The review, led by Muhammad Hassan Saeed and colleagues working across institutions in Pakistan and Somalia, was conducted in accordance with the PRISMA 2020 reporting guideline and prospectively registered in PROSPERO (CRD420251137112). The investigators searched PubMed/MEDLINE, Embase, Scopus, the Cochrane Library, Google Scholar, and ClinicalTrials.gov, completing the original searches in August 2025 and re-searching all information sources on September 21, 2026, to capture any newly published eligible trials. Their objective was precise: to compare the efficacy and safety of bulevirtide monotherapy against bulevirtide combined with Peg-IFNα-2a in adults with chronic hepatitis D. Only randomized controlled trials directly comparing the two strategies were eligible, ensuring that the synthesis rested on the highest tier of interventional evidence rather than observational cohorts or single-arm studies.

The methodological rigor of the analysis deserves particular attention because it reflects a broader shift in how meta-analysts handle small trials. The primary analyses used restricted maximum likelihood (REML) estimation with the Hartung–Knapp–Sidik–Jonkman (HKSJ) adjustment, a random-effects approach that widens confidence intervals when the number of studies is small and between-trial heterogeneity is uncertain, thereby guarding against the overconfident estimates that conventional methods can produce. As sensitivity analyses, the team also ran DerSimonian–Laird (DL) random-effects models with conventional normal-approximation confidence intervals. Outcomes synthesized included trial-defined virological, biochemical, and combined responses, as well as continuous changes in HDV RNA and alanine aminotransferase (ALT), the liver enzyme that serves as a surrogate marker for inflammatory activity. Standardized mean differences were expressed as Hedges’ g, and binary outcomes as risk ratios (RR).

Three randomized controlled trials comprising 225 patients met the inclusion criteria. At week 24, virological response was numerically higher with the combination regimen, but the difference was not statistically significant under the primary analysis (RR 2.07, 95% CI 0.38–11.40), and combined response showed essentially no difference (RR 1.01, 95% CI 0.12–8.48). Combination therapy did, however, produce a significantly greater reduction in HDV RNA (Hedges’ g −0.96, 95% CI −1.75 to −0.18), indicating deeper viral suppression during the first six months of treatment. In contrast, biochemical response favored bulevirtide monotherapy (RR 0.36, 95% CI 0.17–0.78), as did the ALT change (mean difference 45.58 U/L, 95% CI 33.51–57.65), meaning that patients on the combination were less likely to achieve normalized liver enzymes while on treatment.

The picture changed at week 48. By that point, virological response clearly favored the combination regimen (RR 5.33, 95% CI 1.57–18.12), suggesting that the interferon-containing strategy delivers superior viral control with longer exposure, even though biochemical and combined responses did not differ significantly between the groups at this time point. The divergence between viral and biochemical endpoints is scientifically noteworthy: HDV RNA suppression and ALT normalization do not always move in lockstep, and the mechanisms underlying this dissociation—whether interferon’s immunomodulatory effects, bulevirtide’s entry-blocking pharmacodynamics, or trial-specific response definitions—remain an open question for hepatologists designing future trials.

Post-treatment durability emerged as another key dimension. At 24 weeks after the end of treatment (EOT), combined response favored combination therapy (RR 4.15, 95% CI 1.05–16.40), while the virological and biochemical response estimates remained imprecise, with confidence intervals too wide to support firm conclusions. Sustained off-treatment response is the ultimate goal in chronic hepatitis D, since relapse after stopping therapy is common, and the finding that the combination arm maintained an advantage in combined endpoints at this late stage will inform discussions about treatment duration and discontinuation strategies. Nevertheless, the authors emphasize that these longer-term estimates rested on only two randomized trials, limiting the reliability of the post-treatment synthesis.

Safety signals tracked the known toxicity profile of interferon. Regimens containing Peg-IFNα-2a were associated with higher rates of neutropenia and thrombocytopenia, the hematologic adverse effects that have long constrained interferon use in viral hepatitis. Bulevirtide itself has generally been well tolerated in clinical practice, with the most frequently reported adverse effect being injection-site reactions and, in some studies, transient elevations in bile acids. The meta-analysis thus frames the central clinical trade-off: deeper viral suppression and better sustained combined responses with the combination, at the cost of interferon-related hematologic toxicity and less favorable on-treatment liver enzyme responses.

A technically important caveat concerns the statistical method itself. The DL sensitivity analyses yielded narrower confidence intervals for several outcomes and changed the statistical significance of some longer-term estimates, illustrating how method choice can alter conclusions in meta-analyses built on very few trials. The HKSJ adjustment, which the authors treated as primary, is specifically designed for settings with fewer than five studies, where the conventional DL approach with normal-approximation intervals can be anticonservative. The fact that significance flipped for some endpoints between methods is not a flaw of the review but a faithful reflection of the fragility of the underlying randomized evidence, and it underscores why the authors call for cautious interpretation, particularly for longer-term and post-treatment outcomes.

The authors declare no competing interests and received no funding for the study, and the work is published open access under a Creative Commons Attribution 4.0 license. For clinicians managing chronic hepatitis D, the review provides a technically grounded evidence base: bulevirtide plus Peg-IFNα-2a was associated with greater virological suppression at week 48 and better combined responses 24 weeks after treatment ended, whereas bulevirtide monotherapy showed more favorable on-treatment biochemical responses and avoided interferon-related cytopenias. With only three trials and 225 patients, and wide confidence intervals under the primary REML-HKSJ analysis, the randomized evidence remains limited. Larger, longer randomized trials with standardized endpoint definitions will be needed to determine the optimal position of combination therapy in chronic hepatitis D, particularly as bulevirtide dosing strategies, treatment duration, and off-treatment durability continue to evolve in this rare and devastating disease.

Subject of Research: Virology

Article Title: Bulevirtide with or without Peg-IFNα-2a for chronic hepatitis D: a systematic review and meta-analysis of randomized controlled trials

Article References: Saeed, M. H., Lohana, K. C., Kashish, F. N. U., Nadeem, U., Dadabhoy, S. J., Ahsan, A. E., Zafar, H., Rizvi, S. E. Z., Bux, A., Butt, N., Jama, M. M., & Shaikh, M. A. (2026). Bulevirtide with or without Peg-IFNα-2a for chronic hepatitis D: a systematic review and meta-analysis of randomized controlled trials. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14668-7

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14668-7

Keywords: chronic hepatitis D, bulevirtide, pegylated interferon alfa-2a, HDV RNA suppression, NTCP inhibitor, meta-analysis, randomized controlled trials, viral hepatitis, liver disease, antiviral therapy, without, Peg-IFN

The historical roots of hantavirus research trace back to the Korean War in the early 1950s, when thousands of United Nations soldiers developed a severe febrile illness later identified as Korean hemorrhagic fever. The causative agent, Hantaan virus, was not isolated until 1978 near the Hantan River in South Korea, giving rise to the name hantavirus. Since that discovery, numerous hantavirus species have been identified worldwide, each tightly associated with a specific rodent or small mammal reservoir. Today, hantaviruses are classified within the order Bunyavirales and the family Hantaviridae, a taxonomic revision that reflects their unique genetic and biological characteristics compared with other bunyaviruses. The genus Orthohantavirus contains the majority of species pathogenic to humans, including Hantaan virus, Seoul virus, Puumala virus, Dobrava-Belgrade virus, Sin Nombre virus, and Andes virus.

Structurally, hantaviruses form spherical virions measuring approximately 80 to 120 nanometers in diameter, enclosed by a lipid envelope studded with glycoprotein spikes that mediate host cell recognition, attachment, and membrane fusion. The genome consists of three segments of negative-sense single-stranded RNA designated large, medium, and small. The large segment encodes the RNA-dependent RNA polymerase responsible for transcription and replication, the medium segment encodes the precursor of the envelope glycoproteins Gn and Gc, and the small segment encodes the nucleocapsid protein that binds and protects viral RNA. Beyond its structural role, the nucleocapsid protein contributes to immune evasion by interfering with host innate immune responses. Critically, the segmented genome permits genetic reassortment when multiple strains co-infect the same host cell, a mechanism that enhances viral diversity and can generate novel strains with altered virulence and host range.

The replication cycle of hantaviruses is entirely cytoplasmic, distinguishing them from many other RNA viruses that involve nuclear stages. Viral entry is initiated through interactions between surface glycoproteins and host cell receptors, including β3 integrins, β1 integrins, decay-accelerating factor, and protocadherin-1, the latter identified as a particularly critical entry receptor for New World hantaviruses such as Andes virus, Sin Nombre virus, and Laguna Negra virus. Following receptor engagement, virions enter cells predominantly through clathrin-mediated endocytosis, and endosomal acidification triggers conformational changes that facilitate membrane fusion. The viral polymerase employs a cap-snatching mechanism, cleaving short capped fragments from host messenger RNAs to prime viral mRNA synthesis, a strategy that ensures efficient translation while contributing to host protein synthesis shutoff and immune evasion. Virion assembly occurs primarily at the Golgi apparatus before mature particles are released via the secretory pathway.

Epidemiologically, hantaviruses impose two distinct clinical syndromes shaped by geography and viral species. Hemorrhagic fever with renal syndrome, caused by Old World viruses such as Hantaan, Seoul, Puumala, and Dobrava-Belgrade, predominates in Europe and Asia and follows a classically five-phase course encompassing febrile, hypotensive, oliguric, polyuric, and convalescent stages, with mortality ranging from less than 1 percent in mild Puumala infections to roughly 15 percent in severe Dobrava-Belgrade cases. Hantavirus cardiopulmonary syndrome, driven by New World viruses in the Americas, presents with fulminant respiratory compromise, pulmonary edema, and cardiogenic shock, with case-fatality rates reaching 40 to 50 percent despite intensive supportive care. China bears the highest global burden of hemorrhagic fever with renal syndrome, while Puumala virus drives cyclical outbreaks across Northern and Central Europe tied to fluctuations in bank vole populations. In the Americas, Andes virus stands apart as the only hantavirus with well-documented person-to-person transmission, primarily among household members, sexual partners, and caregivers during the prodromal phase when viral loads are high but symptoms remain nonspecific.

