Antiviral Research

Filovirus outbreaks caused by Ebola virus (EBOV) and Marburg virus (MARV), pose severe global health threats characterized by high rates of fatal hemorrhagic fever. While species-specific vaccines and therapeutic monoclonal antibodies are approved for Zaire ebolavirus, broadly-active therapeutics remain unavailable, leaving populations vulnerable to MARV and other pathogenic Ebola species, such as Bundibugyo (BDBV) and Sudan (SUDV) ebolaviruses. Here we report a computationally guided, infectious virus validated screening platform for the rapid discovery of broad-spectrum filovirus antivirals. By leveraging quantitative structure-activity relationship (QSAR) models, we screened 142,382 compounds in silico to prioritize 125 high-potential candidates. Subsequent dose-response and viability profiling identified 23 compounds exhibiting potent, low-micromolar pan-filovirus activity and favorable cytotoxicity profiles. Molecular docking indicates these compounds target conserved structural and functional domains--primarily the VP35 and L proteins--which may disrupt essential viral replication and immune antagonism. Furthermore, systematic combinatorial screening revealed three highly synergistic compound pairs, notably NCGC00113249-01 and NCGC00118008-01, demonstrating robust cross-species efficacy. By targeting conserved vulnerabilities across the filovirus family, this integrated in silico and in vitro pipeline provides a scalable framework to rapidly nominate and optimize synergistic therapeutic regimens against both endemic and emerging viral threats including BDBV. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/737586v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1251baorg.highwire.dtl.DTLVardef@b3a2feorg.highwire.dtl.DTLVardef@191d314org.highwire.dtl.DTLVardef@b8f710_HPS_FORMAT_FIGEXP M_FIG C_FIG

Targeting viral macrodomains (Mac) has emerged as a promising strategy for antiviral drug development, especially after the outbreak of COVID-19 that claimed millions of lives worldwide. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mac1 inhibitors have been reported in the past few years. In the present work, we converted GS-441524 (IC50 of [~]10 M for SARS-CoV-2 Mac1) to KP-S54 (18c), a potent inhibitor of both SARS-CoV-2 Mac1 (IC50: 44 nM) and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 (IC50: 91 nM) through an iterative direct-to-biology approach. This approach leverages efficient amide-coupling reaction and the mix-and-read fluorescence polarization (FP) assays where reaction mixtures could be screened directly without purification. Cocrystal structure of a selected derivative (12p) binding to SARS-CoV-2 Mac1 revealed the binding mode, which will guide future drug development against viral macrodomains.

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3Dpol) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3Dpol, including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3Dpol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3Dpol, including hit-to-lead compound 727590, which displayed an IC50 value of 25 M and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737532v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14a54a6org.highwire.dtl.DTLVardef@fb6621org.highwire.dtl.DTLVardef@ee2e2aorg.highwire.dtl.DTLVardef@118f91d_HPS_FORMAT_FIGEXP M_FIG Created with biorender.com and PyMOL Molecular Graphics System, version 2.5.0. Schrodinger, LLC. C_FIG

