Pub Date : 2025-10-25DOI: 10.1016/j.antiviral.2025.106302
Daniel C. Sanford , Melicia R. Gainey , Cheryl Triplett , Irma M. Grossi , Scott A. Foster , Jeffry D. Shearer , Shantha Kodihalli
Brincidofovir (BCV, commercially known as Tembexa®) is an antiviral drug with broad-spectrum activity against orthopoxviruses. BCV was approved under the FDA Animal Rule for the treatment of human smallpox disease in adult and pediatric patients, including neonates. Rabbitpox (RPXV) and mousepox (ectromelia, ECTV) models were used to evaluate the survival benefit of oral BCV treatment. Progression of clinical disease, including clinical signs of disease, body weight change, body temperature, respiration rate (rabbits only) and viral load, was also compared in BCV-treated and placebo treated animals. In rabbits, oral BCV treatment was administered as 20, 5 and 5 mg/kg doses 48 h apart beginning on RPXV post-inoculation day (PID) 3, 4, 5, or 6. In mice, oral BCV treatment was administered as 10, 5 and 5 mg/kg doses 48 h apart beginning on ECTV PID 4, 5, or 6 or as 20, 5 and 5 mg/kg doses 48 h apart beginning on ECTV PID 4, 5, 6, or 7. BCV prevented death caused by RPXV or ECTV infection when treatment was initiated at the latest PID time point evaluated in each model. BCV treatment did not markedly affect clinical observations compared to placebo in either model. Viral load was reduced at the two earliest PID treatment points in rabbits. Viral load was not evaluated statistically in mice, however terminal liver viral loads tended to be higher with either dose regimen when BCV treatment was delayed. These studies formed the basis for approval of BCV by the FDA Animal Rule.
{"title":"Pivotal animal efficacy studies supporting brincidofovir licensure under the FDA animal rule","authors":"Daniel C. Sanford , Melicia R. Gainey , Cheryl Triplett , Irma M. Grossi , Scott A. Foster , Jeffry D. Shearer , Shantha Kodihalli","doi":"10.1016/j.antiviral.2025.106302","DOIUrl":"10.1016/j.antiviral.2025.106302","url":null,"abstract":"<div><div>Brincidofovir (BCV, commercially known as Tembexa®) is an antiviral drug with broad-spectrum activity against orthopoxviruses. BCV was approved under the FDA Animal Rule for the treatment of human smallpox disease in adult and pediatric patients, including neonates. Rabbitpox (RPXV) and mousepox (ectromelia, ECTV) models were used to evaluate the survival benefit of oral BCV treatment. Progression of clinical disease, including clinical signs of disease, body weight change, body temperature, respiration rate (rabbits only) and viral load, was also compared in BCV-treated and placebo treated animals. In rabbits, oral BCV treatment was administered as 20, 5 and 5 mg/kg doses 48 h apart beginning on RPXV post-inoculation day (PID) 3, 4, 5, or 6. In mice, oral BCV treatment was administered as 10, 5 and 5 mg/kg doses 48 h apart beginning on ECTV PID 4, 5, or 6 or as 20, 5 and 5 mg/kg doses 48 h apart beginning on ECTV PID 4, 5, 6, or 7. BCV prevented death caused by RPXV or ECTV infection when treatment was initiated at the latest PID time point evaluated in each model. BCV treatment did not markedly affect clinical observations compared to placebo in either model. Viral load was reduced at the two earliest PID treatment points in rabbits. Viral load was not evaluated statistically in mice, however terminal liver viral loads tended to be higher with either dose regimen when BCV treatment was delayed. These studies formed the basis for approval of BCV by the FDA Animal Rule.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106302"},"PeriodicalIF":4.0,"publicationDate":"2025-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145413770","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}
Pub Date : 2025-10-25DOI: 10.1016/j.antiviral.2025.106301
Min Wei , Rumeng Ma , Xiaoyang Wang , Chunmei Wang , Mi Wang , Han Bai , Wenchong Ye , Wen Zhou , Keyu Zhang
Japanese encephalitis virus (JEV) is the primary causative agent of viral encephalitis, and Nitazoxanide (NTZ) has been shown to exhibit potent antiviral activity against JEV both in vitro and in vivo. Previous studies have demonstrated that NTZ modulates the expression of succinyl-CoA synthetase GDP forming subunit β (SUCLG2), a GTP-specific succinyl-CoA synthetase, in JEV-infected cells. However, the functional role and underlying mechanism of SUCLG2 in NTZ-mediated antiviral effects against JEV remain poorly understood. In this study, we found that SiRNA-mediated knockdown of SUCLG2 significantly enhanced JEV proliferation in BHK-21 cells, whereas overexpression of SUCLG2 markedly suppressed viral replication. Moreover, SUCLG2 overexpression attenuated ROS production induced by JEV infection and promoted ATP recovery. Furthermore, SUCLG2 activated innate immunity by enhancing IFN-β expression. Additionally, our findings indicate that SUCLG2 exhibits relatively weak direct binding affinity with NTZ. In conclusion, our results demonstrate that SUCLG2 plays a critical role in mediating the antiviral activity of NTZ against JEV, providing novel insights into the molecular mechanisms underlying NTZ's antiviral efficacy.
