVID-19 disease progression by multiparametric analysis of circulating extracellular vesicles with flow cytometry.","authors":"Evelyn Hammer, Charlotte Flynn, Johannes Rößler, Johanna Erder, Rudolf Napieralski, Lisa Fricke, Birgit Campbell, Martin Feuerherd, Felix Esslinger, Albrecht von Brunn, Timm Weber, Siobhan King, Sisareuth Tan, Alain R Brisson, Ulrike Protzer, Gabriele Schricker, Kathrin Gärtner, Gregor Ebert, Allessandra Moretti, Florian Klein, Kevin Knoops, Ron Heeren, Wolfgang Hammerschmidt, Reinhard Zeidler, Olaf Wilhelm, Percy A Knolle, Bastian Höchst","doi":"10.1128/jvi.01189-25","DOIUrl":"10.1128/jvi.01189-25","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are released from all cells of the body. They are considered to mirror the state of the cells from which they are released and circulate in the blood, suggesting a possible use of EV analysis for diagnostic purposes. Here, we report that the analysis of single EVs by flow cytometry can detect infection of cells with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by identifying expression of the SARS-CoV-2 spike (S) protein on the surface of EVs and the cellular origin of EVs by detecting cell-type-specific markers such as troponin (cTNT1) for cardiomyocytes. In coronavirus-associated disease 19 (COVID-19) patients, we detected a direct correlation of the frequencies of circulating S-expressing EVs, but not of cTNT/S-co-expressing EVs, with the subsequent development of a severe disease course. Detection of circulating S-expressing EVs indicates widespread SARS-CoV-2 infection in the body, which may contribute to the immune pathogenesis that triggers tissue and organ damage in COVID-19. Our findings suggest that detecting circulating viral antigen-expressing EVs may provide crucial predictive information on infection-associated disease courses in situations of a future viral pandemic.IMPORTANCEThe ability to predict which patients infected with the SARS-CoV-2 virus will develop severe disease remains a significant clinical challenge. The present study demonstrates that EVs in the peripheral blood, carrying the SARS-CoV-2 spike protein, can be detected by flow cytometry and serve as early biomarkers of disease progression. In contradistinction to PCR or serology, this method provides insight into systemic viral spread and potential organ involvement. The early identification of spike-positive EVs at the time of hospital admission has the potential to facilitate the timely identification of high-risk patients, thereby enhancing the efficacy of triage and subsequent care. This approach may also be of value in terms of facilitating a more rapid and precise response to future virus pandemics.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0118925"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"LPS/TLR4-activated M1-polarized macrophage-derived exosomes enhance IBV vaccine efficacy in chickens.","authors":"Jun Zhou, Shikai Cai, Hongbin Huang, Fan Yang, Kexin Pan, Zhaoyang Sun, Yun Fan, Feng Wen, Limei Qin, Yun Zhang","doi":"10.1128/jvi.01156-25","DOIUrl":"https://doi.org/10.1128/jvi.01156-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Infectious bronchitis virus (IBV) imposes substantial economic losses on poultry production due to extensive serotypic diversity and limited cross-protection conferred by conventional vaccines. This study evaluated exosomes derived from M1-polarized chicken macrophages (HD11&lt;sub&gt;M1&lt;/sub&gt;-exo) as a novel adjuvant for IBV vaccination. HD11&lt;sub&gt;M1&lt;/sub&gt;-exo, isolated from lipopolysaccharide (LPS)-activated HD11 macrophages via ultracentrifugation, demonstrated significant immunomodulatory properties across multiple experimental systems. &lt;i&gt;In vitro&lt;/i&gt; analyses demonstrated that HD11&lt;sub&gt;M1&lt;/sub&gt;-exo enhances macrophage phagocytosis and promotes cellular immune activation via the LPS/TLR4 signaling pathway. &lt;i&gt;In ovo&lt;/i&gt; analyses showed that HD11&lt;sub&gt;M1&lt;/sub&gt;-exo pretreatment upregulates tracheal expression of IL-1β, IL-2, IL-4, IFN-γ, TNF-α, and TLR4 at different time points, thereby enhancing viral resistance and reducing pathological damage. In chickens, HD11&lt;sub&gt;M1&lt;/sub&gt;-exo administration elevated CD80/CD86 and TGF-β4 expression in respiratory tissues and increased secretory immunoglobulin A (IgA) levels in lacrimal fluid. When