Transmission to humans occurs overwhelmingly through inhalation of aerosolized viral particles from contaminated rodent urine, feces, and saliva, particularly in enclosed or poorly ventilated spaces. Secondary routes include contact with contaminated fomites, rodent bites, and consumption of contaminated food or water. High-risk groups encompass farmers, forestry workers, military personnel, laboratory workers, and outdoor enthusiasts. Rodent population dynamics, governed by climatic fluctuations such as El Niño Southern Oscillation events, deforestation, agricultural expansion, and urban encroachment into wildlife habitats, strongly modulate spillover risk. Despite public concern, the review emphasizes that hantaviruses lack efficient airborne transmission comparable to influenza or SARS-CoV-2, and human-to-human spread remains rare and largely restricted to Andes virus, a constraint that limits widespread global dissemination even as localized outbreaks with serious consequences persist.

Pathogenically, hantavirus disease is defined by profound vascular permeability resulting from endothelial dysfunction rather than direct cytopathic injury. The viruses preferentially infect vascular endothelial cells and persist without causing overt cell lysis, meaning that host immune responses, not viral-mediated death, drive tissue injury. Pattern-recognition receptors including Toll-like receptors 3 and 7 and cytosolic sensors RIG-I and MDA5 initiate type I interferon signaling, yet many hantaviruses encode mechanisms to blunt this response, delaying antiviral defenses. Subsequent adaptive immunity, characterized by cytotoxic T lymphocyte expansion and neutralizing antibodies against the Gn and Gc glycoproteins, can clear infection, but dysregulated or excessive responses generate a cytokine storm phenotype that disrupts endothelial barrier integrity. This delicate balance between viral control and immunopathology shapes disease severity and informs therapeutic strategies aimed at restoring antiviral control while tempering hyperinflammation.

Diagnosis relies on a combination of clinical suspicion, exposure history, and laboratory confirmation, with serological assays detecting virus-specific IgM and IgG antibodies remaining the most widely used approach, complemented by reverse transcription polymerase chain reaction for early viral RNA detection and genome sequencing for strain identification. However, nonspecific early symptoms, a narrow window of viremia, serological cross-reactivity among related species, and limited access to molecular diagnostics in resource-constrained settings continue to impede accurate case recognition, particularly in Africa and many low- and middle-income countries. Treatment remains fundamentally supportive, encompassing oxygen supplementation, mechanical ventilation, extracorporeal membrane oxygenation, hemodynamic stabilization, and renal replacement therapy as needed. Ribavirin has shown partial efficacy against certain Old World hantaviruses when administered early, while favipiravir, monoclonal antibodies, and nanotechnology-based delivery platforms represent promising but still investigational avenues. No universally approved vaccine exists; licensed inactivated formulations are restricted to China and South Korea for selected Old World strains, and next-generation platforms including DNA, mRNA, recombinant viral vector, and virus-like particle vaccines are under development to achieve broader cross-protection.

The review operationalizes a One Health framework into concrete prevention and control actions, calling for integrated human-animal-environment surveillance, targeted rodent reservoir monitoring, environmental sampling, and formal intersectoral coordination mechanisms. Primary prevention centers on environmental sanitation, rodent-proofing, proper ventilation of enclosed spaces before cleaning, wet-cleaning methods that minimize aerosol generation, and personal protective equipment for occupationally exposed individuals. Community education campaigns play a critical role in improving risk perception and encouraging early medical consultation after high-risk exposures. Genomic surveillance, combined with climate modeling and ecological forecasting, enables more precise outbreak reconstruction and proactive preparedness, while artificial intelligence-assisted prediction models may further enhance the integration of ecological, climatic, and epidemiological data.

Looking ahead, the author identifies persistent controversies and knowledge gaps, including the determinants of reservoir competence, the sensitivity of current surveillance systems, and the limits of predictive models, alongside unresolved questions about the biological basis of Andes virus transmissibility and whether other hantavirus species harbor undetected human-to-human potential. Although a COVID-19-like global pandemic caused by hantaviruses currently appears unlikely given their transmission dynamics, the review concludes that increasing ecological disruption and global connectivity demand sustained interdisciplinary collaboration among virologists, epidemiologists, ecologists, clinicians, and public health authorities. Strengthening early warning systems, expanding genomic surveillance, developing pan-hantavirus vaccines and broad-spectrum antivirals, and formalizing One Health coordination mechanisms are framed as essential pillars for preventing future high-consequence hantavirus events in an era of accelerating environmental change.

Subject of Research: Hantavirus zoonotic spillover risk, molecular virology, and One Health preparedness

Article Title: Hantaviruses at an inflection point: scientific evidence, spillover risk, and One Health approach

Article References: Zatla, I. (2026). Hantaviruses at an inflection point: scientific evidence, spillover risk, and One Health approach. Frontiers in Virology, 6, Article 1883348. https://doi.org/10.3389/fviro.2026.1883348

Image Credits: AI Generated

DOI: 10.3389/fviro.2026.1883348

Keywords: hantavirus, zoonosis, rodent reservoirs, Andes virus, One Health, hemorrhagic fever with renal syndrome, hantavirus cardiopulmonary syndrome, spillover, genomic surveillance, vaccine development, antiviral therapy, public health preparedness

The research, led by Ziruo Sun, Jinliang Wang, Zhigao Bu and colleagues at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, began with a systematic search for the host machinery that rabies virus depends on to invade cells. In a previous genome-wide screen, the team had used 64,755 small interfering RNAs targeting 21,585 human genes in HEK293 cells to identify host factors required for rabies virus replication. Among the hits were sixteen genes related to potassium channels, and the strongest effect came from silencing KCNMA1, the gene that encodes the pore-forming subunit of the large conductance calcium- and voltage-activated potassium channel known as KCa1.1, a seven-transmembrane protein sitting in the plasma membrane.

To confirm that this channel genuinely matters for infection, the researchers knocked down KCa1.1 expression in both mouse neuroblastoma N2a cells and human HEK293 cells using specific siRNAs. Quantitative PCR verified that the channel’s mRNA was substantially reduced, and when the silenced cells were infected with a recombinant rabies virus strain expressing enhanced green fluorescent protein, the amount of infectious virus released into the culture supernatant dropped significantly at both 24 and 48 hours after infection. The team then turned to paxilline, a selective chemical inhibitor of KCa1.1 that binds the inner pore of the channel and blocks potassium conductance. In N2a, HEK293, and human SK-N-SH neuroblastoma cells, paxilline had no effect on cell viability but suppressed rabies virus replication in a clear dose-dependent manner, and time-course experiments confirmed the inhibition persisted across the tested period.

Because KCa1.1 is expressed primarily at the plasma membrane, the investigators hypothesized that it might act during viral entry rather than during later stages of the viral life cycle. Entry involves two separable steps: attachment of virus particles to the cell surface and their subsequent internalization. By incubating cells with virus at 4 degrees Celsius, a temperature that permits binding but prevents internalization, and then quantifying attached viral RNA, the team showed that neither KCa1.1 silencing nor paxilline treatment changed how much virus bound to cells. When the temperature was shifted to 37 degrees to allow entry, however, and surface-bound virus was stripped away with trypsin before analysis, the amount of viral RNA inside treated or silenced cells fell dramatically. A complementary microscopy assay, which labeled viral glycoprotein under conditions that distinguish particles remaining on the surface from those that had entered, showed that paxilline-treated cells accumulated more virus on their surface while their total particle count stayed the same. Together, these experiments demonstrated that activated KCa1.1 is specifically required for the internalization step of rabies virus entry.

A co-immunoprecipitation assay revealed that KCa1.1 does not physically interact with the rabies virus glycoprotein, the viral surface protein that drives attachment and clathrin-mediated endocytosis. That result pointed to an intermediary. Prior work had shown that KCa1.1 assembles into macromolecular complexes with voltage-gated calcium channels, including Cav1.2, and that calcium influx through these channels can activate KCa1.1. Notably, Cav1.2 was the only calcium channel that emerged from the team’s earlier genome-wide screen. Co-immunoprecipitation confirmed a specific interaction between Cav1.2 and KCa1.1, and silencing Cav1.2 in N2a and HEK293 cells significantly reduced rabies virus titers, establishing that the L-type calcium channel is also required for productive infection.

The mechanistic picture became sharper when the researchers examined the role of the viral glycoprotein itself. Co-immunoprecipitation showed that Cav1.2 specifically interacts with the rabies virus glycoprotein, and a pull-down assay using purified virus particles demonstrated that Cav1.2 captures rabies virions in a dose-dependent manner. Binding assays revealed that Cav1.2 silencing or blockade did not reduce how much virus attached to cells, but internalization assays showed far less viral RNA inside treated cells. In other words, the glycoprotein engages Cav1.2, and this engagement is required to activate the channel and trigger the internalization machinery, even though the virus still docks normally on the cell surface when the channel is disabled.

The team also identified the downstream cellular process that the channel axis manipulates. Rabies virus enters cells through clathrin-mediated endocytosis with the help of actin, and previous work had shown that KCa1.1 promotes influenza virus entry by regulating F-actin polymerization. Using phalloidin staining to visualize filamentous actin, the researchers found that rabies virus entry induces F-actin polymerization in HEK293 cells, and that this polymerization was markedly reduced when Cav1.2 or KCa1.1 were knocked down or when cells were treated with diltiazem or paxilline. The authors therefore propose a sequential model: rabies virus glycoprotein binds Cav1.2 and triggers its activation; activated Cav1.2 then interacts with and activates KCa1.1; and activated KCa1.1 drives F-actin polymerization that powers the internalization of the virus particle.

The therapeutic implications rest on diltiazem, a benzothiazepine-class L-type calcium channel blocker approved in the United States in 1982 and long prescribed for hypertension, angina, and atrial arrhythmias. In cell culture, diltiazem blocked rabies virus infection in a dose-dependent manner without affecting viability, and four other FDA-approved Cav1.2 antagonists, the phenylalkylamine verapamil and the dihydropyridines nifedipine, nicardipine, and nimodipine, also inhibited the virus in vitro. The decisive test came in mice. Six-week-old C57BL/6J mice were inoculated intramuscularly with ten 50 percent mouse lethal doses of the rabies street virus GX/09, and six hours later they began receiving daily intramuscular injections of diltiazem at 25 or 50 milligrams per kilogram, or a phosphate buffer control. All 19 control mice died within 9 to 16 days with typical rabies signs such as body tremors and hind limb paralysis. At the lower dose, 5 of 19 mice survived, roughly 26 percent, while at 50 milligrams per kilogram, 13 of 19 mice survived, approximately 70 percent, and the survivors remained completely asymptomatic throughout a 28-day observation period.

