Human coronaviruses have been primarily associated with upper respiratory tract infections, yet cases of gastrointestinal symptoms in COVID-19 patients have highlighted their potential to cause systemic disease. Here, we detail the infection of intestinal epithelia by an endemic, low-pathogenic human coronavirus, human alphacoronavirus 229E, using patient-derived human intestinal enteroids (HIEs) from donors of various ages. Using fetal, pediatric, and adult HIEs, we investigated how physiologically relevant temperatures: 37 °C and 32 °C, reflecting gastrointestinal and upper-airway conditions, respectively, modulate epithelial responses and viral infection dynamics. We show that there is temperature-dependent transcriptional reprogramming, indicating strong temperature-dependent regulation of virus replication and epithelial responses. Among the seasonal coronaviruses tested, only HCoV-229E productively infects HIEs. At 32 °C, HCoV-229E replicates efficiently in enteroids from all donor ages and releases high titers of infectious progeny. In contrast, at 37 °C, productive replication is largely confined to fetal and a subset of pediatric tissues, revealing a developmental and temperature-sensitive restriction on infection. Confocal and flow cytometry analyses identify enterocytes as the primary target cells for HCoV-229E. Furthermore, we show that camostat, a serine protease inhibitor, significantly reduces HCoV-229E replication in HIEs, confirming a critical role for host serine protease activity. Collectively, these findings establish HIEs as a relevant model for HCoV-229E-host interactions and reveal temperature- and age-dependent determinants governing intestinal permissiveness to this seasonal coronavirus.

RNA viruses represent an integral component of human-associated environments and human health. However, the ecology of environmental RNA viruses remains largely unexplored. Here, we analyzed 2922 metatranscriptomic samples collected from urban and surrounding environments-including human-dense settings (e.g., transit hubs, hospitals, banks), alongside peri-urban settings - across 102 cities in 31 countries and constructed the Urban & Peri-urban RNA Virus Atlas (UPVAtlas), comprising 54,945 RNA viruses, 77% of which had not been previously observed. Phylogenetic reconstruction based on RNA-dependent RNA polymerases from UPVAtlas greatly expanded the evolutionary diversity of RNA viruses, leading to the identification of two potential candidate phyla, one candidate class, and several unclassified clades. Host association analyses further revealed the ecological complexity of environmental RNA viruses, with the diversity of vertebrate-related and ESKAPE pathogen-related viruses underscoring the importance of continued monitoring of urban environments for tracking RNA viral prevalence and dynamics, with direct relevance to future public health.

SARS-CoV-2 non-structural protein 14 (nsp14) is essential for viral mRNA cap guanine-N7 methylation and represents a promising but underexplored antiviral target. Herein we describe a structure-guided campaign based on a hit from a focussed SAM mimetic library. Systematic SAR exploration guided by six X-ray co-crystal structures in complex with SARS-CoV-2 led to compound 26, a bi-substrate inhibitor that bridges the SAM and RNA cap binding sites. Compound 26 achieved nanomolar potency against nsp14 from SARS-CoV-2 (IC50 = 53 nM), SARS-CoV-1, and two alphacoronaviruses, with excellent selectivity over human RNMT and flaviviral MTase. In general, the compounds demonstrated favourable metabolic stability, passive permeability, and no HepG2 cytotoxicity. However, cellular antiviral activity was limited, revealing disconnects between enzyme inhibition and phenotypic response. These findings provide a structural framework for optimizing bi-substrate methyltransferase inhibitors against coronaviruses with a view for pan-coronaviral activity.

The risk of zoonotic spillover of coronaviruses poses a constant threat to humanity, as recently exemplified by SARS‑CoV-2. During the pandemic, the rapid emergence of immune escape mutations led to a loss of efficacy of both therapeutic and humoral anti‑spike antibodies, emphasizing the need for therapeutics that engage binding sites within the spike protein that are both conserved among related viruses and not under selective pressure from immune surveillance. Here, we report the development of a first-in-class fusion inhibitor cyclic peptide that targets such a site. Guided by a deep mutational scan of our earlier mRNA display hit we arrived at an analogue with 5 mutations that retains broad activity within the subgenus sarbecovirus, including against SARS-CoV-1, but shows up to 50-fold improved potency and approximately 25-fold improved solubility. Multimerization further improved IC50 by one order of magnitude to give a tetrameric construct (H4v1Az)4 that inhibits Omicron BA.2.86 replication with an IC50 of approximately 10 nM in cellular assays, including human airway epithelial cell primary cultures. Moreover, (H4v1Az)4 demonstrated significant antiviral efficacy in vivo in a Syrian golden hamster model following combined pre- and post-exposure prophylactic intranasal administration. This multimeric peptide represents a promising broadly active candidate suitable both for treatment of SARS-CoV-2 infection and for preparedness against future emerging sarbecoviruses.

