Virology

Although controversial, limited publications support the notion that HIV-1 can infect CD4-negative cells. The objective of this study was to provide a comprehensive investigation of a universally available CD4-negative cell line model system that can be infected with X4 and R5 HIV-1 to generate integrated proviral DNA and serve to study latent viral reservoirs. The reason that HIV-1 infection of CD4-negative cells has become less investigated is due to a lack of a fully characterized model for the study of this unusual pathway. To address this critical need, human osteosarcoma (HOS) cells, engineered to express either CD4, CCR5 or CXCR4, and easily available from a commercial source were used. CD4 expression was examined using western immunoblot, flow cytometry, anti-CD4 blocking antibody and mRNA expression. Cells were infected with HIV-1 pseudo-enveloped viruses bearing either JR-FL (R5-tropic) or HXB2 (X4-tropic) envelopes, constructed on NL4-3 luciferase/GFP backbone. Infection was monitored by luciferase readout and visualized by GFP immunofluorescence. Raltegravir was used to inhibit integration, and AMD3100 and maraviroc used to block chemokine coreceptors, CXCR4 and CCR5, respectively. Productive versus latent infection was quantified by dual-fluorescence readouts using HI.fate.E. We confirmed that HOS cells lack CD4. HOS cells expressing only CCR5 or CXCR4 supported HIV-1 infection, although infection was significantly lower than in matched CD4-positive controls. Raltegravir treatment blocked proviral integration in all instances. Coreceptor antagonism and envelope-deficient viruses revealed that infection of CD4-negative CXCR4 cells remained CXCR4-dependent, whereas CD4-negative CCR5 cells showed evidence of CCR5-independent infection. Dual-reporter HI.fate.E assays indicated that CD4-negative cells could support both productive and latent infection. These studies establish a universally available cell line model for the study of CD4-negative HIV-1 infection. This cell line model will provide insight into the question of how CD4-negative cells can be infected with HIV-1 and whether CD4-negative cells can provide latent viral reservoirs in HIV/AIDS. Author summarySince the first description of HIV/AIDS in 1981 and the recognition that CD4 was a primary receptor for HIV-1 in 1983, a limited number of reports have suggested that cells lacking CD4 could be infected with HIV-1. These reports continued even when it was shown in 1996 that co-receptors, CXCR4 and CCR5, were also required for HIV-1 infection of CD4 T-helper cells. Indeed, crystallography studies showed that CD4 was required to interact with the HIV-1 envelope gp120 in order to cause conformational changes in the envelope to expose the binding motif for chemokine co-receptor engagement, required for additional conformational changes to expose the gp41 fusion protein, allowing for entry and infection. However, reports continued that cells lacking CD4 could be infected which raised questions as to how this can happen. To address this critical gap, we have identified a cell line, HOS, that is commercially available, having expression of CD4, CXCR4 and/or CCR5. Using these HOS cell lines, we have been able to confirm that HIV-1, either X4 or R5 enveloped viruses, can infect CD4-negative cells. We have also confirmed that infection is productive and allows for latent proviral integration. Our findings provide a system for further studies of the mechanism(s) of HIV-1 infection of CD4-negative cells using a consistent model and may aid in elucidating establishment of viral reservoirs.

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.

High Pathogenicity Avian Influenza (HPAI) H5N1 clade 2.3.4.4b has spread beyond birds to affect seals across the Southern Ocean and sub-Antarctic region, with southern elephant seals (Mirounga leonina) particularly devastated. The virus, likely introduced via spillover from infected migratory birds, has killed tens of thousands of adult seals and pups throughout most of their range, though Macquarie Island remains unaffected so far. We used twenty years of elephant seal movement data from the southern Indian and Pacific oceans to assess whether seal-to-seal transmission could spread HPAI H5N1 between breeding colonies, despite the vast distances separating them (Marion Island, Iles Crozet, Iles Kerguelen, and Macquarie Island). There was substantial overlap in seals' at-sea distributions during their winter post-moult trips, when seals travel for weeks at average speeds of 3.5 km/h. Two transmission pathways were examined: (1) terrestrial "stepping stone" routes, where infected seals could pass the virus between colonies during short intervals to remain infectious were feasible from Marion Island to Kerguelen but not from Kerguelen to Macquarie Island; and (2) at-sea encounters between seals, which occurred frequently enough to enable transmission. The findings suggest that once established at Macquarie Island, the virus could potentially spread further to New Zealand's sub-Antarctic islands and mainland New Zealand. While seal-to-seal transmission appears possible, we conclude this is unlikely. Nonetheless, understanding at-sea contact rates enhances knowledge of H5N1 epidemiology and demonstrates the value of combining long-term population monitoring with movement data to understand wildlife disease dynamics.