Chronic hepatitis B virus (HBV) infection contributes to hepatocellular carcinoma by disrupting host transcription, cell-cycle control, and apoptotic signaling. Isochlorogenic acid A (ICAA), a natural compound with antiviral and hepatoprotective properties, was previously shown to inhibit HBV replication by interfering with multiple steps of the viral life cycle. Because chronic HBV often reflects an imbalance between proliferation and cell death, we investigated how ICAA affects gene expression related to these processes in the presence or absence of HBV. We performed transcriptome analysis using RNA sequencing (RNA-seq) in HepAD38 cells (a HepG2-derived stable HBV-expressing line) and HepG2 control cells (HBV-negative) treated with ICAA or DMSO. HBV caused major differences in gene expression in HepAD38 cells compared with HBV-negative HepG2 cells. Principal component analysis showed that ICAA significantly altered HBV-dependent expression patterns, resulting in 189 differentially expressed genes (DEGs) that were regulated in opposite directions by both HBV and ICAA. Functional enrichment analysis highlighted pathways in viral carcinogenesis, apoptosis, MAPK signaling, and p53 signaling. Annexin V/propidium iodide assays showed apoptotic cells in both treated and untreated HepAD38 cultures, with only minor pattern changes. Mechanistically, in untreated HBV-positive cells caspase-9 cleavage failed to activate PARP, suggesting that induction of intrinsic apoptosis is followed by blocked execution. In contrast, ICAA inhibits caspase-9 cleavage in a dose-dependent manner, while activating PARP. Consistent with this, ICAA treatment increased apoptotic DNA fragmentation in HepAD38, reflecting the proapoptotic potential of ICAA under these conditions facilitating the elimination of HBV-positive cells by apoptosis. These findings highlight the potential therapeutic relevance of this compound in processes associated with HBV pathogenesis, together with its antiviral effect. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/733975v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@38d107org.highwire.dtl.DTLVardef@235a13org.highwire.dtl.DTLVardef@ee988aorg.highwire.dtl.DTLVardef@60cb13_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mammalian hantaviruses are RNA viruses that cause hantavirus cardiopulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome in Eurasia. The cellular entry mechanisms of most hantaviruses remain poorly defined. To examine entry by phylogenetically distinct hantaviruses, we generated replication-competent recombinant vesicular stomatitis viruses (rVSVs) bearing Gn/Gc proteins from Necocli, Sangassou, Thottapalayam, Kenkeme, Nova, Oxbow, and Tula viruses. All these Gn/Gc proteins except Kenkeme supported infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. Except for rVSV-Kenkeme, these rVSVs did not acquire additional mutations beyond pre-engineered rescue-enhancing changes during rescue and passaging. Genetic studies in human cells lacking protocadherin-1 (PCDH1) showed that Necocli, Tula, and Nova viruses use PCDH1 for efficient infection, although the Nova phenotype was weaker. These three Gn/Gc proteins bound soluble PCDH1 with different apparent avidities, and infection by the corresponding rVSVs was inhibited by soluble PCDH1; Necocli and Tula, but not Nova, were also blocked by a PCDH1-targeting monoclonal antibody. Authentic Tula virus infection was similarly reduced in PCDH1 knockout endothelial cells. Finally, the broadly reactive anti-Gn/Gc human monoclonal antibody ADI-42898 efficiently neutralized Necocli, Nova, Sangassou, and Kenkeme rVSVs but showed weak or undetectable activity against Oxbow, Tula, and Thottapalayam rVSVs. Together, these findings expand the range of hantavirus glycoproteins capable of mediating infection of human endothelial cells, broaden the phylogenetic scope of PCDH1-dependent entry, and identify receptor-targeted and viral glycoprotein-targeted strategies with differential activity. ImportanceMany newly discovered hantaviruses are known only from sequence data, leaving their ability to enter human cells and their receptor usage unresolved. Using a BSL2-compatible rVSV system, we show that glycoproteins from several divergent hantaviruses can mediate infection of primary human endothelial cells, indicating that endothelial cell entry is permissive for a broader range of hantaviruses than previously appreciated. We also show that protocadherin-1 (PCDH1), previously linked mainly to New World hantaviruses, is used by Necocli, Tula, and Nova viruses but not universally across the panel, revealing broader but heterogeneous receptor usage. An authentic Tula virus experiment supports this conclusion beyond the surrogate system. Finally, a broadly reactive anti-Gn/Gc antibody neutralizes several, but not all, of these viruses, highlighting both the promise and the limits of broadly protective countermeasures and the utility of these rVSVs for evaluating entry inhibitors.

The lack of effective anti-hepatitis B virus (HBV) therapies highlights the need for a new type of treatment that targets different stages of the viral life cycle. The HBV core protein (HBc) is a critical component of this cycle. Various capsid assembly modulators (CAMs) have been developed to target the HBc and inhibit HBV replication. We recently described a subset of capsid assembly modulators (CAMs) that induce the formation of aberrant structures from the HBc in the nucleus, leading to cell death via annexin A1 (ANXA1)-driven apoptosis. Thus, we further elucidated the mechanism of HBc aggregation in the nucleus, with a particular focus on the interplay between nuclear HBc aggregates and PML nuclear bodies. We found that long-term treatment with CAM-A induced the formation of enlarged PML bodies, approximately 1-2 m in diameter, that accumulated aggregated HBc. PML silencing in HBc-overexpressing HepG2-NTCP cells led to a dramatic increase in apoptosis following CAM-A-induced HBc aggregation, which was associated with elevated ANXA1. Next, we showed that PML nuclear bodies orchestrate proteasomal degradation of nuclear HBc aggregates via sumoylation-dependent recruitment of RNF4. Collectively, our results suggest that PML nuclear bodies act as storage compartments for aggregated HBc proteins in the nucleus, thereby counteracting the apoptotic elimination of cells. Further study of PML function and the targeting of PML nuclear bodies in HBV-infected hepatocytes could reveal new ways to enhance the effectiveness of CAMs.

Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction.

Discovering small molecules targeting proteins represents a major effort in drug development. RNA, however, as a class of macromolecule that carrying out important regulatory roles in the cell as drug target, only received attention recently. Although several methods have been proposed, an easy to operate, fast and robust method is still lacking. We designed a generic florescence screening method by fusing the target RNA with a florescent aptamer (fusion RNA) and then carried out screening using high-throughput format (Fluorescent Aptamer Screening, FAS). In this work, we chose SL5 on SARS-Cov-2 5’UTR as the test target. SL5 is a conserved motif across several corona virus family members whose core is not prone to mutation. We screened 9528 compounds, successfully identified four molecules (Sertraline (hydrochloride), Samuraciclib (hydrochloride), Minocycline (hydrochloride), JG-98 bind direct to the full-length SL5 at micromolar or higher affinity. The design of FAS could be easily adapted to structured RNA motifs without prior knowledge of its 3D structural information. In addition, this work showed the possibility of developing generic drugs for RNA virus by targeting the conserved viral RNA genome and paved a new way for the discovery of small molecule drugs in combating human diseases.

The 2022 Monkeypox virus (MPXV) outbreak renewed interest in vaccines for orthopoxviruses. Initial development efforts focused on well-established antigen targets, especially A35, B6, and M1. However, orthopoxvirus surfaces are complex, displaying many antigens across two infectious forms, mature virions (MV) and extracellular virions (EV) and targets relevant to protection remain to be comprehensively defined. We leveraged advances in orthopoxvirus protein biology and mRNA vaccine technology to compare immunity to all feasible targets. Mice were immunized with mRNAs encoding each antigen, or antigen complex, and neutralizing antibody responses were measured prior to heterologous challenge with vaccinia virus. Among MV antigens, A28 induced potent complement-mediated neutralizing antibodies, and the A17:G10 complex induced neutralizing antibodies and protected from challenge. For EV antigens, A36 induced neutralizing antibodies and protected from challenge. Our results affirm the consensus strategy focusing on key antigens while highlighting additional targets that could enhance updated MPXV mRNA vaccines. SIGNIFICANCEMonkeypox virus, a member of the Orthopoxvirus genus along with variola virus, has been associated with two recent outbreaks of mpox disease leading to a renewed focus on orthopoxvirus vaccine development. We report an agnostic screen of all monkeypox virus surface antigens where we combined recent advances in structural biology and mRNA technology to evaluate these potential new vaccine targets. We confirmed that historically prioritized antigens M1, A35 and B6 were protective but also discovered new antigens of interest including A28, the A17:G10 complex and A36 that can be the targets of protective immune responses. These findings are critical to inform next-generation vaccine designs should novel orthopoxviruses emerge as human pathogens.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

Tuberculosis (TB) remains a leading global cause of infectious mortality due, in part, to the limited efficacy of the Mycobacterium bovis BCG vaccine against pulmonary TB. Previous studies in mice have shown that stimulating type I interferon (IFN) signaling during BCG vaccination can bolster protection against Mycobacterium tuberculosis, yet clinically feasible delivery strategies for this approach are lacking. Adenoviral vectors, which induce potent type I IFN responses and are utilized in approved vaccine platforms, represent a promising adjuvant strategy. To evaluate the host immune response to this combination, bone marrow-derived murine macrophages were co-infected with replication-deficient adenovirus and BCG. Adenovirus-infected macrophages elicited a robust type I IFN response via the cGAS/STING pathway. Compared to BCG infection alone, co-infected macrophages exhibited additive expression of genes with known host-protective roles against M. tuberculosis. Conversely, co-infection with BCG suppressed adenovirus-induced type I IFN signaling and diminished the production of IFN-stimulated genes compared to adenovirus infection alone. Together, these findings reveal a complex regulatory interplay during adenovirus and BCG co-infection. While BCG partially restricts adenoviral IFN induction, the co-infection still drives an enhanced host-protective gene profile, suggesting that adenoviral vectors could serve as a viable platform to modulate innate immunity and improve BCG vaccine efficacy. IMPORTANCETuberculosis (TB) remains the leading cause of death by a single infectious organism with approximately 1.25 million deaths annually. M. bovis BCG remains the only approved vaccine for TB; however, its efficacy against the contagious and most common pulmonary form of the disease is limited. There have been numerous attempts to improve BCG efficacy, but these approaches have not resulted in any clinically approved vaccine. We propose that BCG combined with a replication-deficient adenovirus presents a way to bolster vaccine-conferred protection as the combination may elicit a robust innate immune response and drive a more protective T cell response. Moreover, BCG and replication-deficient adenoviruses have well-assessed safety profiles and decades of studies regarding their use in patients. The significance of our work is in leveraging their complementary immunology to function as a combined vaccine platform. This approach presents a novel and clinically feasible approach to improve the BCG vaccine.