{"title":"SUCLG2 plays a crucial role in the activity of Nitazoxanide against Japanese encephalitis virus","authors":"Min Wei , Rumeng Ma , Xiaoyang Wang , Chunmei Wang , Mi Wang , Han Bai , Wenchong Ye , Wen Zhou , Keyu Zhang","doi":"10.1016/j.antiviral.2025.106301","DOIUrl":"10.1016/j.antiviral.2025.106301","url":null,"abstract":"<div><div>Japanese encephalitis virus (JEV) is the primary causative agent of viral encephalitis, and Nitazoxanide (NTZ) has been shown to exhibit potent antiviral activity against JEV both in vitro and in vivo. Previous studies have demonstrated that NTZ modulates the expression of succinyl-CoA synthetase GDP forming subunit β (SUCLG2), a GTP-specific succinyl-CoA synthetase, in JEV-infected cells. However, the functional role and underlying mechanism of SUCLG2 in NTZ-mediated antiviral effects against JEV remain poorly understood. In this study, we found that SiRNA-mediated knockdown of SUCLG2 significantly enhanced JEV proliferation in BHK-21 cells, whereas overexpression of SUCLG2 markedly suppressed viral replication. Moreover, SUCLG2 overexpression attenuated ROS production induced by JEV infection and promoted ATP recovery. Furthermore, SUCLG2 activated innate immunity by enhancing IFN-β expression. Additionally, our findings indicate that SUCLG2 exhibits relatively weak direct binding affinity with NTZ. In conclusion, our results demonstrate that SUCLG2 plays a critical role in mediating the antiviral activity of NTZ against JEV, providing novel insights into the molecular mechanisms underlying NTZ's antiviral efficacy.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106301"},"PeriodicalIF":4.0,"publicationDate":"2025-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145413769","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}
Pub Date : 2025-10-21DOI: 10.1016/j.antiviral.2025.106300
Mélissa Bessonne , Jessica Morel , Quentin Nevers , Julie Groutsch , Agathe Urvoas , Marie Valerio-Lepiniec , Thibaut Crépin , Philippe Minard , Bernard Delmas
Seasonal epidemics and pandemics caused by influenza A viruses still represent a main public health burden in the world. Influenza viruses replicate and transcribe their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol) through extensive structural rearrangements and differential interactions with host cell factors. To get insights into its functioning, we screened a phage-display library of biosynthetic proteins (named αReps and built on a rigid alpha-helicoidal HEAT-like scaffold) against the structurally invariant FluPol core and several flexibly-linked domains of the FluPol PB2 subunit. Several αReps specific of the cap binding domain [CBD], the 627-domain and the NLS domain of PB2 displayed FluPol inhibitory and virus neutralization activities when transiently expressed in the cytosol. Furthermore, intracellular ectopic inducible expression of the αReps C3 and F3 (specific of the CBD and the 627-domain, respectively) in influenza virus permissive cells blocked multiplication of viruses representative of the H1N1, H3N2 and H7N1 subtypes, even when induced at late times post-infection. Bispecific αReps constructs (C3-F3 and F3-C3) display a higher FluPol inhibitory activity than their monomeric counterparts. These results suggest that interfering with FluPol structural rearrangements may represent a promising strategy to block virus multiplication and to design new types of antivirals such as dual binders targeting distant sites on FluPol. Furthermore, we found that the 627-domain constitutes a new possible target for engineering influenza antivirals.
