Dengue, an Aedes mosquito-borne viral infection, is on the rise with climate and demographic change. In 2023, WHO declared dengue its highest grade of emergency, following the largest number of cases and deaths in recorded history. This emergency highlighted the absence of safe and effective dengue-specific treatments as a gap in the product landscape for care for people with dengue. To accelerate development of dengue-specific treatments for wide implementation and access in all dengue-endemic countries, WHO conducted a landscape analysis of the dengue therapeutics pipeline and convened a global expert and public consultation to develop target product profiles for treatments for non-severe and severe dengue. These target product profiles provide strategic guidance for product developers, regulators, procurement agencies, and funders on the intended use, target populations, and key product characteristics of dengue-specific treatments required to meet public health needs, setting clear targets to drive development of dengue drugs accessible to those who need them the most.

Developing drugs to treat rhinovirus (RV) infections remains a serious and unresolved problem. Pleconaril is the most widely studied broad-spectrum RV inhibitor. Fewer than 10 % of circulating RV types are naturally resistant to pleconaril, which is why researchers continue to study it as a basis for creating new, more active molecules. We conducted a comprehensive study on modifying the pleconaril molecule and demonstrated that the isoxazole ring is crucial for determining anti-RV activity. Based on these findings, we aimed to further study the pleconaril core to discover new therapeutic candidates. We synthesized a large series of new pleconaril derivatives with mono- or di-substituted central phenyl rings, modified linkers, and substitutions on the isoxazole or 1,2,4-oxadiazole moieties. We evaluated the cytotoxicity and inhibitory activity of the obtained compounds using RV-A2 and RV-B14 strains in HeLa cells. To explain the differences in inhibitory profiles, we performed molecular dynamics simulations. Results from the structure-activity relationship analysis revealed that the substituents on the isoxazole and 1,2,4-oxadiazole rings, as well as the linker, play a decisive role in the anti-RV activity profile of monomethylated pleconaril derivatives. We demonstrated that the movement of two loops at the entrance of the VP1 binding pocket of the capsid protein and the inhibitor molecules correlates with the stability of the complex, and therefore, with efficacy against RV. Compounds 1q, 1gg, 2g, and 4c were identified as potent new RV inhibitors from this series of compounds. These compounds are promising new candidates for lead optimization and preclinical studies in the development of antiviral drugs against RV.

Almost half of the world's population is at risk of acquiring dengue virus (DENV) each year. However, no specific licensed prophylactic or antiviral treatment for dengue currently exists. Mosnodenvir, a novel DENV inhibitor, has been shown to inhibit DENV replication in vitro and in animal studies. Here, we provide new insights into the in vivo prophylactic inhibitory effect of mosnodenvir exposure on primary DENV-2 infection by fitting mechanistic within-host models of DENV infection to virological and serological data observed from pre-clinical challenge studies in AG129 mice and rhesus macaques. We estimated a median mosnodenvir concentration achieving 50% of maximal inhibitory effect (IC50) on viral replication of 8.35 (6.82, 9.22) ng ml-1 and 7.61 (5.67, 8.92) ng ml-1 for AG129 mice and rhesus macaques, respectively. A higher concentration is typically required to suppress viral replication in AG129 mice compared with rhesus macaques owing to a higher estimated within-host basic reproduction number (R0) in mice. By integrating multiple data types in a single framework, this study enhances our understanding of the within-host dynamics of primary DENV infection in non-human host species. Furthermore, the methods developed here could possibly assist in quantifying the prophylactic inhibitory effect of mosnodenvir on DENV infections in humans.

Remdesivir (RDV), a nucleoside analog targeting viral RNA-dependent RNA polymerase (RdRp), suffers from critical limitations including low oral bioavailability, plasma instability, and reliance on intravenous administration, hindering its clinical utility. To address these issues, we designed and synthesized 28 novel N-acylated derivatives by modifying the amino group on remdesivir's triazine ring. The lead compound 4f exhibited potent anti-SARS-CoV-2 activity (EC50 = 0.0862 μM) in Vero E6 cells, comparable to remdesivir (EC50 = 0.0459 μM), with reduced cytotoxicity (CC50 = 15.6 μM, SI = 181.0). Notably, 4f displayed broad-spectrum efficacy against human coronaviruses (HCoV-229E and HCoV-OC43), zika virus (ZIKV), and yellow fever virus (YFV), matching remdesivir's potency. Pharmacologically, 4f showed significantly enhanced plasma stability (half-life >8 h vs. 2 h for RDV) and reduced CYP3A4 inhibition (23.8% vs. 57.4% for RDV at 10 μM). In vivo pharmacokinetics confirmed its superior oral bioavailability (45.3% based on active metabolite GS-441524 exposure), efficiently delivering GS-441524 into systemic circulation. This N-acylation strategy overcomes key pharmacokinetic barriers of remdesivir while preserving broad-spectrum antiviral activity, highlighting 4f as a promising oral candidate.