In an era of increasing drug resistance, exploring alternative molecules is crucial for the efficient management and treatment of viral diseases. Nucleic acid aptamers have emerged as highly promising candidates due to their exceptional target specificity, low immunogenicity, and versatile mechanisms for viral blocking. This manuscript describes AptViralDB, a manually curated database providing comprehensive information on experimentally validated antiviral aptamers. It contains 1,768 entries of antiviral aptamers against 40 viral species and 104 molecular targets, compiled from literature and existing databases. Each entry provides detailed annotations, including sequence, aptamer type, target, chemical modifications, binding affinity, antiviral activity, stability, and cytotoxicity. We also provide predicted secondary structures and their corresponding minimum free energy (MFE) values. Additionally, a knowledge graph created using ArcadeDB/openCypher enables users to seamlessly explore connections among aptamers, viruses, molecular targets, and biological activities. Finally, the platform offers advanced search and browsing tools, BLAST-based sequence similarity searches, GC-content analysis, downloadable datasets, and REST API access to support computational applications. (https://webs.iiitd.edu.in/raghava/aptviraldb/).

Endogenous retroviruses (ERVs) constitute a significant fraction of vertebrate genomes and serve as genomic records of past retroviral infections, while also influencing host biology through regulatory co-option and, in some cases, ongoing retrotransposition. Despite extensive examination of ERVs in haplorrhine primates, equivalent analyses in strepsirrhines remain absent, leaving a substantial gap in our understanding of ERV diversity and evolutionary dynamics across the primate order. Here, we present the first comprehensive characterisation of ERVs in a strepsirrhine primate, identifying 15 Loris Endogenous Retrovirus (LERV) families encompassing 34 subfamilies and over 6,000 insertions in the Nycticebus coucang reference genome. Phylogenetic analyses resolved LERVs into three retroviral genera: betaretroviruses (LERV1-4), type-D betaretroviruses (LERV5-9), and gammaretroviruses (LERV10-15). LERV2a shows multiple hallmarks of recent or potentially ongoing retrotransposition, including a median insertion age of zero, a high proportion of identical LTR pairs, dN/dS ratios comparable to the active retrovirus HTLV, and insertional polymorphism between two conspecific genomes. Comparative genomic screening across Lorisidae revealed that LERV subfamily distribution broadly mirrors estimated insertion ages, with progressively fewer subfamilies detected in more distantly related species. These findings establish a detailed foundation for understanding retroviral evolution in Strepsirrhini and reveal that ongoing retroviral activity is not restricted to haplorrhine primates.

Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we implement precision molecular medicine and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high genomic selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive malignancy affecting children and adolescents. We describe the development of a non-viral and programmable polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for selective EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in patient-derived xenografts (PDXs) of EWS. Moreover, silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. To our knowledge, we describe the first non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which can be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

Influenza viruses circulate in humans, causing a substantial burden on global health. Investigation of influenza-host interactions could identify host factors that regulate influenza pathogenicity. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator and regulates crucial cellular processes. S1P lyase (SPL), an enzyme that mediates S1P degradation, was shown to display anti-influenza activity in a cell culture system. Here, we constructed a mouse model to demonstrate the antiviral function of SPL in respiratory epithelial cells during influenza in vivo. Deletion of SPL from lung epithelial cells exacerbated influenza-induced weight loss and mortality. Influenza virus began to propagate more effectively in the absence of SPL at the innate immune stage. Increased virus titers were sustained during influenza and associated with enhanced accumulation of multiple immune cell types in the lungs. Single-cell RNA sequencing was conducted to further define the function of SPL in lung epithelial cells. SPL deletion increased the proportion of alveolar type 1 (AT1) cells compared to alveolar type 2 (AT2) cells with alteration of the related signaling pathways, suggesting a role of SPL in AT1/AT2 programming. Importantly, host innate defense pathways were changed in SPL-deficient lung epithelial cells upon infection, which corroborates the antiviral function of SPL. This study elucidates the host protective function of SPL in lung epithelial cells during influenza and provides gene signature profiles critical for SPL-mediated alleviation of influenza pathogenicity. The findings may contribute to development of host-directed therapeutics to better control influenza.