{"title":"Inhibition of influenza virus replication by artificial proteins (αReps) targeting its RNA-polymerase","authors":"Mélissa Bessonne , Jessica Morel , Quentin Nevers , Julie Groutsch , Agathe Urvoas , Marie Valerio-Lepiniec , Thibaut Crépin , Philippe Minard , Bernard Delmas","doi":"10.1016/j.antiviral.2025.106300","DOIUrl":"10.1016/j.antiviral.2025.106300","url":null,"abstract":"<div><div>Seasonal epidemics and pandemics caused by influenza A viruses still represent a main public health burden in the world. Influenza viruses replicate and transcribe their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol) through extensive structural rearrangements and differential interactions with host cell factors. To get insights into its functioning, we screened a phage-display library of biosynthetic proteins (named αReps and built on a rigid alpha-helicoidal HEAT-like scaffold) against the structurally invariant FluPol core and several flexibly-linked domains of the FluPol PB2 subunit. Several αReps specific of the cap binding domain [CBD], the 627-domain and the NLS domain of PB2 displayed FluPol inhibitory and virus neutralization activities when transiently expressed in the cytosol. Furthermore, intracellular ectopic inducible expression of the αReps C3 and F3 (specific of the CBD and the 627-domain, respectively) in influenza virus permissive cells blocked multiplication of viruses representative of the H1N1, H3N2 and H7N1 subtypes, even when induced at late times post-infection. Bispecific αReps constructs (C3-F3 and F3-C3) display a higher FluPol inhibitory activity than their monomeric counterparts. These results suggest that interfering with FluPol structural rearrangements may represent a promising strategy to block virus multiplication and to design new types of antivirals such as dual binders targeting distant sites on FluPol. Furthermore, we found that the 627-domain constitutes a new possible target for engineering influenza antivirals.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106300"},"PeriodicalIF":4.0,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145353594","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}
Pub Date : 2025-10-12DOI: 10.1016/j.antiviral.2025.106291
Juncheng Yang , Guifeng Yang , Haitang He , Hai Liu , Qunfang Fu , Xiaoting Wu , Ran Meng , Zhuoyue Li , Qian Zhao , Kangxian Luo , Zhihua Liu
Background & aims
To evaluate baseline HBV quasispecies (QS) characteristics as a predictor of HBsAg seroconversion in peginterferon-alfa-2a-treated HBeAg-positive pediatric chronic hepatitis B (CHB).
Methods
25 patients achieved HBsAg seroconversion (SS group) and another 25 did not (SN group) were enrolled, matched for baseline profiles. Full-length HBV genomes were obtained from pretreatment serum via clone sequencing. Twelve untreated carriers (CA group) served as controls.
Results
SN group demonstrated significantly higher genetic distance (GD) and mutation frequency index (MFI) in the preCore/Core (preC/C) gene versus SS and CA groups (p < 0.05). Treatment failure exclusively correlated with G1896A/C1913A/C2078G mutations. Jonckheere-Terpstra test confirmed a CA→SS→SN diversity gradient in preC/C (p < 0.05). Non-invasive fibrosis indices increased across CA→SS→SN (p < 0.05) and correlated positively with QS diversity. Evolutionary analyses revealed divergent haplotype clustering in SN versus star-like radiation patterns in CA/SS. Phylogenetic analysis positioned CA/SS haplotypes at earlier evolutionary phases, while SN haplotypes at a later phase. Notably, haplotypes harboring the three aforementioned mutations were also located at the later phase. Positive selection analysis identified two SN-specific mutations (codon 34[C1913A] and 89[C2078G]). A LASSO-derived predictive model (age, C1913A/C2078G, preC/C MFI) yielded the area under the receiver operating characteristic curve (AUROC) 0.814 (sensitivity 92 %, specificity 68 %).
Conclusions
Lower baseline HBV quasispecies diversity predicts Peginterferon-alfa-2a-induced HBsAg seroconversion in pediatric CHB. The observed diversity continuum, association with fibrosis indices, suggests seroconversion occurs preferentially during earlier hepatitis phases. Pretreatment viral genetic profiling may optimize therapeutic timing, highlighting its prognostic value for childhood HBV management.