Twenty diterpenes (1-20) and a sesquiterpene (21) were isolated from Dictyota fasciola and Dictyota mediterranea collected from the Aegean Sea and evaluated as potential antiviral agents against a panel of viral pathogens. Among the isolated metabolites, six dolabellane diterpenes (1-4, 9, and 12) and one germacrane sesquiterpene (21) are new natural products. The evaluation of the antiviral activity of the isolated compounds was carried out against three viruses of different phylogenetic backgrounds that are characterized by high incidence in human population and cause significant medical threat and economic losses worldwide: herpes simplex virus (HSV-1), human coronavirus (HCoV-NL63), and Zika virus (ZIKV). The antiviral assays demonstrated that some compounds inhibited the viruses within a concentration range of 3-10 µg/mL. The maximal suppression of viral replication was 90% for HSV-1, 99.99% for HCoV-NL63, and 96.8% for ZIKV, highlighting the differential efficacy of the tested compounds against specific viruses. Mechanistic studies revealed that, depending on the compound, antiviral activity occurred either at the virus attachment stage or during a post-entry replication stage. Among the tested compounds, metabolites 6-8, 13, 17, and 18 exhibited considerable antiviral activity against RNA (HCoV-NL63, ZIKV) and DNA (HSV-1) viruses.

Feline calicivirus (FCV) infection causes nasal discharge, oral mucosa inflammation, ulcerations, gingivitis, and conjunctivitis, often progressing to chronic gingivostomatitis, severe pneumonia, and fatal systemic infections. With no antivirals currently available, poly(sodium 4-styrene sulfonate) (PSSNa) was identified in 2019 as a safe inhibitor in vitro. In this preliminary single-center, randomized, double-blind, placebo-controlled field study, we further characterized the PSSNa’s safety profile and tested its efficacy in cats after topical oral application. Twenty-eight cats were enrolled in the study, and they were initially treated with standard dental therapy, followed by adjuvant local oral application of PSSNa or placebo. After 4 weeks, PSSNa demonstrated a favorable safety profile with no adverse effects. The treatment group showed a significant decrease in viral load (p = 0.001) compared to placebo (p = 0.012). Disease symptoms improved significantly, though the oral health index remained unchanged. Additionally, PSSNa showed activity against multiple genetically diverse isolates, indicating a potential, exploratory link between genetic background and treatment outcome. Summarizing, this study presents initial data on the efficacy and tolerability of PSSNa treatment for FCV infections in cats. Nevertheless, several significant limitations should be acknowledged, including inconsistent drug administration by owners, non-sterile housing, sample size, variable oral disease severity, and concurrent treatments.

Background Platelets are small, non-nucleated elements that play an essential role in maintaining homeostasis and modulating tissue responses, carrying a set of translation-competent mRNA molecules. Methods We examined the mRNA content of platelets isolated from blood obtained from hospitalized COVID-19 patients during the acute phase of the disease with pneumonia. Results We report an upregulation in the expression of genes coding for IFITM3 and TIMP1 in the platelets of COVID-19 patients, both of which are involved in antiviral responses. Additionally, we observed increased expression of genes encoding the TREML1 and SPARC, which protect against inflammation and promote tissue regeneration, as well as the HLA-E gene, which modulates lymphocyte functions. Stratification of patients based on "severe" and "non-severe" pneumonia revealed that TREML1, SPARC, and HLA-E expression negatively correlated with disease severity, while IFITM3 and TIMP1 expression were upregulated in both groups. Conclusion Our findings highlight significant differences in blood platelet mRNA content between COVID-19 patients and healthy subjects, which may influence disease progression and serve as potential predictive markers.