Measurements of viral burden supported the idea that diltiazem works by blocking entry before the virus reaches the brain. No viral RNA was detectable in the brains of treated or control mice at day 4, but by day 6, viral RNA was present in all six control animals examined and in only two of six diltiazem-treated animals, and immunohistochemistry at day 8 detected rabies antigen in all control brains but only one of three treated brains. When the researchers delayed diltiazem administration until days 6 and 7, after viral replication was already underway in the brain, they observed no significant reduction in brain viral RNA, indicating that the drug’s protection is unlikely to stem from direct suppression of established central nervous system infection. Notably, the 50 milligram per kilogram mouse dose corresponds, after body surface area normalization under FDA guidelines, to a human equivalent dose of roughly 4 milligrams per kilogram, or about 240 milligrams per day for a 60-kilogram adult, which falls squarely within the range already used clinically for cardiovascular indications.

The authors argue that diltiazem could serve as an affordable, mass-produced complement to existing post-exposure prophylaxis, offering a stopgap where rabies immunoglobulin is unavailable and a potential enhancement where it is not, since it is estimated that only 1 to 10 percent of category III exposure patients currently receive the recommended treatment. The findings also extend a growing body of evidence that ion channels are conserved entry factors for diverse viruses: Cav1.2 has been implicated in SARS-CoV-2 and influenza virus entry, KCa1.1 in influenza virus endocytosis, and diltiazem has shown activity against rotavirus, porcine reproductive and respiratory syndrome virus, and porcine deltacoronavirus. With no approved drugs currently targeting KCa1.1, the potassium channel remains a target for future drug development, while Cav1.2, already served by three structural classes of clinically validated antagonists, stands out as a promising foundation for broad-spectrum antiviral strategies. Further studies will be needed to evaluate the in vivo efficacy of the other calcium channel blockers and to determine whether diltiazem concentrations in the central nervous system reach antiviral levels, but the prospect of repurposing a decades-old cardiovascular pill against one of humanity’s most lethal pathogens gives the global effort to eliminate rabies deaths a genuinely new weapon to test.

Subject of Research: Repurposing the calcium channel blocker diltiazem to block rabies virus entry via the Cav1.2-KCa1.1 ion channel axis

Article Title: Targeting the Cav1.2-KCa1.1 axis prevents rabies virus internalization and protects against lethal infection

Article References: Sun, Z., Wang, J., Wen, Z., Shuai, L., Sun, W., Yang, M., Wang, J., Chen, J., Mi, T., Zhou, X., Zhou, P., Lv, N., Zhao, Z., Ge, J., Chen, W., Wang, X., Wang, C., Bu, Z., & Wang, J. (2026). Targeting the Cav1.2-KCa1.1 axis prevents rabies virus internalization and protects against lethal infection. PLOS Pathogens, 22(10), e1014675. https://doi.org/10.1371/journal.ppat.1014675

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014675

Keywords: rabies virus, diltiazem, Cav1.2, KCa1.1, ion channels, viral entry, endocytosis, drug repurposing, F-actin polymerization, post-exposure prophylaxis, PLOS Pathogens, antiviral therapy

The target in question is the immediate-early protein 1, or IE1, a product of the viral major immediate-early gene that has long been considered undruggable. Through alternative splicing, the immediate-early transcript yields two major isoforms, IE1 and IE2, which together orchestrate the onset of lytic replication. IE1 coordinates the cascade activation of early and late viral genes, neutralizes host translational repressors such as PKR to favor viral protein synthesis, interferes with STAT1/STAT2 signaling to suppress type I interferon responses, and pushes infected cells from the G1 into the S phase of the cell cycle to create a replication-friendly environment. Because IE1 lacks a well-defined catalytic pocket or conventional active site, structure-based small-molecule design has historically been difficult to apply, leaving the protein without direct pharmacological inhibitors despite abundant evidence that disabling it halts viral replication.

A research team led by Wanfeng Liu, Yu Shi, Sunzhong Mao, Xiangyang Xue, and Shiyu Feng at Wenzhou Medical University and collaborators approached this problem through computational drug repurposing. Using the resolved crystal structure of the IE1 central core domain (PDB ID 6TGZ) as the receptor, they docked 890 compounds from an FDA-approved drug library with Discovery Studio 2019 and ranked hits by LibDock score. Six candidates exceeded the score threshold of 150, among them ritonavir, which topped the list with a score of 174.651. A second round of docking using CB-Dock2 for blind surface scanning and AutoDock-GPU for site-specific calculations identified five candidate pockets on the IE1 homodimer, with pocket 1 at the interface of the A and B subunits selected for analysis. Ritonavir again ranked first, showing the most favorable predicted binding energy of −16.16 kcal/mol.

To test whether the predicted interaction was physically plausible, the team ran 100-nanosecond molecular dynamics simulations of both unbound IE1 and the IE1–ritonavir complex in explicit solvent using GROMACS 2024 on the WeMol platform. Analyses of backbone root-mean-square deviation, radius of gyration, residue-level fluctuations, hydrogen-bond counts, solvent-accessible surface area, secondary-structure content, and principal components indicated that the complex remained stable without major perturbation of the global protein fold. MM-PBSA calculations on the equilibrated 50–100 nanosecond trajectory segment suggested energetically favorable binding driven largely by van der Waals contacts, with residue-level decomposition flagging GLU615, LEU523, PHE642, and PRO694 as potential hotspot residues at the predicted interface.

The decisive evidence came from surface plasmon resonance. Using a Biacore 1K system with recombinant IE1 immobilized on a CM5 sensor chip, the researchers injected serial dilutions of ritonavir ranging from 12.5 to 6400 nanomolar and observed concentration-dependent, reversible binding. Global fitting of the sensorgrams to a 1:1 Langmuir model yielded an equilibrium dissociation constant of 3.18 micromolar, while steady-state analysis gave a comparable value of 4.14 micromolar. The authors caution that docking scores were used only for relative ranking within identical simulation systems and cannot reproduce absolute affinities, but the combination of computational prediction and direct biophysical measurement provides the first indication that ritonavir binds IE1.

Binding, however, was only the beginning. In human foreskin fibroblast cells infected with the laboratory-adapted HCMV strain AD169, ritonavir reduced IE1 protein levels in a time- and dose-dependent manner, with a detectable decrease from day 2 post-infection. The same reduction appeared in HeLa cells engineered to express IE1 alone, showing that the effect does not depend on the full viral replication context, and immunofluorescence microscopy confirmed diminished nuclear IE1 signal in infected cells. Crucially, the drug appeared selective: the abundance of host proteins such as GAPDH, β-actin, α-tubulin, and HSP90 was unchanged, global cellular ubiquitination was not increased, and ectopically expressed viral proteins UL96, UL55, UL86, UL50, and UL83 were not appreciably reduced. Reverse-transcription quantitative PCR showed that IE1 mRNA levels were unaffected across five days of treatment, pointing to a post-transcriptional mechanism.

That mechanism turned out to be targeted protein degradation. In a cycloheximide chase assay that blocks new protein synthesis, ritonavir markedly shortened the half-life of IE1, producing roughly 40 percent greater degradation within nine hours compared with controls. Rescue experiments then discriminated between the two major intracellular degradation routes: the proteasome inhibitor MG132 partially reversed the ritonavir-induced loss of IE1, whereas the autophagy inhibitor chloroquine had no effect. Co-immunoprecipitation experiments in cells stably expressing Flag-tagged IE1 showed that ritonavir treatment substantially increased ubiquitin signals attached to IE1, indicating that the drug enhances IE1 ubiquitination and channels the protein into the ubiquitin–proteasome pathway. Although ritonavir is neither a PROTAC nor a molecular-glue degrader, the authors note that its mode of action is conceptually related to targeted protein degradation, an approach increasingly explored as an antiviral strategy.

The functional consequences for the virus were substantial. At concentrations that did not significantly affect host cell viability or apoptosis, ritonavir reduced extracellular viral DNA loads in the supernatant of infected cells from 31.52 to 11.75 copies per milliliter, an inhibition rate of approximately 62.7 percent, and TCID50 assays confirmed a dose-dependent drop in infectious particle production. A recombinant HCMV expressing GFP provided independent confirmation: supernatants from ritonavir-treated cultures produced roughly 2.2-fold lower GFP signals upon reinfection of fresh fibroblasts. Western blotting revealed that prolonged treatment suppressed not only IE1 but also the downstream viral proteins UL44, UL83, and UL84, consistent with collapse of the immediate-early regulatory cascade. Quantifying dose-response relationships, the team measured a half-maximal cytotoxic concentration of 57.36 micromolar and a half-maximal effective concentration of 6.10 micromolar for ritonavir, corresponding to a selectivity index of 9.40, a moderate but meaningful in vitro safety margin.

Perhaps the most clinically suggestive finding concerns combination therapy. When ritonavir was paired with ganciclovir, the two drugs acted synergistically: 5 micromolar ritonavir combined with 2 micromolar ganciclovir achieved 90.83 percent inhibition of viral replication, well above the 73.43 percent predicted by the Bliss independence model, and a 5-by-5 checkerboard analysis yielded an overall Bliss synergy score of 10.443 with a most synergistic area score of 12.10. The combination also suppressed IE1 more strongly than either drug alone. By contrast, ritonavir combined with letermovir showed no meaningful synergy, with observed effects falling below Bliss-predicted values and an overall synergy score of only 1.475. The authors attribute the ganciclovir synergy to the drugs’ distinct mechanisms, with ritonavir dismantling early viral regulation while ganciclovir blocks DNA synthesis, whereas the basis for the absence of synergy with letermovir remains unclear.