The recent outbreak of Oropouche virus (OROV) in Latin-America with unprecedented reports of vertical transmission resulting in congenital malformations and fetal death, redefines the threat posed by this arbovirus. We discover that injection of a low inoculum of the epidemic OROVIRCCS-SCDC_1/2024 strain into the placenta of Swiss mice results in viral replication across embryonic organs, in particular the brain. Infected embryos present with multi-organ pathology, pneumonitis, myocarditis, steatohepatitis and severe neuropathology, including microcephaly, ventriculomegaly and neural loss. Neural cell populations are drastically altered due to massive cell death. OROV-induced neuroinflammation is marked by cytokine upregulation, microglial activation and neutrophil infiltration. When we compare to Zika virus (ZIKV) infection in the same model, a much lower inoculum of OROV than of ZIKV results in embryonic malformations, with markedly distinct neurological developmental and immune responses. Our findings establish a link between congenital OROV infection and neurodevelopmental disease, underscoring its teratogenic potential. Using a mouse model of congenital infection, researchers show that Oropouche virus causes severe brain damage and developmental defects in the fetus, highlighting its potential to cause congenital disease in humans.

Molnupiravir, an oral prodrug of β-D-N4-hydroxycytidine (NHC), is a broad-spectrum antiviral agent. However, its clinical efficacy is hampered by suboptimal plasma stability and inefficient intracellular monophosphorylation. To circumvent these pharmacological limitations, a novel series of NHC dual-prodrug derivatives was designed and synthesized by integrating 5'-ProTide and N4-ester modification strategies. Structure-activity relationship (SAR) analysis revealed that derivatives functionalized with short-chain N4-esters achieved a more favorable balance between antiviral potency and cytotoxicity. Specifically, 5b exhibited favorable anti-SARS-CoV-2 activity with low cytotoxicity, displaying anti-SARS-CoV-2 activity (EC50 = 3.22 μM) with negligible cytotoxicity. Subsequent mechanistic studies supported that the ProTide moiety mediates rapid enzymatic activation, effectively bypassing the rate-limiting first phosphorylation step to release the nucleoside monophosphate. Furthermore, pharmacokinetic evaluations revealed a tunable profile: 4 demonstrated significantly enhanced metabolic stability in human plasma, suggesting its potential as a long-circulating systemic reservoir, whereas 5b displayed favorable in vitro antiviral activity, likely associated with its balanced physicochemical properties and rapid esterase-mediated conversion to the more plasma-stable ProTide intermediate 4. Consequently, these findings demonstrate prodrugs 4 and 5b represent promising antiviral agents with favorable metabolic stability and high potency.

Harbingers of infectious viral pandemics, such as the H1N1 influenza, SARS, Zika, Ebola, MERS, and SARS-CoV-2, caused major outbreaks in the first two  decades of the 21st century. Despite warnings, therapeutic tools that could be rapidly and sustainably scaled at a global level when SARS-CoV-2 emerged were lacking. Small-molecule antivirals can play a crucial role in both individual patient care and broader public health strategies for controlling and mitigating the impact of viral diseases. Despite their utility, the lack of R&D investment in this class of intervention has prevented the world from reaping the benefits they can deliver. The INTREPID Alliance 2025 publication of the Antiviral Clinical and Preclinical Development Landscape-4th Edition, revealed significant gaps in the development pipeline. No antivirals are in clinical development for 4 of the 13 viral families designated by the World Health Organization as viral families of pandemic and endemic concern.