{"title":"Pretreatment viral quasispecies characteristics and evolutionary phases correlate with HBsAg seroconversion in peginterferon-alfa-2a-treated children with HBeAg-positive chronic hepatitis B","authors":"Juncheng Yang , Guifeng Yang , Haitang He , Hai Liu , Qunfang Fu , Xiaoting Wu , Ran Meng , Zhuoyue Li , Qian Zhao , Kangxian Luo , Zhihua Liu","doi":"10.1016/j.antiviral.2025.106291","DOIUrl":"10.1016/j.antiviral.2025.106291","url":null,"abstract":"<div><h3>Background & aims</h3><div>To evaluate baseline HBV quasispecies (QS) characteristics as a predictor of HBsAg seroconversion in peginterferon-alfa-2a-treated HBeAg-positive pediatric chronic hepatitis B (CHB).</div></div><div><h3>Methods</h3><div>25 patients achieved HBsAg seroconversion (SS group) and another 25 did not (SN group) were enrolled, matched for baseline profiles. Full-length HBV genomes were obtained from pretreatment serum via clone sequencing. Twelve untreated carriers (CA group) served as controls.</div></div><div><h3>Results</h3><div>SN group demonstrated significantly higher genetic distance (GD) and mutation frequency index (MFI) in the preCore/Core (preC/C) gene versus SS and CA groups (p < 0.05). Treatment failure exclusively correlated with G1896A/C1913A/C2078G mutations. Jonckheere-Terpstra test confirmed a CA→SS→SN diversity gradient in preC/C (p < 0.05). Non-invasive fibrosis indices increased across CA→SS→SN (p < 0.05) and correlated positively with QS diversity. Evolutionary analyses revealed divergent haplotype clustering in SN versus star-like radiation patterns in CA/SS. Phylogenetic analysis positioned CA/SS haplotypes at earlier evolutionary phases, while SN haplotypes at a later phase. Notably, haplotypes harboring the three aforementioned mutations were also located at the later phase. Positive selection analysis identified two SN-specific mutations (codon 34[C1913A] and 89[C2078G]). A LASSO-derived predictive model (age, C1913A/C2078G, preC/C MFI) yielded the area under the receiver operating characteristic curve (AUROC) 0.814 (sensitivity 92 %, specificity 68 %).</div></div><div><h3>Conclusions</h3><div>Lower baseline HBV quasispecies diversity predicts Peginterferon-alfa-2a-induced HBsAg seroconversion in pediatric CHB. The observed diversity continuum, association with fibrosis indices, suggests seroconversion occurs preferentially during earlier hepatitis phases. Pretreatment viral genetic profiling may optimize therapeutic timing, highlighting its prognostic value for childhood HBV management.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106291"},"PeriodicalIF":4.0,"publicationDate":"2025-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145290807","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}
Pub Date : 2025-10-11DOI: 10.1016/j.antiviral.2025.106299
Mira C. Patel , Ha T. Nguyen , Vasiliy P. Mishin , Philippe Noriel Q. Pascua , Chloe Champion , Mercedes Lopez-Esteva , Angiezel Merced-Morales , Alicia Budd , Marie K. Kirby , Benjamin Rambo-Martin , Jennifer Laplante , Allen Bateman , Kirsten St. George , Maureen Sullivan , John Steel , Rebecca J. Kondor , Larisa V. Gubareva
Antiviral susceptibility monitoring is integral to influenza surveillance conducted by CDC in collaboration with partners. Here, we outlined the algorithm and methods used for assessing antiviral susceptibility of viruses collected during 2023–2024 season. Virus specimens were provided by public health laboratories in the United States (US) and by laboratories in other countries that belong to the Pan American Health Organization. In the US, antiviral susceptibility surveillance conducted nationally is strengthened by sequence-only analysis of additional viruses collected at a state level. Viral genome sequence analysis was the primary approach to assess susceptibility to M2 blockers (n = 5123), neuraminidase (NA) inhibitors (n = 6874), and a polymerase acidic protein (PA) inhibitor (baloxavir, n = 6567). Over 99 % of type A viruses had M2-S31N that confers resistance to M2 blockers. Although oseltamivir-resistant viruses carrying NA-H275Y (N1 numbering) were rare (0.35 %), a cluster of four such viruses was identified in Haiti. Viruses with other NA mutations conferring reduced inhibition by NA inhibitor(s) were also detected sporadically. This includes a cluster of three influenza B viruses in Texas that shared a new mutation, NA-A245G conferring reduced inhibition by peramivir. Three viruses with reduced baloxavir susceptibility were identified, which had PA-I38T, PA-Y24C or PA-V122A; the latter two new mutations identified through augmented approach to sequence analysis. To monitor baseline susceptibility, supplementary in vitro testing was conducted on approximately 7 % of viruses using NA inhibition assay and cell culture-based assay IRINA. Implementation of Sequence First approach provided comprehensive and high throughput methodology for antiviral susceptibility assessment and reduced redundant phenotypic testing.