co-administered with the H120 vaccine, HD11&lt;sub&gt;M1&lt;/sub&gt;-exo significantly improved both humoral immunity (elevated serum IgY and mucosal IgA) and cellular responses (increased CD80/CD86 expression), outperforming commercial adjuvants in efficacy. Following the viral challenge, HD11&lt;sub&gt;M1&lt;/sub&gt;-exo + H120-immunized chickens exhibited significantly reduced viral loads and attenuated histopathological lesions compared to controls. These results collectively suggest that exosome-based formulations may serve as promising adjuvants for enhancing the immunogenicity and protective efficacy of poultry vaccines.IMPORTANCEInfectious bronchitis virus (IBV) causes significant global economic losses in the poultry industry despite extensive vaccination programs. Current vaccines often fail to elicit sufficient mucosal and cellular immunity, which are critical for protection against the virus. Although commercial adjuvants have been employed to enhance vaccine efficacy, many exhibit limitations in eliciting comprehensive immune responses. In this study, we comprehensively evaluated HD11&lt;sub&gt;M1&lt;/sub&gt;-exo as a novel adjuvant for IBV vaccines across &lt;i&gt;in vitro&lt;/i&gt;, &lt;i&gt;in ovo&lt;/i&gt;, and &lt;i&gt;in vivo&lt;/i&gt; models for the first time. Our results demonstrate that HD11&lt;sub&gt;M1&lt;/sub&gt;-exo enhances macrophage function via lipopolysaccharide (LPS)/TLR4 signaling, upregulates key cytokines and immune markers in embryonic tissues, and significantly boosts cellular, humoral, and mucosal immunity when co-administered with live-attenuated IBV vaccines, outperforming commercial adjuvants. Importantly, this adjuvant strategy significantly enhanced protective efficacy in challenged chickens. This study provides a foundation for developing exosome-based adjuvants that could advance poultry vaccination strategies against IBV and other avian respiratory pa","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0115625"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145125032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction for Oom et al., \"The two-dose MVA-BN mpox vaccine induces a nondurable and low avidity MPXV-specific antibody response\".","authors":"Aaron L Oom, Kesi K Wilson, Miilani Yonatan, Stephanie Rettig, Heekoung Allison Youn, Michael Tuen, Yusra Shah, Ashley L DuMont, Hayley M Belli, Jane R Zucker, Jennifer B Rosen, Ramin Sedaghat Herati, Marie I Samanovic, Ralf Duerr, Angelica C Kottkamp, Mark J Mulligan","doi":"10.1128/jvi.00872-25","DOIUrl":"10.1128/jvi.00872-25","url":null,"abstract":"","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0087225"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455918/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144992909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biographical Feature: Diane E. Griffin and Ann Palmenberg-luminaries in RNA virology.","authors":"Robert F Kalejta, Paul D Friesen, Andrew Pekosz, Stacey Schultz-Cherry","doi":"10.1128/jvi.01164-25","DOIUrl":"10.1128/jvi.01164-25","url":null,"abstract":"","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0116425"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455961/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The 5'-end segment-specific noncoding region of influenza A virus regulates both competitive multi-segment RNA transcription and selective genome packaging during infection.","authors":"Zining Liu, Lei Zhang, Wenyu Zhang, Yuerong Lai, Tao Deng","doi":"10.1128/jvi.00328-25","DOIUrl":"10.1128/jvi.00328-25","url":null,"abstract":"<p><p>The 3' and 5' termini of the eight RNA segments of the influenza A virus (IAV) genome contain segment-specific or subtype-specific (in HA and NA segments) noncoding regions (ssNCRs), located between the highly conserved terminal promoter sequences and the coding start or stop codons. These ssNCRs vary significantly in both length and sequence among segments and have been implicated in selective genome packaging. Beyond their established role in packaging, we previously demonstrated that the 3'-end H1-ssNCR, together with its adjacent coding region, supports optimal replication of HA viral RNA (vRNA) in a multi-segment context. In this study, we discovered that truncation of the 5'-end H1-ssNCR, unlike its 3' counterpart, resulted in a reduction in HA mRNA levels in a template-competitive manner and