COVID-19, caused by SARS-CoV-2, has led to significant morbidity and mortality worldwide. The pandemic has sparked extensive efforts to develop therapeutic strategies targeting either the virus itself or human proteins involved in the infection, resulting in hundreds of potential drugs and numerous clinical trials. In the search for new anti-SARS-CoV-2 drug candidates, we focussed on natural products. Using the cyanobacteria as a source of new structures due to their unique metabolism, two spumigins were isolated from the Baltic cyanobacterium Nodularia spumigena and tested for their activity against SARS-CoV-2. These compounds efficiently reduced SARS-CoV-2 infection in human A549ACE2/TMPRSS2 cells and fully differentiated primary human airway epithelium cell systems, in which viral entry depends on activation of the viral spike protein by the cellular serine protease, such as TMPRSS2. In contrast, the compounds did not inhibit viral replication in Vero cells, which lack TMPRSS2 but have high levels of cysteine protease cathepsin L, which may serve as an alternative for spike protein activation. Biochemical assays showed that spumigins inhibit TMPRSS2 with an EC50 ranging from 17 to 85 nM at which no toxicity is observed. Good parameters of identified inhibitors prove that cyanobacteria may serve as a rich source for new scaffolds reaching beyond the canonical chemical combinatorial space.

SARS-CoV-2 entry into host cells depends on proteolytic activation of the spike protein by host proteases, a process shaped by spike mutations that influence viral specificity and infectivity. Using human airway epithelial models, this study investigated how different SARS-CoV-2 variants interact with host serine proteases. The Delta variant exhibited enhanced and stable binding to Hepsin through stronger ionic and hydrophobic interactions, promoting efficient spike activation and cell entry. In contrast, Omicron BA.1 showed weaker Hepsin binding and relied more on TMPRSS2 or cathepsins, depending on the cellular context. These findings reveal how variant-specific differences in protease usage are linked to spike protein mutations and cleavage site evolution. By illuminating the dynamic interplay between viral adaptation and host protease specificity, this work provides insights into mechanisms that influence viral transmission and immune evasion, with implications for developing targeted antiviral strategies and understanding the evolution of emerging SARS-CoV-2 variants.

The Rev-dependent nuclear export of unspliced and singly-spliced transcripts of human immunodeficiency virus type 1 (HIV-1) constitutes a critical yet poorly characterized post-transcriptional event essential for effective viral replication. In this study, we engineered a dual-fluorescent HIV-1-based cellular reporter system to elucidate the mechanisms underpinning Rev-dependent export. By generating multiple stably integrated inducible cellular clones, we ensured the expression of two distinct fluorescent proteins, mKO2, and ECFP, from unspliced (Rev dependent) and multiply spliced (Rev independent) HIV-1 transcripts, respectively. Utilizing flow cytometry, we performed quantitative analyses of dual-fluorescent cell populations. The developed tool enables precise assessment of the Rev-dependent export, and we validated it using known inhibitors of this pathway (leptomycin D), as well as targeted depletion of MATR3, an essential cofactor of Rev, and CRNKL1, a repressor of unspliced HIV-1 RNA export. IMPORTANCE The developed dual-fluorescent reporter system represents a powerful and handy tool for the identification and characterization of novel molecular players involved in the Rev-dependent export pathway. This system not only holds promise for advancing our understanding of human immunodeficiency virus type 1 (HIV-1) biology but also serves as an invaluable platform for high-throughput drug screening aimed at targeting post-transcriptional HIV-1 RNA processes, particularly nuclear export. Consequently, this study offers significant implications for the development of novel therapeutic strategies to eradicate the virus.

The coronavirus membrane protein (M) is the main organizer of coronavirus assembly1-3. Here, we report on an M-targeting molecule, CIM-834, that blocks the assembly of SARS-CoV-2. CIM-834 was obtained through high-throughput phenotypic antiviral screening followed by medicinal-chemistry efforts and target elucidation. CIM-834 inhibits the replication of SARS-CoV-2 (including a broad panel of variants) and SARS-CoV. In SCID mice and Syrian hamsters intranasally infected with SARS-CoV-2, oral treatment reduced lung viral titres to nearly undetectable levels, even (as shown in mice) when treatment was delayed until 24 h before the end point. Treatment of infected hamsters prevented transmission to untreated sentinels. Transmission electron microscopy studies show that virion assembly is completely absent in cells treated with CIM-834. Single-particle cryo-electron microscopy reveals that CIM-834 binds and stabilizes the M protein in its short form, thereby preventing the conformational switch to the long form, which is required for successful particle assembly. In conclusion, we have discovered a new druggable target in the replication cycle of coronaviruses and a small molecule that potently inhibits it.