{"title":"Antiviral susceptibility monitoring: testing algorithm, methods, and findings for influenza season, 2023–2024","authors":"Mira C. Patel , Ha T. Nguyen , Vasiliy P. Mishin , Philippe Noriel Q. Pascua , Chloe Champion , Mercedes Lopez-Esteva , Angiezel Merced-Morales , Alicia Budd , Marie K. Kirby , Benjamin Rambo-Martin , Jennifer Laplante , Allen Bateman , Kirsten St. George , Maureen Sullivan , John Steel , Rebecca J. Kondor , Larisa V. Gubareva","doi":"10.1016/j.antiviral.2025.106299","DOIUrl":"10.1016/j.antiviral.2025.106299","url":null,"abstract":"<div><div>Antiviral susceptibility monitoring is integral to influenza surveillance conducted by CDC in collaboration with partners. Here, we outlined the algorithm and methods used for assessing antiviral susceptibility of viruses collected during 2023–2024 season. Virus specimens were provided by public health laboratories in the United States (US) and by laboratories in other countries that belong to the Pan American Health Organization. In the US, antiviral susceptibility surveillance conducted nationally is strengthened by sequence-only analysis of additional viruses collected at a state level. Viral genome sequence analysis was the primary approach to assess susceptibility to M2 blockers (n = 5123), neuraminidase (NA) inhibitors (n = 6874), and a polymerase acidic protein (PA) inhibitor (baloxavir, n = 6567). Over 99 % of type A viruses had M2-S31N that confers resistance to M2 blockers. Although oseltamivir-resistant viruses carrying NA-H275Y (N1 numbering) were rare (0.35 %), a cluster of four such viruses was identified in Haiti. Viruses with other NA mutations conferring reduced inhibition by NA inhibitor(s) were also detected sporadically. This includes a cluster of three influenza B viruses in Texas that shared a new mutation, NA-A245G conferring reduced inhibition by peramivir. Three viruses with reduced baloxavir susceptibility were identified, which had PA-I38T, PA-Y24C or PA-V122A; the latter two new mutations identified through augmented approach to sequence analysis. To monitor baseline susceptibility, supplementary <em>in vitro</em> testing was conducted on approximately 7 % of viruses using NA inhibition assay and cell culture-based assay IRINA. Implementation of Sequence First approach provided comprehensive and high throughput methodology for antiviral susceptibility assessment and reduced redundant phenotypic testing.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106299"},"PeriodicalIF":4.0,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145285446","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}
Pub Date : 2025-10-11DOI: 10.1016/j.antiviral.2025.106298
Priscila Sutto-Ortiz , Barbara Selisko , François Ferron , Jean-Pierre Sommadossi , Adel Moussa , Steven Good , Bruno Canard , Etienne Decroly
Human respiratory syncytial virus (RSV) causes pediatric bronchiolitis and severe respiratory illness in the elderly. Despite recent advancements in vaccines and antibody therapies, the search for antiviral agents remains a significant public health challenge. We designed nucleotide analogs (NAs) with ribose modifications to assess their incorporation by the RSV polymerase. Biochemical assays and structural modelling revealed that these NAs effectively disrupt RNA synthesis elongation. They act as chain-terminators via a unique mechanism mediated by the 4′-modification, whereas 2′-F alone has no effect and 1′-modification slows-down RNA synthesis. We evaluated the ability of the polymerase to discriminate between natural nucleotides and NAs through incorporation efficiency/competitive assays, correlating these findings with RSV replication inhibition in infected cell cultures. Our ranking of compounds indicates that cytidine analogs demonstrate the strongest antiviral activity, due to their phosphorylation efficiency and intracellular concentration relative to natural nucleotides as well as their ready incorporation into the growing RNA chain. 4′-modifications are accepted by the RSV polymerase due to structural differences between the active sites of (+) and (−) RNA virus polymerases.