The study has limitations that the authors acknowledge openly. Their screening pipeline relied on conventional LibDock and AutoDock docking rather than newer AI-driven frameworks, without retrospective benchmarking using decoy-set enrichment or ROC-AUC evaluation. Mechanistically, MG132 only partially rescued IE1 levels, the responsible E3 ubiquitin ligase remains unidentified, and binding-site mutagenesis and IE1 rescue experiments are still needed to establish a definitive causal link between IE1 degradation and antiviral activity. Most experiments used laboratory cell lines and the AD169 strain rather than clinical isolates, drug-resistant strains, or animal models, and whether the effective concentrations are achievable in patients remains to be determined. Even so, the work validates IE1 as a druggable target, reveals a previously unknown direct antiviral activity of a widely used HIV protease inhibitor, and suggests a combination strategy that could matter particularly for patients co-infected with HIV and HCMV. Because the IE1 core domain is highly conserved among humans, rhesus macaques, and rats, the approach may also open the door to cross-species, broad-spectrum antiviral development built on pharmacological destabilization of essential viral regulators.

Subject of Research: Repurposing ritonavir to induce proteasomal degradation of the HCMV immediate-early protein IE1 as an antiviral strategy

Article Title: Repurposing ritonavir to induce proteasomal degradation of IE1 for inhibition of human cytomegalovirus replication

Article References: Liu, W., Shi, Y., Guo, X., Lou, C., Xie, Z., Shen, Y., Zhang, L., Mao, S., Xue, X., & Feng, S. (2026). Repurposing ritonavir to induce proteasomal degradation of IE1 for inhibition of human cytomegalovirus replication. PLOS Computational Biology, 22(10), e1014867. https://doi.org/10.1371/journal.pcbi.1014867

Image Credits: AI Generated

DOI: 10.1371/journal.pcbi.1014867

Keywords: human cytomegalovirus, ritonavir, drug repurposing, IE1 protein, ubiquitin-proteasome pathway, molecular docking, surface plasmon resonance, ganciclovir, antiviral therapy, targeted protein degradation, synergy, molecular dynamics simulation

The drug in question is favipiravir, a broad-spectrum nucleoside analogue that inhibits viral RNA-dependent RNA polymerases and is already approved for influenza treatment in several countries. The research team began by characterizing its activity against Oropouche virus in cultured Vero E6 cells, comparing it directly with ribavirin, another broad-spectrum antiviral. Favipiravir proved substantially more potent, with a mean half-maximal effective concentration of 28.4 micromolar compared with 115.7 micromolar for ribavirin. Untreated infected cells developed marked cytopathic effects, and ribavirin offered only limited protection, whereas favipiravir preserved cellular morphology in a dose-dependent fashion and robustly reduced infectious virus production. Time-of-addition experiments pinpointed the drug’s stage of action: treatment significantly reduced viral titers when administered at the time of infection or during early replication, but not at the maturation stage, consistent with favipiravir’s established mechanism of interfering with viral RNA synthesis during the post-entry replication phase.

The decisive test came in vivo. The researchers used a lethal Syrian hamster model, infecting animals intraperitoneally with 5.4 × 10⁵ plaque-forming units of the OROV TRVL 9760 strain. All vehicle-treated animals succumbed to infection by three days post-infection, without preceding overt clinical symptoms or weight loss—a fulminant course that underscores how aggressive lethal Oropouche disease can be in this model. In stark contrast, animals receiving high-dose favipiravir at 600 milligrams per kilogram per day, given orally twice daily beginning one hour before infection and continuing for six days, survived the entire 26-day observation period without any clinical signs and steadily gained weight. Lower doses of favipiravir and both tested doses of ribavirin conferred only partial protection, and notably, some animals in the ribavirin groups developed neurological symptoms, including ataxia, hindlimb weakness, and paralysis, after treatment ceased.

Virological measurements explained these divergent outcomes. Using plaque assays and reverse transcription quantitative PCR, the team tracked viral loads in serum, brain, liver, and spleen at multiple time points. Vehicle-treated animals carried high viral burdens in all tissues at two days post-infection. High-dose favipiravir, by contrast, suppressed viral replication so completely that no viral RNA or infectious virus was detectable in any tissue at any time point examined. The suboptimal regimens told a more cautionary story: although low-dose favipiravir and ribavirin reduced infectious virus in serum, liver, and spleen, infectious virus persisted in the brains of these animals at six and eight days post-infection, indicating incomplete clearance from the central nervous system that correlated with the delayed neurological disease observed after treatment withdrawal. The message is clear—insufficient antiviral pressure allows viral neuroinvasion, and neuroinvasion drives fatal disease.

Perhaps the most clinically encouraging finding concerns timing. In outbreak settings, patients rarely present before infection is already established, so a drug that works only as prophylaxis would be of limited value. The researchers therefore tested delayed treatment, initiating favipiravir at one hour before infection, at the time of infection, or at 6, 24, or 48 hours after infection. Strikingly, animals that began treatment as late as 24 hours post-infection were fully protected, with 100 percent survival and complete suppression of viral replication in systemic organs and the central nervous system. Even at 48 hours post-infection, when the therapeutic window was narrowing, favipiravir still conferred partial protection, with 50 percent survival and consistent weight gain among survivors. This therapeutic window, demonstrated in a model where untreated infection is uniformly lethal within days, substantially strengthens the case for favipiravir as a practical candidate for human Oropouche disease.

Beyond efficacy, the study’s second major contribution is mechanistic. To define the host responses associated with Oropouche virus pathogenesis and their modulation by antiviral therapy, the team performed tissue-resolved transcriptomic profiling—RNA sequencing of liver and brain collected at two days post-infection. Because the Syrian hamster genome remains less comprehensively annotated than the mouse genome, hamster genes were functionally annotated through their mouse orthologs. Principal component analysis revealed a clear transcriptional separation between infected and uninfected animals in both tissues, while favipiravir-treated animals clustered near uninfected controls, indicating that the infection-driven transcriptional upheaval was largely suppressed by the drug. This is, to the authors’ knowledge, the first in vivo, tissue-resolved transcriptomic framework of Oropouche virus infection.

The transcriptomic signatures in infected animals were distinctive and, in places, ominous. In the liver, where the virus replicated robustly, infection induced upregulation of genes involved in chemotaxis, cytokine signaling, myeloid cell differentiation, and interferon responses, alongside downregulation of metabolic pathways governing energy production and fatty acid metabolism. In the brain, antiviral and inflammatory programs were induced even more strongly, accompanied by pathways promoting immune cell recruitment. Quantitative PCR validation of selected interferon-stimulated genes—Cxcl10, Rsad2, and Isg15—confirmed strong induction at two days post-infection in both tissues of untreated animals, which was suppressed by both favipiravir and ribavirin at that early stage. At later time points, however, Rsad2 and Isg15 expression tended to remain elevated in ribavirin-treated animals, correlating with their persistent brain viral loads, whereas expression stayed low in favipiravir-treated animals. Interestingly, the canonical inflammatory cytokines Il6, Il10, and Tnf-alpha showed no significant changes at two days post-infection and did not track with viral replication, suggesting they are unlikely to be major drivers of Oropouche disease in this model.

The metabolic findings deserve particular attention. Although liver involvement is not a prominent feature of typical Oropouche fever, recent fatal human infections have been associated with markedly elevated aminotransferase levels and liver injury biomarkers, and independent studies have demonstrated hepatic infection and inflammatory responses in human liver organoids and an experimental mouse model. The simultaneous enrichment of pathways associated with positive regulation of innate immune responses and negative regulation of defense responses in high-burden animals suggests a complex, potentially dysregulated host response, in which sustained antiviral and inflammatory signaling coexists with regulatory mechanisms that constrain excessive immune activation. When viral replication remains high, such regulation appears insufficient. The concurrent downregulation of energetic, lipid metabolic, and mitochondrial pathways in the liver implies that cellular stress during high-burden infection may contribute to tissue dysfunction and injury, meaning that Oropouche pathogenesis likely reflects not only direct viral replication but also the interplay between persistent viral burden, dysregulated immunity, and disrupted metabolic homeostasis.

The authors are candid about the study’s limitations. The reliance on mouse orthologs for gene annotation means hamster-specific transcriptional features and genes lacking confident mouse orthologs may have been underrepresented. Additionally, because all vehicle-treated animals reached the humane endpoint before six days post-infection, direct comparisons of gene expression between untreated and treated animals at later time points were impossible, leaving some uncertainty about whether the later profiles reflect favipiravir’s effects or the absence of ongoing infection. These caveats notwithstanding, the study extends earlier work by Rodrigues and colleagues, who established the Syrian hamster as a relevant model of Oropouche pathogenesis, by integrating tissue-resolved transcriptomics with therapeutic intervention in a single experimental framework.

Translational considerations will shape what happens next. Favipiravir offers genuine advantages over other polymerase-targeting candidates such as 4′-fluorouridine, including oral bioavailability and prior clinical use against multiple viral diseases, and its demonstrated efficacy with delayed initiation enhances its relevance for outbreak settings where early diagnosis is difficult. Yet the drug carries known safety liabilities: teratogenic and embryotoxic effects, and an association with elevated serum uric acid and hyperuricemia. These concerns are especially pertinent for Oropouche virus, given the growing recognition of congenital and pregnancy-associated disease. The authors emphasize that further studies addressing pharmacokinetics, optimal dosing, treatment duration, and safety in relevant populations will be required before clinical application. Even so, the findings provide strong preclinical evidence that timely, potent antiviral intervention can prevent central nervous system invasion, suppress pathogenic inflammatory and metabolic programs, and avert lethal Oropouche disease—transforming the outlook for a pathogen that, until now, medicine could only watch.