BACKGROUND:Current latency-reversing agents (LRAs) employed in the "shock-and-kill" strategy primarily focus on relieving epigenetic and transcriptional blocks to reactivate the latent HIV-1. However, their clinical efficacy is limited, partly due to their inability to fully reverse latency and the lack of LRAs specifically targeting post-transcriptional mechanisms. N6-methyladenosine (m6A) modification in HIV-1 RNA is emerging as an important post-transcriptional regulator of HIV-1 gene expression, yet its role in latency and reactivation remains largely unrecognized. Here, we explored the potential of small chemical compounds targeting the m6A pathway, specifically investigating the inhibition of ALKBH5 and its effect on latent HIV-1 reactivation mediated by the LRA romidepsin. METHODS:We used four in vitro cellular models of latency, primary model of CD4+ T cells HIV-1 infection and ex vivo cultures of CD8+-depleted PMBCs from ART-treated HIV+ patients. We measured latent viral reactivation by evaluating the expression of reporter protein GFP by flow cytometry, viral production by CA-p24 ELISA, and viral transcripts by RT-qPCR. CRISPR/Cas9 method was used to deplete ALKBH5. MeRIP and immuno-RNA FISH were used to address the m6A methylation levels on HIV-1 RNA upon ALKBH5 inhibition. RESULTS:We showed that ALKBH5 inhibitor 3 (ALKi-3) potentiated romidepsin-mediated viral reactivation in in vitro models of latency, primary model of CD4+ T cells infected with HIV-1 as well as in ex vivo cultures of CD8+-depleted PBMCs from ART-treated HIV+ patients. CRISPR/Cas9-mediated depletion of ALKBH5 mimicked the effects of ALKi-3. ALKi-3 increased levels of m6A-methylated HIV-1 RNA as shown by meRIP and immuno-RNA FISH. CONCLUSION:Our study provides a proof-of-concept for the modulation of the m6A pathway in enhancing HIV-1 reactivation. This approach represents a promising adjunct to existing reactivation protocols and provides a concept of "dual-kick", aiming to target transcriptional and post-transcriptional steps in HIV-1 reactivation from latency.

The continued emergence and zoonotic threat posed by coronaviruses highlight the urgent need for effective antiviral strategies with broad reactivity to counter new emerging strains. Nanobodies (or single-domain antibodies) are promising alternatives to traditional monoclonal antibodies, due to their small size, cost-effectiveness and ease of bioengineering. Here, we describe 7F, a llama-derived nanobody, targeting the spike receptor binding domain of sarbecoviruses and SARS-like coronaviruses. 7F demonstrates potent neutralization against SARS-CoV-2 and cross-neutralizing activity against SARS-CoV and SARS-like CoV WIV16 pseudoviruses. Structural analysis reveals 7F's ability to induce the formation of spike trimer dimers by engaging with two SARS-CoV-2 spike RBDs, targeting the highly conserved class IV region, though concentration dependent. Bivalent 7F constructs substantially enhance neutralization potency and breadth, up to more recent SARS-CoV-2 variants of concern. Furthermore, we demonstrate the therapeutic potential of bivalent 7F against SARS-CoV-2 in the fully differentiated 3D tissue cultures mirroring the epithelium of the human airway ex vivo. The broad sarbecovirus activity and distinctive structural features of bivalent 7F underscore its potential as promising antiviral against emerging and evolving sarbecoviruses.

HIV-1 latency is mainly characterized at transcriptional level, and little is known about post-transcriptional mechanisms and their contribution to reactivation. The viral protein Rev controls the nucleocytoplasmic export of unspliced and singly-spliced RNA that is central to proviral replication-competence and is therefore a prerequisite for efficient viral reactivation during the "shock-and-kill" cure therapy. Here we show that during infection and reactivation, unspliced HIV-1 RNA is a subject to complex and dynamic regulation by the Rev cofactor MATR3 and the MTR4 cofactor of the nuclear exosome. MATR3 and MTR4 coexist in the same ribonucleoprotein complex functioning to either maintain or degrade the RNA, respectively, with Rev orchestrating this regulatory switch. Moreover, we provide evidence of nuclear retention of unspliced HIV-1 RNA in ex vivo cultures from 22 ART-treated people with HIV, highlighting a reversible post-transcriptional block to viral RNA nucleocytoplasmic export that is relevant to the design of curative interventions.