Developing antiviral agents for RNA viruses is crucial due to their global health impact, high mutation rates, and pandemic potential. Most RNA virus infections still lack vaccines or specific antiviral treatments. New spirooxindole-benzothiazole hybrids (4a-e and 5a-m) incorporating a trifluoromethyl moiety were synthesized from treating 1H-indole-2,3-diones with 2-amino-4-(trifluoromethyl)benzenethiol in ethanol. Structures of compounds 4a-e and 5a-m were confirmed by analytical and spectral data and single crystal X-ray analysis. The antiviral effects of the compounds were tested against Chikungunya virus (CHIKV), Parainfluenza virus 3 (PIV3), Zika virus (ZIKV), and Enterovirus 71 (EV-A71). Compounds 4a-c, 5c-h, and 5j-m against CHIKV, and compounds 4a, 5d, 5g, and 5l against PIV3 were selected for further evaluation. R1= methyl and R2= 5,7-dichlorine substituted compound 5m was found to have significant antiviral activity against CHIKV (EC50= 0.86 mu M, SI= 10-20). The three-dimensional structures of compounds 5l and 5m were determined by single crystal X-ray analysis, and combined molecular docking and molecular dynamics simulation studies were performed to predict their interactions with PIV3 and CHIKV.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus primarily infecting equines, causing severe illness or death. The virus can also infect humans causing severe neurological complications. There are no antivirals or human vaccines available. Although major outbreaks have been relatively rare in recent years, there is a need for antiviral drugs to combat this virus, both as a precaution against epidemics and for biological defense. In this study, we developed a rapid, fluorescence-based antiviral assay using recombinant VEEV TC-83 expressing the reporter Azurite. Two different strategies were compared (i) the nsP3–Azurite fusion construct (VEEV-nsP3-Azurite), in which the reporter was inserted in-frame within the nsP3 gene, and (ii) the extra subgenomic promoter construct (VEEV-SINVpr-Azurite), in which the Azurite reporter was expressed under the control of an additional Sindbis virus (SINV) subgenomic promoter as an independent open reading frame. While VEEV-nsP3-Azurite rapidly lost fluorescence in cell culture, the clone selection allowed the VEEV/SINVpr-Azurite maintained reporter expression for at least two passages, enabling enrichment to ~ 50% Azurite-positive plaques. Viral replication kinetics of VEEV/SINVpr-Azurite closely matched parental TC-83 in BHK-21 J. For quantitative readout, a BHK-21 J cell line expressing nuclear-localized miRFP670 was used to enable automated nuclear segmentation and cell counting during high-content image analysis. Optimized assay conditions resulted in robust viral reporter signal (Z′ >0.5) within 18 h post-infection. Validation with reference antivirals demonstrated dose-dependent inhibition comparable to the activity against the WT virus, providing a reliable assay for antiviral evaluation.

Orthoflaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), constitute a significant public health concern with billions of people at risk of infection. Climate change and the expanding geographical distribution of mosquito vectors transmitting orthoflaviviruses have increased their potential to cause large-scale disease outbreaks. The frequency and severity of disease outbreaks highlight the urgent need for a broad-spectrum antiviral agent targeting orthoflaviviruses. In this work, we conducted a comprehensive morphological profiling of approximately 200,000 small molecules through a fluorescence-based high-content imaging platform, which led to the identification of a singular small molecule exhibiting broad-spectrum activity against orthoflaviviruses. Subsequent hit deconvolution against DENV serotype 2 (DENV-2) revealed NS2A protein as a novel therapeutic target. Mechanistically, JNJ-1953 inhibits viral RNA synthesis, as demonstrated by robust reductions in intracellular viral RNA and infectious virus production. Additional experiments show that JNJ-1953 further impacts viral RNA packaging and interferes with the interaction between NS2A and prM, rendering the molecule a multimodal inhibitor.

Recurring outbreaks of filoviruses, including Ebola virus and Sudan virus, threaten to cause large epidemics. No treatment or vaccine is available for Sudan virus and how protective immunity is achieved remains unknown. To unravel mechanisms contributing to protection, we used a surrogate mouse model of Sudan virus infection amenable to deep functional analysis. Mice vaccinated with a live viral vector based on live-attenuated YF17D expressing Sudan virus glycoprotein were protected against lethal challenge with a surrogate virus, a chimeric recombinant vesicular stomatitis virus expressing Sudan virus glycoprotein, despite lacking virus-neutralizing antibodies. Glycoprotein-specific humoral responses associated with antibody-mediated neutrophil phagocytosis and natural killer cell activation suggested Fcγ receptor (FcγR) effector involvement. However, protection was not compromised in FcγR-deficient mice. By contrast, targeted depletion and reconstitution experiments identified antigen-experienced interferon-γ (IFNγ)-secreting CD4+ T cells, particularly short-lived effector cells and regulatory T cells as key players for survival. Multiple complementary vaccination-induced effector mechanisms may contribute to Sudan virus immunity; however, only proliferative antigen-specific CD4+ T cells and sustained IFNγ production, orchestrating an acute antiviral response, appear required for protection.