Subject of Research: Favipiravir efficacy and host transcriptomic responses in lethal Oropouche virus infection

Article Title: Integrated in vivo and transcriptomic analyses of lethal Oropouche virus infection reveal suppression of pathogenic host responses by antiviral therapy

Article References: Sousa Moraes, C., Gonzalez, G., Sato, A., Miki, S., Inoue, A., Tabata, K., Kranrod, J. W., Kabamba, C. F., Ohnuma, A., Matsuno, K., Harada, R., Saito, S., Akimoto, N., Sasaki, M., Orba, Y., Hall, W. W., Sawa, H., & Itakura, Y. (2026). Integrated in vivo and transcriptomic analyses of lethal Oropouche virus infection reveal suppression of pathogenic host responses by antiviral therapy. PLOS Pathogens, 22(10), e1014647. https://doi.org/10.1371/journal.ppat.1014647

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DOI: 10.1371/journal.ppat.1014647

Keywords: Oropouche virus, favipiravir, antiviral therapy, arbovirus, neuroinvasion, Syrian hamster model, transcriptomics, interferon response, ribavirin, PLOS Pathogens, emerging infectious disease, RNA virus

A team of hematologists and oncologists affiliated with the Bavarian Cancer Research Center (BZKF) conducted a multi-center retrospective analysis of lymphoma patients who contracted COVID-19 at five tertiary care centers in Germany between March 2020 and March 2022. The centers involved span some of the country’s largest university hospitals, including LMU University Hospital Munich, TUM University Hospital, University Hospital Erlangen, University Hospital Regensburg, and University Hospital Augsburg. By pooling anonymized routine clinical data across these institutions, the investigators assembled a cohort large enough and sufficiently granular to compare baseline characteristics, treatment decisions, and clinical outcomes across distinct pandemic waves. The study, led by Stefanie Forkl and Johannes C. Hellmuth of LMU Munich, was published as a brief report in the journal Annals of Hematology and received ethics approval from the medical faculty of LMU Munich.

The central finding is striking in its clarity. In the most recent pandemic wave covered by the analysis — defined as infections occurring after December 27, 2021, when the Omicron variant dominated transmission in Germany — severe COVID-19 courses were recorded in only 4 of 40 lymphoma patients. In the earlier waves, by contrast, 26 of 73 patients experienced severe disease. This difference was statistically significant, with a p-value of 0.03. Mortality followed the same pattern: two of the 40 patients in the late wave died, compared with 14 of the 73 patients infected earlier in the pandemic, a difference that also reached statistical significance at p = 0.039. In proportional terms, the case fatality rate fell from roughly 19 percent in the earlier period to 5 percent in the most recent wave, while the rate of severe disease dropped from about 36 percent to 10 percent.

The authors attribute this improvement not to any single intervention but to the convergence of several protective factors that changed the clinical landscape between 2020 and early 2022. Vaccination coverage among lymphoma patients rose dramatically over the study period. In the final pandemic wave, 88 percent of the infected lymphoma patients had received at least partial vaccination before their SARS-CoV-2 infection, a level of coverage that was essentially absent during the first waves of 2020. This matters even in a population known to mount attenuated antibody responses, because cellular immunity, hybrid immunity from prior infection, and residual humoral responses all contribute to protection against severe disease even when neutralizing antibody titers are suboptimal.

Therapeutic options expanded in parallel. Once infected, 57 percent of the lymphoma patients in the most recent wave were treated with neutralizing monoclonal antibodies, and 35 percent received antiviral agents. Neither class of drug was available in the early months of the pandemic. Monoclonal antibodies such as those directed against the SARS-CoV-2 spike protein offered passive immunization that could compensate for the blunted endogenous antibody responses typical of patients treated with CD20-depleting agents like rituximab, which eliminate the B cells responsible for antibody production. Antiviral drugs, meanwhile, provided a mechanism of protection independent of the patients’ own immune competence, directly inhibiting viral replication during the critical early phase of infection.

The technical significance of this study lies in its wave-by-wave comparison within a single, well-characterized patient population. Many earlier analyses of COVID-19 in hematologic malignancies pooled cases across the entire pandemic or focused on a single center, making it difficult to separate the effects of evolving viral variants, changing patient management, and the natural heterogeneity of lymphoma subtypes. By stratifying outcomes by calendar period and documenting vaccination status and pharmacologic treatment for each case, the BZKF registry provides a longitudinal view of how the intersection of a vulnerable immune state and a shifting pandemic environment determined clinical risk. The consistency of the improvement across both severity and mortality endpoints strengthens the conclusion that the change is real rather than a statistical artifact.

At the same time, the investigators are careful about causal attribution. Because vaccination, monoclonal antibody therapy, antiviral treatment, and the transition to the less virulent Omicron variant all occurred roughly simultaneously, the retrospective design cannot cleanly disentangle the relative contribution of each factor to the observed improvement. A patient infected in early 2022 differed from one infected in 2020 in nearly every relevant dimension: the circulating variant, the immune history, the drugs available, and the accumulated clinical experience of the treating teams. The authors explicitly acknowledge this limitation, noting that while they cannot clearly distinguish the individual effects of each intervention, the aggregate data demonstrate that severity and mortality of COVID-19 in this vulnerable population have recently declined.

For clinicians managing lymphoma patients, the practical implications are nonetheless substantial. The authors conclude that their data largely support a return to pre-pandemic treatment recommendations and protocols for this patient group. During the acute phases of the pandemic, many cancer centers deferred or modified lymphoma therapy, delayed rituximab-containing regimens, and imposed restrictive isolation measures out of concern that immunosuppression would amplify COVID-19 risk. The new findings suggest that such extraordinary caution is no longer proportionate to the residual risk, provided that patients are vaccinated and that breakthrough infections are treated promptly with the antiviral and antibody-based agents now available. This does not mean vigilance can be abandoned entirely — a 5 percent mortality rate among infected patients remains far higher than in the general population — but it marks a meaningful recalibration of the risk-benefit calculus that governed oncology practice during the pandemic’s peak.

The study also underscores the enduring value of disease-specific registries during public health emergencies. The COVID-19-lymphoma registry of the BZKF was built on the infrastructure of an established regional cancer research consortium, allowing rapid, coordinated data collection across five centers without the delays that typically accompany prospective trials in rare populations. Lymphoma patients are heterogeneous — the category encompasses indolent and aggressive B-cell malignancies, T-cell lymphomas, Hodgkin lymphoma, and patients at vastly different points in their treatment trajectories — and only registry-scale efforts can capture enough cases to draw meaningful conclusions about outcomes in subgroups defined by both malignancy and pandemic timing. Similar registry models have proven their worth for other immunocompromised populations and represent a template for future infectious disease threats.

As the pandemic recedes from daily attention, studies like this one serve as a quantitative record of how clinical outcomes evolved in the populations hit hardest. For lymphoma patients in Bavaria and, by extension, in comparable health systems across Europe and North America, the trajectory from 2020 to 2022 was one of genuine improvement: vaccination rates approaching 90 percent, widespread access to passive and antiviral therapies, and a decline in severe disease and death to a fraction of earlier levels. The residual risks are real and warrant continued attention to vaccination schedules, pre-exposure prophylaxis where appropriate, and early treatment of breakthrough infections. But the central message of the BZKF registry is ultimately an encouraging one — that the convergence of immunization, targeted therapeutics, and clinical experience substantially tamed what was once one of the most feared complications facing patients with cancer of the immune system.

Subject of Research: COVID-19 outcomes in lymphoma patients across pandemic waves

Article Title: Change in COVID-19 outcomes in lymphoma patients across pandemic waves: results of the COVID-19-lymphoma registry of the Bavarian Cancer Research Center (BZKF) 2020–2022

Article References: Forkl, S., Freudenberger, F., Jacobs, B., Einhell, S., Hirschbuehl, K., Claus, R., Hellwig, D., Mougiakakos, D., Dreyling, M., Illert, L., Heidegger, S., Weigert, O., & Hellmuth, J. C. (2026). Change in COVID-19 outcomes in lymphoma patients across pandemic waves: results of the COVID-19-lymphoma registry of the Bavarian Cancer Research Center (BZKF) 2020–2022. Annals of Hematology, 105(9), Article 401. https://doi.org/10.1007/s00277-026-07094-x

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DOI: 10.1007/s00277-026-07094-x

Keywords: COVID-19, lymphoma, BZKF registry, vaccination, monoclonal antibodies, antiviral therapy, rituximab, CD20-depleting therapy, immunocompromised patients, Omicron variant, Annals of Hematology, hematologic malignancies

A new review published in MicrobiologyOpen examines whether plant-derived compounds could help fill this gap, not by mimicking existing antivirals but by targeting the host signaling pathways that influenza exploits. The work is timely because severe influenza is often driven less by direct viral damage than by immune dysregulation, particularly the excessive cytokine production known as a cytokine storm, which contributes substantially to pulmonary pathology and mortality. Central to this process are key signaling cascades, including the Toll-like receptors (TLRs), the retinoic acid-inducible gene I (RIG-I) pathway, the mitogen-activated protein kinase (MAPK) cascades, and nuclear factor kappa B (NF-κB), which orchestrate antiviral responses through interferon and cytokine induction but can also drive immunopathology when aberrantly activated. The review, which surveyed literature from 2000 to 2025 and focused specifically on human IAV subtypes H1N1 and H3N2, argues that phytochemicals capable of both inhibiting viral replication and fine-tuning these pathways could serve as adjunctive therapies.

The evidence base begins at the level of innate immune recognition. TLR3, TLR7, and TLR8 detect viral RNA species generated during IAV replication, while TLR4 is activated indirectly by host-derived damage-associated molecular patterns released during infection-induced oxidative stress. TLR3 signaling through the adaptor TRIF activates TBK1 and IRF3 to induce IFN-β, yet overexpression of this interferon can itself contribute to cytokine storm and lung damage. Autopsy studies of fatal influenza cases have revealed prolonged TLR4 and MyD88 expression in lung tissue, illustrating that the TLR4/MyD88/NF-κB axis is a double-edged sword, essential for initiating antiviral immunity but potentially detrimental when dysregulated. Against this backdrop, several flavonoids have shown the ability to dampen TLR signaling. Quercetin reduced H1N1-induced cell death and activation of the TLR7/NF-κB p65 axis in a human bronchial epithelial co-culture model, while dihydromyricetin inhibited TLR3 and TRIF expression in infected lung epithelial cells, lowering pro-inflammatory cytokines such as CCL5, IP-10, and MIG.