{"title":"Mapping the impact of 1′-, 2′- and 4′-nucleotide modifications on the Respiratory Syncytial Virus RNA-dependent RNA polymerase","authors":"Priscila Sutto-Ortiz , Barbara Selisko , François Ferron , Jean-Pierre Sommadossi , Adel Moussa , Steven Good , Bruno Canard , Etienne Decroly","doi":"10.1016/j.antiviral.2025.106298","DOIUrl":"10.1016/j.antiviral.2025.106298","url":null,"abstract":"<div><div>Human respiratory syncytial virus (RSV) causes pediatric bronchiolitis and severe respiratory illness in the elderly. Despite recent advancements in vaccines and antibody therapies, the search for antiviral agents remains a significant public health challenge. We designed nucleotide analogs (NAs) with ribose modifications to assess their incorporation by the RSV polymerase. Biochemical assays and structural modelling revealed that these NAs effectively disrupt RNA synthesis elongation. They act as chain-terminators via a unique mechanism mediated by the 4′-modification, whereas 2′-F alone has no effect and 1′-modification slows-down RNA synthesis. We evaluated the ability of the polymerase to discriminate between natural nucleotides and NAs through incorporation efficiency/competitive assays, correlating these findings with RSV replication inhibition in infected cell cultures. Our ranking of compounds indicates that cytidine analogs demonstrate the strongest antiviral activity, due to their phosphorylation efficiency and intracellular concentration relative to natural nucleotides as well as their ready incorporation into the growing RNA chain. 4′-modifications are accepted by the RSV polymerase due to structural differences between the active sites of (+) and (−) RNA virus polymerases.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106298"},"PeriodicalIF":4.0,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145285456","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}
Pub Date : 2025-10-09DOI: 10.1016/j.antiviral.2025.106288
Timothy M. Block, Dimitar Gotchev, Yanming Du
Small-molecule HBV RNA destabilizing agents, such as the dihydroquinolizinones (DHQs), were first disclosed in a patent filing in 2015 and in peer reviewed literature in 2018. These compounds inhibit Poly-adenylating Polymerases 5 and 7 (PAPD5/7) and represent a novel antiviral strategy and their ability to degrade hepatitis B surface antigen (HBsAg) in cell culture and animal models generated considerable excitement and commercial interest. However, extrahepatic toxicity observed in preclinical and Phase I studies led to the discontinuation of several development programs. The subsequent emergence of liver-targeted PAPD5/7 inhibitors with improved safety profiles has rekindled interest in this therapeutic approach. Yet, with the apparent success of other investigational antivirals in reducing HBsAg levels, such as siRNAs, antisense oligonucleotides, and in at least one example, capsid assembly modulators (CAMs), questions remain as to whether RNA destabilizers still have a role in managing chronic hepatitis B (CHB). This review describes the current status of PAPD5/7 inhibitor development, evaluates the advantages and limitations of the approach, and considers potential strategies for integrating this class of molecules with other HBV therapies.
{"title":"Small molecule HBV RNA destabilizing drugs: Drugs of the future or compounds from the past?","authors":"Timothy M. Block, Dimitar Gotchev, Yanming Du","doi":"10.1016/j.antiviral.2025.106288","DOIUrl":"10.1016/j.antiviral.2025.106288","url":null,"abstract":"<div><div>Small-molecule HBV RNA destabilizing agents, such as the dihydroquinolizinones (DHQs), were first disclosed in a patent filing in 2015 and in peer reviewed literature in 2018. These compounds inhibit Poly-adenylating Polymerases 5 and 7 (PAPD5/7) and represent a novel antiviral strategy and their ability to degrade hepatitis B surface antigen (HBsAg) in cell culture and animal models generated considerable excitement and commercial interest. However, extrahepatic toxicity observed in preclinical and Phase I studies led to the discontinuation of several development programs. The subsequent emergence of liver-targeted PAPD5/7 inhibitors with improved safety profiles has rekindled interest in this therapeutic approach. Yet, with the apparent success of other investigational antivirals in reducing HBsAg levels, such as siRNAs, antisense oligonucleotides, and in at least one example, capsid assembly modulators (CAMs), questions remain as to whether RNA destabilizers still have a role in managing chronic hepatitis B (CHB). This review describes the current status of PAPD5/7 inhibitor development, evaluates the advantages and limitations of the approach, and considers potential strategies for integrating this class of molecules with other HBV therapies.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106288"},"PeriodicalIF":4.0,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145257217","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}
Pub Date : 2025-10-09DOI: 10.1016/j.antiviral.2025.106290
Anil Pant , Djamal Brahim Belhaouari , Lara Dsouza , D.M. Nirosh Udayanga , Zhengqiang Wang , Zhilong Yang
Poxviruses remain a significant global health concern, necessitating the development of novel antiviral strategies. Through high-throughput screening, we previously identified ciclopirox (CPX), an FDA-approved antifungal, as a hit that inhibits vaccinia virus (VACV) replication. Here, we further characterized its antiviral activity and mechanism of action using human primary fibroblasts. CPX significantly reduced VACV titers without reducing host cell viability, with an EC50 in the sub-micromolar range and a CC50 > 500 μM. Rescue experiments demonstrated that CPX inhibits viral replication primarily through chelation of intracellular Fe3+ and, to a lesser extent, Fe2+, as evidenced by partial restoration of viral replication with ferric ammonium citrate supplementation. Furthermore, overexpression of the iron-dependent enzymes RRM2 and the VACV-encoded F4L reduced the inhibitory effect of CPX, indicating that these host and viral proteins are affected by CPX treatment. Moreover, CPX treatment suppressed cowpox virus and monkeypox (mpox) virus replication in vitro. It also reduced VACV titers in ex vivo mouse lung tissue. These findings highlight host iron metabolism as a critical determinant of poxvirus replication and identify CPX as a promising antiviral candidate against multiple orthopoxviruses.