BackgroundHerpesviruses are a group of double-stranded DNA viruses known to infect a wide range of vertebrates and establish life-long latent infections. While bats serve as natural reservoir hosts for numerous viral families, relatively few bat herpesviruses have been successfully isolated. In this study, we report the isolation and characterization of two novel alphaherpesvirus strains obtained from Rousettus aegyptiacus bats in Coastal Kenya. MethodsThe samples of oral and rectal swabs were collected from three different species of bats from coastal Kenya between October 2024 and April 2025; the bat species collected include Hipposideros spp., Coleura afra, and Rousettus aegyptiacus. Virus isolation was performed by inoculation of samples in Vero E6 cells and subsequent monitoring for cytopathic effects (CPE). Total nucleic acids were extracted from CPE positive cultures and subjected to library preparation to enable unbiased detection of both RNA and DNA viruses. The libraries were sequenced using next-generation sequencing with Illumina MiSeq platform. Subsequently, bioinformatic analysis was carried out to identify the virus, generate consensus genomes as well as phylogenetic analysis to determine the placement of identified viruses. ResultsTwo samples from R. aegyptiacus (KIK_460_O and KIK_465_O) induced typical CPE within five days. Sequencing and assembly yielded partial consensus sequences of approximately 60 kb (KIK_460_O) and 70 kb (KIK_465_O), representing extended genomic data for a bat-associated alphaherpesvirus. This virus has a genome of about 140kb, indicating that our partial assemblies account for about 43-50% of the total genome. Both isolates were found to be closely related to Dzifa herpesvirus, an alphaherpesvirus previously identified in Kilifi, Kenya. Alphaherpesvirus was identified based on partial sequencing of UL19 (3,787bp) and UL30 (2,846bp) genes. The two isolates were found to be identical at the UL19 gene, showing that they belonged to the same virus strain. Phylogenetic analysis showed that the novel alphaherpesvirus belongs to primate alphaherpesviruses under the subfamily Alphaherpesvirinae. ConclusionThis study reports the isolation and genomic characterization of a novel fruit bat alphaherpesvirus from Kenyan Rousettus aegyptiacus bats. The partial genome assembly (60-70 kb) represent the first extended genomic data for this virus, covering approximately 43-50% of the estimated 140 kb complete genome. The phylogenetic placement of this alphaherpesvirus near primate viruses, especially Pteropodid alphaherpesvirus 1, suggests bat-association and needs further investigation into its zoonotic potential.