impaired incorporation of HA vRNA into progeny virions. Interestingly, upon passaging of a recombinant virus carrying the most severely truncated 5'-end H1-ssNCR, an adaptive mutation occurred seven nucleotides upstream of the truncation site. This mutation restored both HA mRNA expression and HA vRNA packaging, thereby rescuing viral replication. Further analysis revealed that these regulatory effects may be mediated by RNA secondary structures formed around the 5'-end H1-ssNCR. Collectively, our findings reveal, for the first time, a dual regulatory role of the 5'-end segment-specific noncoding regions of influenza A virus RNA segments in modulating multi-segmental RNA transcription and selective genome packaging during infection.IMPORTANCEThe 3' and 5' segment-specific noncoding regions (ssNCRs) of influenza A virus (IAV) have long been recognized as critical signals for selective genome packaging. However, their potential roles in other regulatory processes remain largely unexplored. We previously reported that the 3'-end H1-ssNCR, together with its adjacent coding region, primarily determines optimal HA vRNA accumulation in a multi-segment environment. In this study, we further demonstrate that the 5'-end ssNCR plays a dual role in regulating viral RNA transcription in a template-competitive manner and governing vRNA incorporation into progeny virions. These findings reveal previously underappreciated levels of complexity, highlighting that ssNCRs contribute not only to genome packaging but also to the fine-tuning of viral RNA synthesis in multi-segmented negative-strand RNA viruses.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0032825"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455950/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784612","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cathepsin L and transmembrane serine protease 11E mediate trypsin-independent entry of porcine deltacoronavirus into Huh7 cells.","authors":"Wenwen Xiao, Yuanxiang Xiong, Yuchen Wang, Ting Li, Chaoqun Chen, Yuting Shi, Guanning Su, Yanrong Zhou, Shaobo Xiao, Liurong Fang","doi":"10.1128/jvi.01055-25","DOIUrl":"10.1128/jvi.01055-25","url":null,"abstract":"<p><p>Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus with zoonotic potential. Previous studies showed that PDCoV productive infection is dependent on exogenous trypsin in porcine-derived cells. In this study, we found that trypsin appears to be dispensable for PDCoV infection in human-derived cell lines. Using Huh7 cells as a model, we investigated the potential mechanisms underlying this phenomenon. Our results demonstrated that exogenous trypsin has no significant impact on PDCoV proliferation in human-derived cells. Furthermore, the culture supernatants collected from Huh7 cells can promote PDCoV infection in porcine-derived cells under trypsin-free conditions. By utilizing protease inhibitors, we identified that PDCoV enters Huh7 cells via endosomal and plasma membrane fusion pathways. Further investigations revealed that cathepsin L (CTSL) and transmembrane serine protease 11E (TMPRSS11E) mediate these pathways, respectively. Mechanistically, CTSL and TMPRSS11E cleave the PDCoV spike (S) protein, activating membrane fusion and promoting viral entry into Huh7 cells. Additionally, our results indicated that the highly expressed furin in Huh7 cells also plays a critical role in the late stages of PDCoV replication cycle, facilitating virion maturation and release. Taken together, these findings elucidate the detailed mechanisms of PDCoV infection in human-derived cells, highlighting the critical roles of CTSL and TMPRSS11E in trypsin-independent entry.</p><p><strong>Importance: </strong>PDCoV can be isolated from human plasma samples and infect various human-derived cells, raising significant concerns regarding its potential for cross-species transmission. Coronavirus invasion involves receptor binding and spike (S) protein cleavage by proteases. While human aminopeptidase N (APN) has been confirmed as a receptor that mediates PDCoV infection, the specific proteases involved in infections of human-derived cells remain incompletely understood. Here, we investigated the mechanisms by which PDCoV enters human-derived cells and demonstrated that PDCoV infection in these cells is independent of exogenous trypsin. Furthermore, we identified two critical proteases, CTSL and TMPRSS11E, which facilitate PDCoV entry into Huh7 cells via endosomal and plasma membrane fusion pathways, respectively. Additionally, we discovered that furin promotes the maturation and release of virions. This study reveals the infection mechanisms of PDCoV in human-derived cells, highlighting the roles of CTSL, TMPRSS11E, and furin in viral entry and release.