Notably, dihydromyricetin also demonstrated genuine dual activity, reducing H1N1 and H3N2 plaque formation in a concentration-dependent manner with IC50 values between 11.67 and 23.33 micromolar, and binding the PB2 subunit of the viral polymerase in mini-replicon and surface plasmon resonance assays. Epigallocatechin gallate (EGCG), the major green tea catechin, targets an early stage of the replication cycle of both H1N1 and H3N2 with EC50 values of 5.7 to 17.3 micromolar, and oral administration reduced TLR4 and NF-κB protein levels in a mouse model of H9N2-induced lung injury. Curcumin, the polyphenol from turmeric, engaged TLR, MAPK, and NF-κB signaling concurrently while directly inactivating virions and blocking viral adsorption, and in mice it reduced lung viral titers, inflammatory cytokines, and mortality. Carvacrol, a phenolic compound from Mosla chinensis, downregulated TLR7, RIG-I, MyD88, and NF-κB in infected mice while reducing lung viral RNA load and pulmonary injury.

The RIG-I pathway presents a more nuanced picture. This cytosolic receptor detects viral RNA and, through the mitochondrial adaptor MAVS, activates IRF3 and NF-κB to drive interferon production and chemokine release, yet some studies suggest RIG-I-mediated recruitment of monocyte-derived dendritic cells can paradoxically enhance viral replication in infected lungs. The flavonoid apigenin suppresses this pathway by promoting RIG-I degradation: it disrupts the stabilizing interaction between RIG-I and the chaperone Hsp90α, enhancing ubiquitination by the E3 ligase RNF125 and proteasomal destruction of the receptor. This reduces the cell’s capacity to sense viral RNA and mount type I interferon responses, potentially limiting virus-induced inflammation, and apigenin also dose-dependently decreased expression of viral proteins NS1, NP, M1, and M2 in A549 cells. Similarly, the triterpenoid pterodontic acid reduced viral propagation by inhibiting viral ribonucleoprotein export while lowering RIG-I expression and downstream inflammatory mediators, and the phytosterol β-sitosterol disrupted RIG-I-mediated STAT1 activation, reducing IL-6, IL-8, TNF-α, and IP-10 production.

The MAPK pathway illustrates perhaps the most mechanistically interesting dimension of phytochemical action, because different compounds manipulate the same kinases in opposite directions with antiviral benefit in both cases. ERK1 signaling facilitates the nuclear export of viral ribonucleoprotein complexes, a critical step in the IAV replication cycle, so blocking ERK phosphorylation directly restricts propagation. The isoquinoline alkaloid berberine blocked H1N1 growth in macrophages and epithelial cells by interfering with ERK1-mediated vRNP nuclear export, while patchouli alcohol, a sesquiterpene from Pogostemonis herba, suppressed ERK1/2 activation with clear anti-influenza activity across multiple H1N1 strains. Conversely, EGCG and the compound DMO-CAP activate rather than inhibit MAPK cascades to enhance antiviral host programs: EGCG induces human β-defensin 3 and IFN-λ2 expression through p38, ERK, and JNK activation, while DMO-CAP triggers the Nrf2/HO-1 axis, which amplifies interferon-stimulated genes. This apparent paradox is resolved by recognizing that ERK inhibition disrupts pro-viral vRNP export whereas ERK activation promotes antiviral interferon induction, both mediated through the same kinase.

NF-κB adds a further layer of complexity because it serves the virus directly as well as the host. Beyond driving inflammatory gene expression, NF-κB signaling is required for viral genomic RNA synthesis and induces pro-apoptotic factors such as TRAIL and Fas ligand, which facilitate caspase-mediated nuclear export of vRNPs. Pterodontic acid exploits this dependency by inhibiting TRAIL and FasL expression, downregulating caspase-3/7 activity and impairing vRNP export. The flavonoid cirsimaritin reduced NF-κB p65 nuclear phosphorylation in infected THP-1 cells while suppressing multiple IAV subtypes with IC50 values of 5.8 to 11.1 micrograms per milliliter, and andrographolide, a diterpenoid from Andrographis paniculata, increased survival and reduced viral loads in a lethal H1N1 mouse model, with a combination study showing that pairing it with a viral entry inhibitor produced greater survival benefit than either compound alone. Among extracts, total flavonoids from Mosla scabra reduced lung viral load and inflammatory infiltration in mice more effectively than amantadine at the highest dose tested, and total flavones from Abelmoschus manihot suppressed viral RNA by day three post-infection in vivo.

The review is candid about the field’s weaknesses, and these are substantial. Many studies assess cytokine markers without measuring infectious viral titers, making it impossible to distinguish direct antiviral effects from indirect immunomodulation that merely creates a less permissive environment. Molecular binding targets are rarely identified, and the concentrations used in cell culture consistently exceed what is pharmacokinetically achievable in human lung tissue. Quercetin’s oral absorption in humans ranges from only 3 to 17 percent, with peak plasma levels rarely exceeding 1 to 2 micromolar, far below the 10 to 50 micromolar concentrations used in vitro. Berberine’s oral bioavailability falls below 5 percent due to P-glycoprotein efflux and hepatobiliary re-excretion, β-sitosterol’s is approximately 0.41 percent, and EGCG undergoes extensive gastrointestinal degradation. No pharmacokinetic data exist for any compound reviewed in bronchoalveolar lavage fluid or lung tissue, the primary site of replication, and safety concerns such as EGCG-associated hepatotoxicity at high supplemental doses and CYP enzyme inhibition that could affect co-prescribed drugs add further complications.

Despite these barriers, the review identifies a clear path forward. Because TLR and RIG-I signaling converge on shared components such as TRAF6, TAK1, and IRF3, compounds like miquelianin and carvacrol can engage multiple pathways through a single molecular target, functioning as modulators of an integrated innate immune network rather than selective inhibitors of discrete cascades. The authors recommend that molecular target identification through thermal shift assays and docking validation become standard practice, that pharmacokinetic evaluation be extended to respiratory tissue rather than plasma alone, that human airway organoid models serve as physiologically relevant intermediates, and that crude extracts undergo bioassay-guided fractionation to define active constituents. Clinical translation to date has involved complex extracts such as standardized black elderberry and Cistus incanus preparations, with limited or inconsistent results, and none of the defined phytochemicals discussed has yet entered a dedicated anti-influenza trial. If the recommended mechanistic rigor is adopted, the structural diversity and broad immunomodulatory activity of these plant compounds represent a genuine and underexploited opportunity for host-directed adjunctive therapy against a virus that continues to outpace the limited drugs available against it.

Subject of Research: Immunomodulatory and antiviral effects of phytochemicals on host signaling pathways during influenza A virus infection

Article Title: Dual Antiviral and Immunomodulatory Effects of Phytochemicals in Influenza A Virus Infection: Targeting Key Host Signaling Pathways

Article References: Chowdhury, D., Vanderven, H. A., Wangchuk, P., & Sarker, S. (2026). Dual Antiviral and Immunomodulatory Effects of Phytochemicals in Influenza A Virus Infection: Targeting Key Host Signaling Pathways. MicrobiologyOpen, 15(5), Article e70424. https://doi.org/10.1002/mbo3.70424

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DOI: 10.1002/mbo3.70424

Keywords: influenza A virus, phytochemicals, TLR signaling, RIG-I, MAPK, NF-κB, cytokine storm, flavonoids, antiviral therapy, immunomodulation, host-directed therapy, natural products

The research team set out to determine whether dampening this kinase pathway could influence the course of infection with A/Puerto Rico/8/1934, the laboratory H1N1 strain universally abbreviated as PR8. Their experimental design was deliberately multi-layered. In cell culture, they infected susceptible cells with PR8 and treated them with GSK583 at defined concentrations, then quantified viral RNA and viral protein accumulation inside the cells. To measure the production of infectious progeny, they performed plaque-forming assays on culture supernatants, a classical virological technique in which diluted samples are overlaid onto fresh monolayers of cells and each infectious particle gives rise to a visible zone of cytopathic effect. In parallel, they moved to a mouse model of PR8 infection, treating animals early after inoculation and tracking body weight, disease severity, pulmonary viral load, and inflammatory markers over the course of the experiment.

The cellular results were consistent and pointed in a single direction. GSK583 reduced both the amount of viral RNA and the abundance of viral protein within infected cells, and supernatants collected from treated cultures contained significantly fewer plaque-forming units, indicating that the compound suppressed the release of infectious virions rather than merely distorting intracellular readouts. Importantly, the investigators used time-of-addition experiments to pinpoint where in the viral life cycle the drug exerted its effect. In this approach, the compound is added at different times relative to infection — before the virus is introduced, during the attachment phase, or only after adsorption has been completed. If a drug directly inactivates virions or blocks their binding to cell surface receptors, it must be present early to show activity. GSK583 failed to directly inactivate PR8 and did not measurably inhibit viral attachment under the conditions tested; instead, its antiviral effect became detectable only after the virus had adsorbed to cells. This pattern is characteristic of a compound that interferes with post-entry stages of replication or with host signaling pathways that the virus co-opts.

Having established antiviral activity in vitro, the team turned to the mouse model, where the stakes for host-directed therapy are higher because the immune system contributes both to viral clearance and to tissue damage. When GSK583 was administered early after infection, treated animals experienced alleviated disease severity compared with untreated controls. Quantitative assessment of lung tissue revealed reduced signals for the viral nucleoprotein — both its RNA transcripts and the antigen itself — indicating that viral replication in the respiratory tract was genuinely blunted, not simply redistributed. Alongside the reduction in viral load, the treated mice showed lower inflammatory readouts in the lung. This dual effect is notable because excessive inflammation is a hallmark of severe influenza, contributing to acute lung injury and, in the worst cases, acute respiratory distress syndrome, a syndrome that the authors explicitly reference in framing the clinical significance of their work.

The mechanistic core of the study concerns the RIPK2–MKK7–JNK signaling module. RIPK2 is a kinase with well-documented roles in innate immunity, particularly as an adaptor that transduces signals from NOD-like pattern recognition receptors, and the mitogen-activated protein kinase cascade it engages includes MKK7, a dual-specificity kinase that phosphorylates and activates JNK. JNK, in turn, is a stress-activated protein kinase whose phosphorylation status is a sensitive indicator of inflammatory and stress signaling intensity. Using protein interaction assays, the investigators demonstrated that the physical association between RIPK2 and MKK7 was linked to PR8 replication-related readouts under their experimental conditions, and that JNK phosphorylation tracked with viral replication. Genetic knockdown experiments and pharmacological inhibition corroborated the involvement of this axis, providing convergent lines of evidence rather than relying on a single technique.