Background and Aims:Hepatitis E virus (HEV) infections are a growing threat to global public health. To obtain an in-depth understanding of HEV infections in untreated and ribavirin-treated rats, we characterized the early HEV viral kinetics using rat HEV (rHEV) as a surrogate model and using two routes of virus inoculation: intravenous (I.V.) or oral infection. Approach and Results:We frequently collected feces, serum, and tissue samples up to 60 days after infection in both infection models to characterize the rHEV viral kinetics. A ~2-week delay in quantifiable RNA levels in feces was observed in the oral versus the I.V. infection model. Early rHEV viral kinetics in feces were found to be multiphasic and showed good concordance with those in the various tissue compartments studied. Comparison of the viral kinetics in these samples also revealed that the liver may serve as the initial site of rHEV replication, followed by replication in the intestine and spleen. While a dosage of 60 mg/kg/day ribavirin was found optimal to maintain rHEV RNA levels at (nearly) undetectable in feces, levels were detectable in the liver and increased both in feces and liver after treatment discontinuation. Conclusions:We found that the two rHEV infection models share similar multiphasic viral kinetics with the liver as the main site of viral replication. Additionally, the rHEV RNA load in feces could be used as a reliable proxy for that in the liver, spleen, and intestine. We also show that ribavirin at 60 mg/kg/day was partially effective in preventing viral rebound. These findings may aid in exploring the correlation between the infection phases and antiviral efficacy, ultimately guiding therapeutic decisions.

Hantaviruses are zoonotic threats that can cause hemorrhagic fever with renal syndrome and severe cardiopulmonary syndrome. Despite disease severity, there are no approved vaccines or therapeutics available for the prevention or management of hantavirus infections. Here, we discuss advances in the development of vaccines, neutralizing antibodies, and small-molecule antiviral therapeutics.

Orthoflaviviruses continue to pose a growing global health burden, as climate shifts, expanding urban environments, and diagnostic limitations drive record case numbers of dengue, Zika, and yellow fever virus infections. Despite the scale of these outbreaks, no small‑molecule antiviral has yet been approved for orthoflavivirus infections. 2′C‑methyl nucleoside analogues exhibit broad antiviral potential, although their clinical utility is hindered by poor cellular uptake and inefficient first mono-phosphorylation. To address these limitations, we designed and synthesized a series of Anchimeric HINT1 Activatable (AHA) ProTides based on 2′C‑methyl nucleosides. This strategy was intended to overcome shortcomings associated with classical McGuigan ProTides, including limited efficacy against non‑hepatotropic viruses following oral dosing and rapid degradation in rodent plasma driven by esterase-mediated activation. Building on our earlier findings, we designed AHA ProTides incorporating both purine and pyrimidine nucleobases. While the rate of anchimeric activation did not differ between nucleobases, it was strongly influenced by the chain length of the thermolytic thioether masking group (~5 h for AHA-Et vs ~15 h for AHA-Bt analogues). A clear structure–activity relationship (SAR) emerged in which only purine analogues showed consistent activity across assay formats, whereas 2′C-α-F‑pyrimidine derivatives were inactive in Vero cells. The antiviral activity of these compounds aligned with HINT1 substrate preferences. Among the series, AHA ProTide 5f exhibited the most potent inhibition of DENV (22.2 ± 1.3 µM) and ZIKV (3.23 ± 1.99 µM) with no cytotoxicity at 100 µM, identifying it as a promising lead for further development. The insights gained from this work provide practical guidance for designing next‑generation phosphoramidate ProTides that rely on HINT1‑mediated activation without dependence on carboxylesterase unmasking. A direct comparison of AHA ProTide 5b with the FDA‑approved ProTide Sofosbuvir further demonstrated superior stability in mouse plasma, although cytochrome P450 (CYP)‑mediated oxidative metabolism remains a current limitation.