Herpes simplex virus 1 (HSV-1) is a highly prevalent DNA virus with a major impact on human health. The HSV-1 genome is assembled into silenced stable chromatin and minimally transcribed during latency or assembled into permissive highly dynamic chromatin and highly transcribed during lytic infections. It is unclear how HSV-1 genomes transition between chromatin states, but epigenetics, including chromatin dynamics, have been proposed to play a major role. Chromatin remodeling complexes regulate cellular chromatin dynamics and contribute to DNA transcription, replication, and repair. The BAF family of chromatin remodeling complexes includes three ubiquitously expressed complexes (cBAF, PBAF, and GBAF) and several cell type-specific ones. Some common BAF subunits interact with two HSV-1 proteins, VP16 or ICP8. Three subunits shared by all BAF complexes and a unique subunit from each cBAF, PBAF, and GBAF were enriched in herpes nuclear domains (HND), the novel nuclear domains formed during lytic infection in which HSV-1 genomes are transcribed, replicated, and packaged. The shared ATPase SMARCA4 bound, directly or indirectly, to HSV-1 genomes. Bromodomains bind to acetylated histones and may thus be involved in this binding. However, none of four structurally unrelated inhibitors of BAF bromodomains drastically affected the recruitment of BAF subunits to HND, and neither of four commonly acetylated histone residues recognized by BAF bromodomains was enriched in the HND. BAF complexes are thus recruited to the HND by their interactions with VP16, which activates viral transcription, and ICP8. Surprisingly, the BAF complexes recruited by VP16 and ICP8 participate in inhibition of immediate-early, early, and early-late HSV-1 transcription, but not DNA replication or late transcription. We propose that BAF complexes are recruited to the HND by VP16 and ICP8, independently of their bromodomains, to inhibit viral transcription early in infection, thus contributing to the regulated cascade of gene expression. These findings also have implications to epigenetic anticancer drugs, in that it should be considered whether their use may reactivate latent herpes simplex viruses. Author SummaryHerpes simplex virus 1 (HSV-1) infects over two-thirds of the world population. HSV-1 establishes latency in neurons, resulting in life-long infection. Although most infections are asymptomatic, reactivation can produce a wide range of clinical manifestations, including cold sores, stromal keratitis, and encephalitis. Available treatments do not prevent reactivation or eliminate latent viral reservoirs, as no viral proteins are expressed during latency. Epigenetic regulation plays a role during the lytic and latent cycles. Lytic HSV-1 chromatin is highly dynamic whereas latent chromatin is stable. Chromatin dynamics are regulated by multiple factors, including the chromatin remodeling complexes. Here we show that the BAF chromatin remodeling complexes regulate HSV-1 transcription during lytic infection in primary fibroblast and transformed epithelial human cells. Although these complexes are recruited to the viral genomes by viral proteins, they counterintuitively downregulate viral transcription before the onset of DNA replication. We propose that BAF complexes play a major role in the regulation of the orchestrated cascade of viral gene expression and propose to consider the potential for reactivation of herpes simplex viruses when using epigenetic inhibitors in the treatment of cancer.

Several mammarenaviruses (MaAv) cause severe and often life-threatening disease in humans and represent major public health threats in their endemic regions. Lassa (LASV) and Junin (JUNV) MaAv, endemic to Western Africa and the Argentine Pampas, respectively, are etiologic agents of viral hemorrhagic fevers associated with high morbidity and mortality. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen capable of causing severe congenital disease and fatal infections in immunocompromised individuals. Despite their public health importance, no FDA-approved vaccines or virus-specific antiviral therapies exist to prevent and treat human MaAv infections. Current treatment relies on the off-label use of ribavirin whose therapeutic efficacy remains controversial. These findings underscore the urgent need to develop effective antiviral strategies against human pathogenic MaAv. Here, we investigated the impact of LCMV infection on host lipid metabolism using an integrated transcriptomic and lipidomic approach. Our data reveal extensive time-dependent remodeling of the cellular lipid landscape, with particularly prominent alterations in sphingolipid and fatty acid metabolic pathways. Functional interrogation of these pathways using pharmacological inhibitors identified acetyl-CoA carboxylase (ACC) and neutral sphingomyelinase 2 (nSMase2) as host factors contributing to efficient viral replication. Notably, inhibition of nSMase2 reduced infectious virus production by 2 logs of infectious virus. Our findings showed that LCMV reprograms host lipid metabolism to facilitate infection and identified sphingolipid turnover as a promising target for host-directed antiviral strategies against MaAv infections.

Identification of acyclovir-resistant herpes simplex virus (HSV) infections is critical for directing appropriate antiviral therapy, particularly for immunocompromised patients where resistance rates can reach 30%. In 2020, the University of Washington Clinical Virology Laboratory launched the first clinical genotypic HSV drug resistance test in the United States. While genotypic testing offers significantly faster turnaround times than traditional phenotypic assays, interpretation depends on established mutational databases and remains challenging when novel variants are identified. Here, we retrospectively reviewed all HSV acyclovir resistance Sanger sequencing tests performed from January 2020 to November 2025 at this primary national reference laboratory. Mutations identified via clinical sequencing were compared against published databases of HSV UL23 mutations to determine their phenotypic effects. Over the nearly six-year study period, 136 samples were sequenced with a median turnaround time of 10.6 days. Among these, 65 samples (47.8%) harbored acyclovir resistance mutations, including 45 frameshift mutations. Notably, across the 100 samples (73.5%) displaying mutations not known to cause acyclovir resistance at the time of clinical testing, we identified 56 distinct mutations, including 23 without prior characterization. Our national experience demonstrates that genotypic testing accelerates actionable results in clinical practice and confirms that frameshift mutations remain a primary driver of acyclovir resistance. Furthermore, by uncovering these 23 novel variants, this work provides critical targets for future biochemical and phenotypic characterization of HSV UL23 mutations.