Against this mechanistic backdrop, the effect of GSK583 became interpretable. Treatment with the compound was accompanied by a reduction in the RIPK2–MKK7 interaction and a corresponding decrease in JNK phosphorylation, findings consistent with the hypothesis that the drug works by loosening a signaling complex that influenza virus either requires or exploits. The authors are careful in their language here: they describe the data as consistent with a potential link between the signaling module and the observed antiviral effect, rather than claiming definitive causation. This restraint is scientifically appropriate, because RIPK2 has pleiotropic functions and inhibitor specificity is never absolute, a caveat the team acknowledges directly when they call for further studies to define the mechanistic specificity of the pathway in influenza infection.

Beyond the JNK arm, the study documented effects on a second major inflammatory pathway. GSK583 treatment reduced the phosphorylation of NF-κB p65, the canonical subunit of the transcription factor that drives expression of a large suite of pro-inflammatory genes, and it lowered the transcript levels of selected inflammatory cytokines in infected cells. The intersection of these two pathways is significant for influenza biology. The JNK and NF-κB cascades are both activated during influenza infection, both contribute to cytokine storm pathology, and both have been implicated in supporting various stages of the viral life cycle in prior literature. A single compound that dampens phosphorylation events in both cascades while simultaneously reducing viral replication therefore offers a pharmacologically attractive profile, at least in the models examined.

The translational implications deserve careful framing. GSK583 was developed in an industrial setting as a RIPK2 inhibitor, and its repurposing for influenza represents the kind of cross-indication thinking that host-directed antiviral research increasingly encourages. Because the target is a host protein, the theoretical barrier to viral escape is much higher than for virus-targeted drugs: the virus cannot simply mutate its own polymerase to render a host-directed compound obsolete. Moreover, a host-directed agent could in principle retain activity against multiple influenza subtypes and even against unrelated respiratory viruses that co-opt overlapping signaling networks. However, host-directed approaches carry their own risks, since interfering with innate immune signaling can impair genuine host defense, and the therapeutic window must be established empirically. The mouse data showing alleviated disease severity with early treatment are encouraging on this point, but early intervention windows are a known limitation of antiviral therapy in practice, because patients frequently present after peak viral replication has occurred.

The study’s methodological rigor is worth noting for readers assessing the strength of the conclusions. Cytotoxicity considerations were built into the design, with the abstract referencing the half-maximal cytotoxic concentration, the half-maximal inhibitory concentration, and the selectivity index — standard pharmacological metrics that distinguish genuine antiviral activity from nonspecific toxicity. Statistical analyses employed Student’s t-tests, analysis of variance, and log-rank tests as appropriate to the data structure, and all animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. The authors also flag that the paper was released as an accepted manuscript under accelerated sharing, subject to final editorial production, and they explicitly position their findings as proof-of-concept rather than as a validated therapeutic strategy.

What, then, does this work contribute to the field? It extends the map of host factors that influenza virus interacts with, placing RIPK2-associated signaling squarely within the network of pathways whose manipulation affects viral replication. It provides a concrete pharmacological tool — a compound with an existing development history — that the community can use to probe this pathway further. And it demonstrates, in both cellular and animal systems, that attenuating this signaling module can simultaneously reduce viral load and inflammatory output, addressing the two faces of influenza pathology in a single intervention. The open questions are equally clear: whether the RIPK2–MKK7 interaction is a direct requirement of the virus or an indirect consequence of altered cellular physiology, whether the effect generalizes beyond PR8 to circulating human strains and other subtypes, and whether the therapeutic window in mammals is wide enough to support clinical development. The authors’ call for further studies on mechanistic specificity and broader applicability is the appropriate next step, and the present findings supply a well-documented starting point for that work.

Subject of Research: Host-directed antiviral activity of the RIPK2 inhibitor GSK583 against A/PR/8 H1N1 influenza virus infection and associated inflammatory signaling

Article Title: GSK583 attenuates viral and inflammatory readouts in an A/PR/8 H1N1 infection model

Article References: Kuang, J., Zhou, J., Xiang, J., Ren, F., Fu, J.-J., Zhu, G., Li, Z., Ruan, H., Tan, X., Zhang, W., Zhou, M., Xu, J., Yang, Q., & Shang, Y. (2026). GSK583 attenuates viral and inflammatory readouts in an A/PR/8 H1N1 infection model. Virology Journal. https://doi.org/10.1186/s12985-026-03283-2

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03283-2

Keywords: GSK583, RIPK2, influenza A virus, H1N1, JNK signaling, MKK7, NF-κB, host-directed antivirals, PR8 mouse model, inflammation, viral replication, antiviral therapy

The hepatitis B virus establishes its persistence through a peculiar molecular intermediate known as covalently closed circular DNA, or cccDNA. After the virus enters a hepatocyte, its partially double-stranded circular genome travels to the nucleus, where host repair enzymes convert it into a fully double-stranded, covalently closed circle. This minichromosome behaves remarkably like a cellular chromosome: it is wrapped in histone proteins, carries epigenetic marks, and serves as the template for all of the viral RNA transcripts that drive the production of new virions and, critically, of hepatitis B surface antigen, the viral protein that accumulates to enormous levels in the blood of chronically infected patients and is thought to exhaust and dysregulate antiviral immune responses. Because cccDNA is extraordinarily stable and can persist in dividing and non-dividing hepatocytes alike, clearing it has been the elusive goal of hepatitis B research for decades.

Existing treatments manage the consequences of cccDNA activity rather than the reservoir itself. Nucleoside and nucleotide analogues, such as tenofovir and entecavir, potently inhibit the viral polymerase and prevent new rounds of replication, but they leave cccDNA untouched, which is why discontinuation almost universally leads to viral rebound. Pegylated interferon-alpha can produce functional control in a minority of patients, likely through a combination of direct antiviral and immunomodulatory effects, but its tolerability is poor and its cure rates remain low. The result is that most patients face lifelong daily therapy, and the dream of a finite treatment course that leaves the liver free of active viral transcription has remained out of reach. Epigenetic silencing offers a conceptual alternative: if the viral minichromosome can be rendered permanently or durably transcriptionally silent, the virus may be functionally inactivated even though its DNA physically remains.

The investigational therapy described in the new study is built on this premise. It is designed as an epigenetic silencer, a construct that targets the hepatitis B viral DNA and recruits repressive chromatin-modifying machinery to the viral genome, thereby converting the active cccDNA minichromosome into a heterochromatic, transcriptionally inert state. In practical terms, the therapy aims to deposit repressive histone marks and promote DNA methylation patterns at the viral regulatory elements that normally drive transcription of all the viral RNAs, including the pregenomic RNA that seeds new replication and the abundant subgeniral RNAs that encode surface and core proteins. By shutting down transcription at its source, the approach addresses both the production of infectious virus and the antigenemia that blunts immune recovery.

The engineering challenge at the heart of such a therapy is specificity. The human liver contains roughly two hundred billion hepatocytes, and the silencing machinery must find the small fraction of cells harboring transcriptionally active cccDNA while leaving the host genome and its epigenetic landscape untouched. The reported system couples a DNA-binding module that recognizes sequences within the hepatitis B genome to effector domains capable of recruiting repressive complexes. Because the viral regulatory region, including the core promoter and enhancer elements, contains sequence features that are conserved across hepatitis B genotypes but absent from the human genome in the same configuration, the design exploits a natural sequence boundary between pathogen and host. Delivery to hepatocytes is achieved with a liver-directed formulation intended to concentrate the payload in the organ where more than ninety percent of viral replication occurs.

Epigenetic silencing of viral genomes is not an entirely new idea, but translating it into a viable therapy has required overcoming several biological realities. The first is that cccDNA is not a static target: it exists in variable copy numbers per cell, ranges from fully active to partially silenced states even within a single liver, and can be replenished from integrated viral DNA in some patients. The second is reversibility. Epigenetic marks are, by definition, modifiable, and a silenced minichromosome could in principle be reactivated if repressive marks decay or if the cell encounters inflammatory or proliferative signals that remodel chromatin. The investigators therefore designed the therapy not merely to deposit transient repressive marks but to establish a self-reinforcing silenced state, in which repressive chromatin at the viral promoter recruits further repressive machinery, creating a memory of inactivation that persists across time and, in dividing cells, across cell generations.

The functional consequences of such silencing would extend beyond simply lowering viral load. Hepatitis B surface antigen is now understood to be a principal driver of immune dysfunction in chronic infection, with circulating subviral particles reaching concentrations of micrograms per milliliter and contributing to the exhaustion of natural killer cells, T cells, and B cells that would otherwise clear infected hepatocytes. A therapy that transcriptionally inactivates cccDNA would be expected to produce a steep and sustained decline in surface antigen, removing this immunosuppressive burden and creating the conditions under which the patient’s own immune system can recognize and eliminate residual infected cells. In this sense, epigenetic silencing is best understood not as a standalone cure but as the keystone of a combination strategy, in which silencing of the reservoir is followed or accompanied by therapeutic vaccination, immune checkpoint modulation, or other immunorestorative interventions that consolidate the gain.

Safety considerations loom large for any therapy that deliberately manipulates chromatin. Broadly acting epigenetic drugs, such as the histone deacetylase inhibitors developed in oncology, carry systemic toxicity profiles that make them unattractive for chronic liver disease. The investigational silencer is therefore designed to act locally and selectively, with its repressive activity confined to the viral sequences it is targeted to. Another concern is the fate of integrated hepatitis B DNA, which in many chronic infections is scrambled into host chromosomes and cannot be circularized or fully silenced by the same mechanism; integrated DNA is a known source of surface antigen production and, in some contexts, of insertional mutagenesis that contributes to hepatocellular carcinoma. The therapy’s developers and independent commentators alike emphasize that silencing cccDNA addresses the replicative reservoir but does not by itself excise integrated sequences, underscoring that long-term monitoring for surface antigen rebound and for liver cancer risk will remain necessary even in successfully treated patients.