{"title":"Ciclopirox suppresses poxvirus replication by targeting iron metabolism","authors":"Anil Pant , Djamal Brahim Belhaouari , Lara Dsouza , D.M. Nirosh Udayanga , Zhengqiang Wang , Zhilong Yang","doi":"10.1016/j.antiviral.2025.106290","DOIUrl":"10.1016/j.antiviral.2025.106290","url":null,"abstract":"<div><div>Poxviruses remain a significant global health concern, necessitating the development of novel antiviral strategies. Through high-throughput screening, we previously identified ciclopirox (CPX), an FDA-approved antifungal, as a hit that inhibits vaccinia virus (VACV) replication. Here, we further characterized its antiviral activity and mechanism of action using human primary fibroblasts. CPX significantly reduced VACV titers without reducing host cell viability, with an EC<sub>50</sub> in the sub-micromolar range and a CC<sub>50</sub> > 500 μM. Rescue experiments demonstrated that CPX inhibits viral replication primarily through chelation of intracellular Fe<sup>3+</sup> and, to a lesser extent, Fe<sup>2+</sup>, as evidenced by partial restoration of viral replication with ferric ammonium citrate supplementation. Furthermore, overexpression of the iron-dependent enzymes RRM2 and the VACV-encoded F4L reduced the inhibitory effect of CPX, indicating that these host and viral proteins are affected by CPX treatment. Moreover, CPX treatment suppressed cowpox virus and monkeypox (mpox) virus replication <em>in vitro</em>. It also reduced VACV titers in <em>ex vivo</em> mouse lung tissue. These findings highlight host iron metabolism as a critical determinant of poxvirus replication and identify CPX as a promising antiviral candidate against multiple orthopoxviruses.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106290"},"PeriodicalIF":4.0,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145257245","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}
Pub Date : 2025-10-09DOI: 10.1016/j.antiviral.2025.106289
C. Gourin , T. Flores , C. Lefèvre , S. Alain , G. Ligat , S. Hantz
Human cytomegalovirus is an opportunistic pathogen responsible for severe infections in immunocompromised patients, the leading cause of congenital infections worldwide, and potentially implicated in carcinogenesis. The HCMV terminase complex (pUL56-pUL89-pUL51) has emerged as a key target for antiviral drug development. Letermovir, an antiviral agent targeting this complex, inhibits viral DNA packaging, but resistance-associated mutations have already been identified within subunits. Moreover, the precise mechanism of action of letermovir remains incompletely understood.
We investigated interactions among terminase subunits in presence or absence of letermovir. Wild-type and mutant forms of these proteins (including resistance mutations V236M, L241P, L257I, C325Y, R369M in pUL56 and A95V in pUL51) were cloned into NanoBiT® PPI and pCI-neo vectors. Letermovir was added after transfection in HEK293T cells, and protein-protein interactions were assessed.
Our results show letermovir does not disrupt interactions between wild-type terminase subunits. Resistance-associated mutations modulate the strength of these interactions, with certain mutations (such as pUL56 V236M and L257I) significantly enhancing or reducing binding. Notably, double mutants exhibited synergistic effects. Structural analysis using the AlphaFold3 platform revealed differences between the mutation site of pUL56 and its HSV-1 counterpart pUL28. A hypothetical 3D analysis based on the cryo-EM structure of the HSV-1 terminase complex showed that resistance mutations were oriented outside the complex.
These findings suggest letermovir does not act by directly inhibiting interactions among HCMV terminase subunits. Analysis of resistance-associated mutations provides insight into the molecular basis of HCMV resistance to letermovir and may inform development of novel antiviral strategies targeting the terminase complex.