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0105525"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12456011/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144816963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TGF-β inhibitor SB431542 suppresses SARS-CoV-2 replication through multistep inhibition.","authors":"Assim Verma, Himanshu Kamboj, Garvit Kumar, Nitin Khandelwal, Benjamin E Mayer, Jitender Rathee, Yogesh Chander, Alka Nokhwal, Shweta Dhanda, Ram Kumar, Ramesh Kumar Dedar, Sandeep Kumar Bejjanki, Deepti Parashar, Gayathri Pananghat, Bhupendra Nath Tripathi, Riyesh Thachamvally, Shalini Sharma, Naveen Kumar","doi":"10.1128/jvi.00529-25","DOIUrl":"10.1128/jvi.00529-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;The coronavirus disease 2019 (COVID-19) pandemic highlighted the critical need for broad-spectrum antivirals with high resistance barriers. Here, we demonstrate that SB431542, a selective TGF-β receptor I (ALK5) inhibitor, exhibits potent antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through unprecedented multitargeted mechanisms. Through comprehensive &lt;i&gt;in vitro&lt;/i&gt;, isothermal titration calorimetry, and &lt;i&gt;in silico&lt;/i&gt; analyses, we identified that SB431542 directly binds to SARS-CoV-2 ORF3a and disrupts its canonical function in inhibiting autophagosome-lysosome fusion. This interaction restored lysosomal acidification and normalized perinuclear LAMP-1 localization, significantly impairing virion assembly as evidenced by disrupted nucleocapsid-RNA association and reduced intracellular viral titers. Additionally, SB431542 downregulated the CLEAR network genes responsible for lysosomal biogenesis, further restricting viral egress pathways. Our temporal analyses revealed that at later infection stages (36-48 hours post-infection [hpi]), SARS-CoV-2 exploits TGF-β-induced lysosomal membrane permeabilization (LMP) and apoptosis for viral release-processes effectively inhibited by SB431542 through suppression of GADD45b and BAX expression. These multiple mechanisms resulted in an exceptional EC&lt;sub&gt;50&lt;/sub&gt; of 751.8 nM against SARS-CoV-2. &lt;i&gt;In vivo&lt;/i&gt; efficacy was demonstrated in embryonated chicken eggs, where SB431542 conferred dose-dependent protection against lethal infectious bronchitis virus (IBV) challenge, with a favorable therapeutic index of 34.54. Remarkably, sequential passaging of SARS-CoV-2 for 50 generations under SB431542 selection pressure failed to generate resistant variants, contrasting sharply with the rapid resistance emergence typical of direct-acting antivirals. These findings establish SB431542 as a promising broad-spectrum coronavirus inhibitor with a unique triple-mechanism approach that simultaneously targets viral entry via TGF-β/Smad modulation, disrupts ORF3a-mediated lysosomal dysfunction affecting assembly, and attenuates TGF-β-induced apoptosis during late-stage infection, collectively imposing multiple selective constraints that impede escape mutation development.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;The COVID-19 pandemic highlighted the urgent need for antiviral drugs with high barriers to resistance. This study reveals that SB431542, a drug previously developed to inhibit TGF-β signaling, exhibits remarkable effectiveness against SARS-CoV-2 through an unprecedented triple-mechanism approach. Unlike conventional antivirals that target a single viral component, SB431542 simultaneously disrupts viral entry, assembly, and release by binding to the viral ORF3a protein and modulating host cellular processes. Most importantly, SARS-CoV-2 failed to develop resistance against SB431542 even after 50 generations of exposure-a significant advantage over current therapeutics tha","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0052925"},"PeriodicalIF":3.8,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455996/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144958984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}