The therapeutic efficacy of oncolytic viruses is often limited by the presence of tumor cells that resist virus-mediated killing. Here, we investigated the molecular mechanisms underlying resistance to Vesicular Stomatitis Virus (VSV) in PC3 cells, an aggressive metastatic prostate cancer (PrCa) cell line, using the VSV-sensitive LNCaP cell line as a comparator. RNA sequencing revealed that, relative to untreated cells, VSV-infected PC3 cells upregulated both pro-apoptotic genes, including BIM, PUMA, and NOXA, and anti-apoptotic and antiviral genes, including A20 and RIG-I. In addition, genes associated with antiviral and pro-survival pathways, including NF{kappa}B and PI3K-Akt signaling, were more highly expressed in PC3 cells than in LNCaP cells. At baseline, PC3 cells also exhibited elevated expression of multiple pro-survival genes, including BCL-xL, MCL1, and CK2, compared with LNCaP cells. Complementary proteomic analyses identified enhanced activation of NF{kappa}B, PI3K-Akt, and MSK1 signaling in VSV-infected PC3 cells relative to infected LNCaP cells. Furthermore, pharmacological inhibition of BCL-2 family proteins or NF{kappa}B signaling restored sensitivity to VSV-induced cell death in PC3 cells. Collectively, these findings identify NF{kappa}B-centered pro-survival signaling networks as key contributors to the resistant phenotype of PC3 cells and suggest that combining oncolytic virotherapy with targeted inhibitors may improve therapeutic efficacy in resistant prostate cancers.

Background: The gut environment is hostile to life, yet the human virome, dominated by bacteriophages, persists. Adaptations to the major capsid protein (MCP) may explain this. Phage MCPs conserve the HK97 fold, ideal for detecting convergent features across phage populations. Prior capsid stability research focused on individual phages, limiting broader pattern identification. Methods: MCPs from the Gut Phage Database (GPD) (n=8,478) and INPHARED (n=4,905) were predicted using ProtPhage + Phold and clustered using MMseqs2 (GPD=902 vs INPHARED=606). Structural predictions, conservation analysis, and capsomere modeling were used to characterize cysteine environments. Results: Biochemical analysis identified cysteine enrichment in GPD MCPs. Phylogenetic mapping was consistent with convergent evolution of high-cysteine MCPs. Over 50% of cysteines were [&ge;]90% conserved within and between clusters. Simulated capsomeres showed 83% of cysteines are buried (RSA <10%). Conclusions: These findings suggest gut phages may have convergently evolved cysteine-based capsid stabilization, with implications for engineering therapeutic phages.

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.

Classical swine fever (CSF) is a highly contagious disease caused by Classical swine fever virus (CSFV), posing a serious threat to the global swine industry. This study aimed to investigate the effect of CSFV on differential genes of histone lactylation at the H3K18 site in the PI3K-AKT signaling pathway. The site with the most significant change in histone lactylation antibody level was screened by Western blot. Omics analysis was performed using CUT&Tag technology to identify differential genes in the PI3K-AKT pathway between the CSFV-infected group and the mock group, followed by validation using RT-qPCR. Functional analysis of significantly differential proteins was conducted, and the protein expression level of THBS4 was detected by Western blot. The results showed that after CSFV infection of 3D4/21 cells, the H3K18la site exhibited the most significant difference in antibody level. A total of 8,859 differential genes at the H3K18la site were identified by CUT&Tag analysis, including 6,349 up-regulated genes and 2,510 down-regulated genes. Further focusing on the PI3K-AKT signaling pathway, 10 differential genes were identified, comprising 6 up-regulated genes and 4 down-regulated genes. Compared with the control group, the mRNA expression levels of CD19, LAMA1, PDGFRA, BDNF, ANGPT4, and THBS4 were up-regulated in the CSFV-infected group, while FOXO3 and NRTN were down-regulated. Western blot results showed that the protein expression level of THBS4 increased after CSFV infection. These findings lay an important foundation for understanding the molecular mechanisms regulating viral replication and immune evasion, and have significant scientific implications and potential application value.

Rodents are an important reservoir of zoonotic viruses and are ubiquitously present in densely populated urban areas. Betacoronaviruses in the Embecovirus lineage are well known to infect both humans and animals and have established rodent reservoirs. Here three Betacoronavirus gravedinis genomes were sequenced and characterized in white footed mice (Peromyscus leucopus, commonly white footed mice) collected in New York City, the second most populous city in North America. The genomes were distinct from mouse hepatitis virus (MHV), the prototype mouse betacoronavirus, and highly similar and identical in one case to previously characterized B. gravedinis sequences from white footed mice in Connecticut. Codon aware evolutionary models were used to identify specific sites under positive selection within the spike protein of B. gravedinis. A novel method was developed to predict the probable geographic distribution of the virus using publicly available data from the Global Biodiversity Information Facility to generate a weighted distribution map highlighting overlapping potential host ranges based on the evolutionary distance using a high resolution cytocrome B (CYTB) phylogeny of rodent species with potentially overlapping ranges. Our models predict three current hotspots of circulation in North America under different possible transmission regimes, and an additional fourth hotspot was predicted to arise in a warming future. This study highlights the continued need for biodiversity-informed surveillance of potential zoonotic pathogens in rodents.