ABSTRACT Ebola outbreaks continue to expand across Central Africa, yet available countermeasures remain limited and virus-specific. Whether available vaccines developed against the related Ebola virus (EBOV) and Sudan virus (SUDV) can confer cross-protection against Bundibugyo virus (BDBV) is incompletely understood. Here, we developed a BDBV surrogate challenge model and evaluated cross-protective immunity conferred by a set of monovalent vaccine candidates representing all three Orthoebolavirus species affecting humans. A yellow fever 17D-vectored BDBV vaccine (YF-BDB) expressing the BDBV glycoprotein (GP) as antigen conferred homologous protection in Ifnar − / − mice, preventing systemic viral dissemination and providing survival following surrogate BDBV challenge. Intriguingly, also vaccination with YF-EBO or YF-SUD protected mice from such BDBV surrogate challenge. Conversely, while YF-BDB and YF-EBO fully protected against respective heterologous EBOV or BDBV infection, they conferred only partial protection against lethal challenge in a comparable SUDV model. Serological analyses revealed comparable titers of cross-reactive antibodies across all vaccine groups. However, neutralization, especially cross-neutralization, was limited suggesting a major role for non-neutralizing mechanisms in protection. These findings demonstrate asymmetry in cross-protective immunity among Orthoebolavirus vaccines and indicate that SUDV GP-based antigens may provide broader protection than EBOV- or BDBV-targeted candidates. However, our data add to the preliminary yet limited evidence that EBOV GP-based vaccines might offer at least some degree of protection against BDBV, the virus driving current Public Health Emergency of International Concern (PHIEC) in the Central-East Africa region.

Dengue virus (DENV) and chikungunya virus (CHIKV) remain major global health threats, yet approved antiviral therapies are lacking. Antiviral sugar baits (AVSBs) represent a novel transmission-blocking strategy that exploits the natural sugar-feeding behaviour of mosquitoes to deliver antiviral compounds directly to the mosquito vector. In this study, we evaluated the antiviral efficacy of AVSBs containing JNJ-A07, β-D-N4-hydroxycytidine (NHC), molnupiravir (MPV), and 4′-fluorouridine (4′FlU) in Aedes aegypti mosquitoes. An ex vivo mosquito gut model was used to select antiviral concentrations and to assess the ability of 4′FlU to reach the mosquito midgut following sugar feeding. AVSBs were subsequently evaluated for their effects on mosquito attractiveness, longevity, fecundity, and fertility, as well as their ability to suppress DENV and CHIKV infection in vivo. 4′FlU potently inhibited CHIKV replication in ex vivo mosquito guts and retained antiviral activity following sugar-bait administration. None of the antiviral compounds affected mosquito attraction to the bait, while only modest and compound-specific effects on mosquito fitness were observed. AVSBs containing JNJ-A07 significantly reduced DENV infection and dissemination, whereas 4′FlU-containing AVSBs significantly reduced CHIKV infection and viral loads. In contrast, NHC- and MPV-containing AVSBs did not exhibit antiviral activity in vivo. Collectively, these findings provide proof-of-concept for AVSBs as a novel strategy to reduce arbovirus transmission by targeting viral replication within the mosquito vector, suggesting that AVSBs could complement existing arbovirus control measures.

Tracking of viral replication and tissue tropism by in vivo imaging can help to unveil how live-attenuated vaccines such as the yellow fever 17D (17D) work, and likewise, to understand how adverse effects develop. Here we validate 17D-TK, a reporter virus derived from 17D that expresses herpes virus thymidine kinase (TK) that specifically converts nucleoside analogues such as Ganciclovir (GCV) to induce cell death, or difluoro-EdU (dF-EdU) for bioorthogonal labelling of infected cells by Click chemistry. 17D-TK induces a cytopathic effect in infected cell cultures, as well as mortality in intracranially inoculated mouse pups in a GCV dependent manner. Preferential phosphorylation of difluoro-EdU (dF-EdU) in 17D-TK infected cells allows to selectively stain cells that support 17D replication. Prospectively, 17D-TK can be used in combination with radiolabeled tracers for real-time detection and localization of sites of active viral replication in living animals using positron emission tomography (PET).

Pregnant women are at higher risk of severe COVID-19, with vaccine access and hesitancy remaining a challenge. Here, we use a pregnant female hamster model of COVID-19 to explore the effects of maternal infection on pregnancy, revealing a significant increase in intrauterine growth restriction (IUGR) due to placental inflammation. Viral infection causes bronchopneumonia and weight loss in infected dams, but no vertical transmission occurs. IUGR is instead linked to placental damage, characterized by fibrin deposition, thrombosis, and elevated placental expression of IP10, IL6, and IL10, irrespective of fetal sex. Enoxaparin treatment reduces placental damage and improves fetal outcomes, while vaccination enhances viral clearance, protects the placenta, and reduces the risk of IUGR. These findings underscore placentitis as a key driver of fetal complications upon SARS-CoV-2 infection and highlight the potential of vaccination and anticoagulant therapy to protect both mother and child.

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