The O'nyong-nyong virus (ONNV) is a member of the Alphavirus genus isolated for the first time in 1959 in Gulu (Uganda). The main symptoms of O'nyong-nyong fever are nonspecific and flu like; a proper diagnosis at the early stage is challenging. Until today, the first ONNV epidemic in 1959-1962 remains one of the largest arbovirus outbreaks ever recorded (over 2 million cases). No specific therapy is available thus far. Capsid protease (CP) is a chymotrypsin-like serine protease that catalyzes the autoproteolytic maturation of the structural polyprotein and undergoes self-inactivation upon the release of the mature capsid (C) protein. Despite the discovery of this virus more than 60 years ago, many aspects of CP remain understudied, including substrate specificity. Herein, the structures of the first phosphonic-type inhibitors and activity-based probes specific to the ONNV CP are presented.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

The main protease (Mpro) of SARS-CoV-2 is critical in the virus's replication cycle, facilitating the maturation of polyproteins into functional units. Due to its conservation across taxa, Mpro is a promising target for broad-spectrum antiviral drugs. Targeting Mpro with small molecule inhibitors, such as nirmatrelvir combined with ritonavir (Paxlovid™), which the FDA has approved for post-exposure treatment and prophylaxis, can effectively interrupt the replication process of the virus. A key aspect of Mpro's function is its ability to form a functional dimer. However, the mechanics of dimerization and its influence on proteolytic activity remain less understood. In this study, we utilized biochemical, structural, and molecular modelling approaches to explore Mpro dimerization. We evaluated critical residues, specifically Arg4 and Arg298, that are essential for dimerization. Our results show that changes in the oligomerization state of Mpro directly affect its enzymatic activity and dimerization propensity. We discovered a synergistic relationship influencing dimer formation, involving both intra- and intermolecular interactions. These findings highlight the potential for developing allosteric inhibitors targeting Mpro, offering promising new directions for therapeutic strategies.

HIV-1 reservoirs display heterogeneous nature, lodging both intact and defective proviruses. Recent evidence has shed light on their difference, particularly in the context of immune-mediated selection. To deepen our understanding of such heterogeneous HIV-1 reservoirs and their functional implications, we pioneered the integration of basic concepts of graph theory to characterize the composition of HIV-1 reservoirs. Our analysis revealed noticeable topological properties in networks, featuring immunologic signatures enriched by genes harboring intact and defective proviruses, when comparing antiretroviral therapy (ART)-treated HIV-1-infected individuals and elite controllers. The key variable, the rich factor, played a pivotal role in classifying distinct topological properties in networks. The host gene expression strengthened the accuracy of classification between elite controllers and ART-treated patients. Overall, our work provides a prime example of leveraging genomic approaches alongside mathematical tools to unravel the complexities of HIV-1 reservoirs. ### Competing Interest Statement The authors have declared no competing interest.

The continued emergence and zoonotic threat posed by coronaviruses highlight the urgent need for effective antiviral strategies with broad reactivity to counter new emerging strains. Nanobodies (or single-domain antibodies) are promising alternatives to traditional monoclonal antibodies, due to their small size, cost-effectiveness and ease of bioengineering. Here, we describe 7F, a llama-derived nanobody, targeting the spike receptor binding domain of sarbecoviruses and SARS-like coronaviruses. 7F demonstrates potent neutralization against SARS-CoV-2 and cross-neutralizing activity against SARS-CoV and SARS-like CoV WIV16 pseudoviruses. Structural analysis reveals 7F’s ability to induce the formation of spike trimer dimers by engaging with two SARS-CoV-2 spike RBDs, targeting the highly conserved class IV region. Bivalent 7F constructs substantially enhance neutralization potency and breadth, up to more recent SARS-CoV-2 variants of concern. Furthermore, we demonstrate the therapeutic potential of 7F against SARS-CoV-2 in the fully differentiated 3D tissue cultures mirroring the epithelium of the human airway ex vivo. The broad sarbecovirus activity and distinctive structural features of 7F underscore its potential as promising antiviral against emerging and evolving sarbecoviruses. ### Competing Interest Statement ID is an employee of Thermo Fisher Scientific. The remaining authors declare that they have no competing interests.

Clinical data indicate that COVID-19 causes cardiovascular complications, regardless of the severity of the disease. In this work, we have shown that SARS-CoV-2 infection causes vascular dysfunction due to the modification of endothelial cell elasticity. We used human pulmonary endothelial cells (HPAECs) expressing the ACE2 receptor as a model of the endothelium. This system mimics in vivo conditions, as it allows virus entry but not replication. As a reference, we used A549 epithelial cells, a well-described model that supports productive replication of SARS-CoV-2. We show that the infection of HPAECs results in loss of cell elasticity, which correlates with increased polymerization of actin filaments and induction of the inflammatory response. On the contrary, A549 epithelial cells supporting viral replication showed increased elasticity. We also showed that the endothelial cell elasticity were impaired after infection with Alpha, Beta and Delta variants. Consequently, we believe that nonproductive SARS-CoV-2 infection associated with loss of the endothelium elasticity may be clinically relevant and result in dysfunction and damage to this tissue.