{"title":"Deciphering letermovir's mode of action and resistance mutation effects","authors":"C. Gourin , T. Flores , C. Lefèvre , S. Alain , G. Ligat , S. Hantz","doi":"10.1016/j.antiviral.2025.106289","DOIUrl":"10.1016/j.antiviral.2025.106289","url":null,"abstract":"<div><div>Human cytomegalovirus is an opportunistic pathogen responsible for severe infections in immunocompromised patients, the leading cause of congenital infections worldwide, and potentially implicated in carcinogenesis. The HCMV terminase complex (pUL56-pUL89-pUL51) has emerged as a key target for antiviral drug development. Letermovir, an antiviral agent targeting this complex, inhibits viral DNA packaging, but resistance-associated mutations have already been identified within subunits. Moreover, the precise mechanism of action of letermovir remains incompletely understood.</div><div>We investigated interactions among terminase subunits in presence or absence of letermovir. Wild-type and mutant forms of these proteins (including resistance mutations V236M, L241P, L257I, C325Y, R369M in pUL56 and A95V in pUL51) were cloned into NanoBiT® PPI and pCI-neo vectors. Letermovir was added after transfection in HEK293T cells, and protein-protein interactions were assessed.</div><div>Our results show letermovir does not disrupt interactions between wild-type terminase subunits. Resistance-associated mutations modulate the strength of these interactions, with certain mutations (such as pUL56 V236M and L257I) significantly enhancing or reducing binding. Notably, double mutants exhibited synergistic effects. Structural analysis using the AlphaFold3 platform revealed differences between the mutation site of pUL56 and its HSV-1 counterpart pUL28. A hypothetical 3D analysis based on the cryo-EM structure of the HSV-1 terminase complex showed that resistance mutations were oriented outside the complex.</div><div>These findings suggest letermovir does not act by directly inhibiting interactions among HCMV terminase subunits. Analysis of resistance-associated mutations provides insight into the molecular basis of HCMV resistance to letermovir and may inform development of novel antiviral strategies targeting the terminase complex.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106289"},"PeriodicalIF":4.0,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145257234","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}
Pub Date : 2025-10-08DOI: 10.1016/j.antiviral.2025.106287
Bonan Lv , Xingran Wang , Ying Zhou , Zihan Su , Yidan Sun , Yingying Yang , Yang Lu , Zishu Pan , Xiao-Feng Tang , Chao Shen
Positive-sense RNA virus infections induce vesicle formation within host cells to support RNA replication. By extracting and purifying foot-and-mouth disease virus (FMDV) replication complexes and comparing host cells and replication complexes through targeted lipidomics and proteomics analyses, we found that FMDV enriches host cell proteins and polyunsaturated fatty acids in viral replication complexes (VRCs) to facilitate their formation. On the basis of these findings, we propose a model in which VRCs progress from single-membrane vesicles to multi-membrane vesicles (MMVs) during FMDV replication, a process that requires coordinated contributions of host cell proteins and organelle membranes derived from multiple organelles. Our study showed that, as infection advances, FMDV converts single-membrane vesicles (SMVs) into MMVs, which aggregate to expand the surface area of the replication platform and enhance replication efficiency. These membrane structures function in FMDV replication; the endoplasmic reticulum undergoes curling and folding to support VRC assembly. Additionally, some VRCs possess outward-facing openings that permit material exchange. These findings reveal unexpected similarities between FMDV and distantly related positive-strand RNA viruses, suggesting that shared host cellular pathways are exploited to construct membrane-bound replication factories.
{"title":"Composition, three-dimensional structure and formation mechanism of the foot and mouth disease virus replication complexes","authors":"Bonan Lv , Xingran Wang , Ying Zhou , Zihan Su , Yidan Sun , Yingying Yang , Yang Lu , Zishu Pan , Xiao-Feng Tang , Chao Shen","doi":"10.1016/j.antiviral.2025.106287","DOIUrl":"10.1016/j.antiviral.2025.106287","url":null,"abstract":"<div><div>Positive-sense RNA virus infections induce vesicle formation within host cells to support RNA replication. By extracting and purifying foot-and-mouth disease virus (FMDV) replication complexes and comparing host cells and replication complexes through targeted lipidomics and proteomics analyses, we found that FMDV enriches host cell proteins and polyunsaturated fatty acids in viral replication complexes (VRCs) to facilitate their formation. On the basis of these findings, we propose a model in which VRCs progress from single-membrane vesicles to multi-membrane vesicles (MMVs) during FMDV replication, a process that requires coordinated contributions of host cell proteins and organelle membranes derived from multiple organelles. Our study showed that, as infection advances, FMDV converts single-membrane vesicles (SMVs) into MMVs, which aggregate to expand the surface area of the replication platform and enhance replication efficiency. These membrane structures function in FMDV replication; the endoplasmic reticulum undergoes curling and folding to support VRC assembly. Additionally, some VRCs possess outward-facing openings that permit material exchange. These findings reveal unexpected similarities between FMDV and distantly related positive-strand RNA viruses, suggesting that shared host cellular pathways are exploited to construct membrane-bound replication factories.</div></div>","PeriodicalId":8259,"journal":{"name":"Antiviral research","volume":"244 ","pages":"Article 106287"},"PeriodicalIF":4.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145273387","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}
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