The publication arrives at a moment of unusual momentum in hepatitis B cure research. Dozens of candidates are advancing through clinical pipelines, including entry inhibitors, capsid assembly modulators, RNA interference therapeutics that degrade viral transcripts, surface antigen secretion inhibitors, and therapeutic vaccines. RNA interference drugs in particular have demonstrated that reducing viral antigen is feasible and well tolerated, and several have advanced to late-stage trials, though rebound after discontinuation has highlighted the persistence of the cccDNA reservoir that RNA-based approaches cannot touch. An epigenetic silencer occupies a distinctive position in this landscape because it acts on the reservoir itself, at the level of the minichromosome, rather than on the RNA or protein products that flow from it. If its silenced state proves durable, it could provide the reservoir control that RNA interference and direct-acting antivirals lack, while complementing the immune-directed agents needed to finish the job.

Much work remains before such a therapy could reach patients. The path from proof of concept in laboratory and animal models of hepatitis B infection to demonstrated safety and efficacy in humans is long, and epigenetic therapies will face scrutiny from regulators accustomed to evaluating small molecules and antibodies. Questions about the completeness of silencing across the enormous heterogeneity of cccDNA populations, the behavior of the therapy in patients with high viral loads and advanced fibrosis, the potential for rare escape variants in the targeted viral sequences, and the reversibility of the silenced state over years rather than weeks will all need rigorous answers. Nevertheless, the development of an investigational therapy designed to transcriptionally inactivate viral DNA marks a conceptual milestone: it treats the hepatitis B minichromosome not as an untouchable fixture of chronic infection but as an epigenetic state that can be rewritten. For the hundreds of millions of people living with chronic hepatitis B, the prospect of a finite course of treatment that leaves the virus silent, the immune system restored, and the liver protected represents a goal that has never been closer to serious experimental pursuit.

Subject of Research: Development of an investigational epigenetic silencer therapy that transcriptionally inactivates hepatitis B viral DNA in chronic hepatitis B

Article Title: Development of an investigational epigenetic silencer therapy to transcriptionally inactivate viral DNA in chronic hepatitis B

Article References: Anglero-Rodriguez, Y., Xiong, Q., Cappelluti, M. A., Voytek, S. B., Acosta, G., Choo-Wing, R., Hoffman, L., Khadka, A., Medina, J. A., Mugambwa, C., Pantano, C., Harel, S., DiPiazza, A., Abraham, S., Hildebrand, E. M., Ramirez, R. N., Zhai, A., Guo, X., Abubucker, S., … Marlowe, J. L. (2026). Development of an investigational epigenetic silencer therapy to transcriptionally inactivate viral DNA in chronic hepatitis B. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-026-01802-8

Image Credits: AI Generated

DOI: 10.1038/s41551-026-01802-8

Keywords: chronic hepatitis B, hepatitis B virus, cccDNA, epigenetic silencing, viral DNA, hepatitis B surface antigen, antiviral therapy, chromatin modification, functional cure, liver disease, RNA interference, transcriptional repression

Yet the numbers remain sobering. According to the review, even with the best currently available regimens, the functional cure rate among patients with chronic hepatitis B stays below 30 percent, and in many treatment settings it is far lower. Nucleos(t)ide analogues such as entecavir and tenofovir suppress viral replication with remarkable potency and an excellent safety profile, but they act on the reverse transcriptase step of the hepatitis B virus life cycle and leave the intracellular reservoirs of viral genetic material untouched. Pegylated interferon-alpha, the other pillar of standard therapy, can reinvigorate antiviral immune responses in a subset of patients, but response rates are limited and tolerability issues frequently force discontinuation. The result is a therapeutic plateau: viral suppression is easy to achieve, but genuine immune-mediated control of the infection remains the exception rather than the rule.

At the heart of this plateau, the authors identify the persistence of covalently closed circular DNA, or cccDNA, as the central virological obstacle. cccDNA is the circular, episodic form of the hepatitis B virus genome that resides in the nucleus of infected hepatocytes and serves as the transcriptional template for all viral RNAs, including the pregenomic RNA from which new viral particles are assembled. Because cccDNA is stable, long-lived and present in minute quantities per cell, it evades both the cytoplasmic degradation pathways triggered by nucleos(t)ide analogues and the surveillance of the immune system, which struggles to recognize infected hepatocytes that produce little or no viral antigen. Clearing cccDNA from the liver would require either eliminating every infected hepatocyte or inducing selective degradation of the minichromosome itself, and neither approach is currently achievable with acceptable safety in the overwhelming majority of patients.

Compounding the cccDNA problem is a second form of viral persistence that the review highlights in detail: the integration of hepatitis B virus DNA into the genome of the host hepatocyte. Integration occurs frequently during the chronic phase of infection, driven by the_error-prone nature of viral replication and the continual turnover of infected cells. Integrated viral sequences are typically rearranged and replication-defective, but they can act as persistent factories for hepatitis B surface antigen, sustaining high circulating levels of HBsAg even when cccDNA transcription has been effectively silenced. This has two major consequences. First, integrated DNA means that surface antigen loss cannot automatically be equated with the elimination of the viral reservoir, complicating the interpretation of treatment endpoints. Second, chromosomal integration is a recognized mutagenic event, and insertional disruption of host genes such as TERT has been implicated in the development of hepatocellular carcinoma, linking viral persistence directly to the most feared clinical outcome of chronic infection.

The review then turns to the immunological face of the problem, describing how prolonged exposure to elevated levels of viral antigens — above all hepatitis B surface antigen and hepatitis B e antigen — induces a state of immune tolerance that is sculpted from the very earliest stages of infection. In patients infected at birth or in early childhood, the developing immune system encounters enormous quantities of circulating antigen during the critical windows in which tolerance mechanisms are established. High antigen loads drive the deletion or functional silencing of HBV-specific T cells, promote the differentiation of regulatory T cells and exhaust antigen-presenting dendritic cells, so that by the time chronicity is established the immune system has been trained, in effect, to ignore the virus. Maternal antigen passage and the unique tolerogenic environment of the liver, which is continuously exposed to gut-derived material and normally biased toward immune non-responsiveness, reinforce this state and help explain why infection acquired early in life so rarely resolves spontaneously.

Even in patients whose T cells do persist, the review documents a landscape of immune exhaustion and dysfunction operating at multiple regulatory levels. HBV-specific CD8 T cells in chronic hepatitis B display the classic molecular signature of exhaustion: sustained expression of inhibitory receptors such as PD-1, CTLA-4, TIM-3 and LAG-3, shortened telomeres, impaired cytotoxic function and a shift toward a terminally differentiated, senescent phenotype. CD4 helper function is similarly compromised, weakening the support that CD8 cells require for sustained antiviral activity. Beyond the T cell compartment, natural killer cell function is dysregulated, B cell responses are blunted with defective production of neutralizing anti-HBs antibodies, and the intrahepatic cytokine milieu — enriched in suppressive mediators such as interleukin-10 and indoleamine 2,3-dioxygenase — actively restrains inflammatory responses. Exhausted T cells, the authors emphasize, are not simply passive casualties; they represent a partially reversible differentiation state, which is precisely why they have become the focal point of new immunotherapeutic strategies.

It is against this mechanistic backdrop that the review surveys the emerging arsenal of immunotherapy. Therapeutic vaccines aim to present HBV antigens in an immunogenic context that can break tolerance and prime new antiviral T cell responses. Checkpoint inhibitors, most notably antibodies directed against PD-1 or PD-L1, seek to reverse the exhausted state of HBV-specific T cells, an approach supported by incidental observations of HBsAg decline in cancer patients treated with such agents. Agonists of pattern-recognition receptors, including Toll-like receptor 7 and 8 agonists, are designed to reawaken innate antiviral defenses and restore interferon production within the liver. Additional strategies under investigation include engineered T cell technologies, cytokine modulation, and combinations that pair direct-acting antivirals with immune interventions to first reduce antigen load and then rebuild antiviral immunity. The authors stress, however, that no single agent has yet demonstrated the capacity to induce functional cure reliably, and that the field’s most promising direction lies in rational combination: reducing the antigenic burden, releasing the brakes on T cell function, and providing the innate cytokine signals needed to coordinate a durable multi-layered response.

The review also underscores why the liver’s unique immunobiology makes this task so difficult. The hepatic environment is constitutively tolerogenic, populated by liver sinusoidal endothelial cells, Kupffer cells and stellate cells that favor the induction of regulatory rather than effector T cell responses. Any curative strategy must therefore overcome not only the viral reservoirs and the exhaustion of the adaptive immune system, but also an organ-specific architecture that has evolved to suppress inflammation. Biomarker development is identified as an equally pressing need: quantitative HBsAg levels, hepatitis B RNA signatures and emerging measures of immune restoration are all being explored as tools to identify which patients are most likely to benefit from which combination, and to shorten the lengthy trials that current endpoints demand.

By synthesizing virological persistence mechanisms, the layered failure of antiviral immunity and the emerging immunotherapeutic toolkit into a single framework, Lin, Yu and Sun aim to provide both a theoretical foundation and practical critical perspective for the clinical translation of functional cure strategies. Their central message is one of mechanistic realism: the obstacles to curing chronic hepatitis B functionally are not a single barrier but an interlocking system of viral reservoirs, integrated DNA, antigen-driven tolerance and multi-level immune exhaustion. Understanding how these pieces reinforce one another — and designing combinations that dismantle them in the correct sequence — is, the authors conclude, the surest path toward turning a goal achieved by fewer than a third of patients today into a routine clinical outcome tomorrow.

Subject of Research: Immunological mechanisms and barriers to achieving a functional cure for chronic hepatitis B

Article Title: The core obstacles and immunological mechanisms in the study of functional cure for Chronic Hepatitis B

Article References: Lin, L., Yu, D., & Sun, S. (2026). The core obstacles and immunological mechanisms in the study of functional cure for Chronic Hepatitis B. Virology Journal. https://doi.org/10.1186/s12985-026-03310-2

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03310-2

Keywords: chronic hepatitis B, functional cure, HBV, cccDNA, HBsAg loss, immune exhaustion, viral integration, immunotherapy, T cell dysfunction, immune tolerance, liver disease, antiviral therapy