Virus-like particles (VLPs) are an abundant component of microbiomes with critical ecological roles such as population control through viral predation and horizontal gene transfer. Studying the collection of viruses in microbiomes (the virome) through metagenomics has provided important insights into the composition and functions of VLPs in different environments. However, the current gold-standard method for VLP purification, CsCl density gradient ultracentrifugation (CsCl), is low throughput, time consuming and suffers from biases which limits the ability to study viromes in larger sample sets and can interfere with data interpretation. Here we present an anion exchange (AEX) chromatography-based approach for the purification of VLPs from microbiome samples that allows for significant increases in throughput and reproducibility while achieving VLP purity levels similar to or higher than CsCl. We used microbiome samples of known composition to first establish and evaluate the AEX approaches and compare them to CsCl. We implemented the AEX approach both for fast performance liquid chromatography (FPLC) and in multi-well plates. We compared the VLPs purified with CsCl and AEX using shotgun metagenomic sequencing and found that AEX performs similarly to or better than CsCl for purification of VLPs. AEX purified VLP-fractions captured significantly more viral DNA compared to CsCl. We also found that both AEX and CsCl were capable of capturing viruses present at extremely low relative abundances (<0.001%). Additionally, we found that DNase digestion and CsCl may bias against filamentous phage morphologies. Finally, we purified VLPs from conventional murine feces using AEX and CsCl. AEX purified murine fecal VLPs had a much higher viral DNA content (85%) than CsCl (41%). While there were some differences in viral contigs assembled from AEX and CsCl VLP metagenomes, these method unique viral contigs made up only small proportions (<8%) of the relative abundance in the VLP metagenomes. AEX, particularly in the multi-well format, enables the ultrapurification of VLPs from tens to hundreds of samples in a single day thus facilitating virome studies with the large sample numbers needed for translational and clinical research.

Bunyamwera virus (BUNV) is the prototypical member of the Peribunyaviridae family of arthropod-borne viruses and possesses a genome comprising three segments of negative-sense RNA, named small, medium and large. The medium segment encodes a polyprotein that is processed to form Gn and Gc spikes and a non-structural protein, NSm. The role of NSm during replication in mammalian cells is poorly characterized, although it associates with a Golgi-derived structure called the virus factory (VF), the site of BUNV genome replication and virion assembly. To further define NSm function, we generated an epitope-tagged BUNV and used co-immunoprecipitation and quantitative proteomics to identify host interacting partners. NSm interacted with BCL-2 interacting protein 1 (BNIP1), a SNARE protein involved in COPI vesicle trafficking, with the importance of this interaction demonstrated by siRNA-mediated knockdown of BNIP1 expression, which significantly reduced BUNV gene expression and virion production. Interestingly, NSm also interacted with components of the NRZ complex, involved in COPI vesicle tethering in association with BNIP1, and inhibition of COPI complex formation resulted in loss of NSm expression. Taken together, our results identify BNIP1 as a host cell factor necessary for efficient BUNV replication and suggest the cellular localization of NSm at the VF is COPI-dependent.

Lone star virus (LSV) is a bandavirus first isolated from Amblyomma americanum ticks in the United States (U.S.) and is phylogenetically related to severe fever with thrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV), and Bhanja virus, each of which has been associated with severe human disease. In contrast to these better-characterized bandaviruses, LSV remains poorly studied, and its pathogenic potential is not well defined. Recent detection of LSV RNA in cerebrospinal fluid from an immunocompromised patient in Idaho, U.S., with fatal meningoencephalitis further highlights the need for experimental systems to investigate LSV biology. Here, we rescued recombinant (r) LSV from cloned cDNA and used it to characterize LSV. rLSV replicated similarly to the parental isolate in mammalian cells and caused rapid, systemic, and lethal disease in IFNAR-/- mice, with widespread detection of viral (v) RNA across multiple tissues, hepatic and splenic pathology, and induction of inflammatory cytokines. In contrast, C57BL/6J mice controlled infection and exhibited no clinical disease. To place LSV within a broader comparative framework, we generated rSFTSV from cloned cDNA and compared rLSV, rSFTSV, and HRTV in cell culture and IFNAR-/- mice. Our studies revealed distinct disease kinetics among these related tick-borne bandaviruses and showed that HRTV-induced immunity protected against homologous HRTV rechallenge and heterologous rSFTSV challenge, but not rLSV challenge. Together, these findings establish reverse-genetics platforms and small-animal models for comparative bandavirus studies, define key features of LSV pathogenesis, and place this neglected virus within a framework of related bandaviruses that differ in virulence and immunological overlap.

Abstract Background: The Chikungunya virus (CHIKV), a re-emerging mosquito-borne alphavirus, is responsible for acute febrile illness and severe polyarthralgia. Although both innate and adaptive immune responses influence the disease outcomes, the detailed cellular immunopathogenesis of CHIKV in peripheral blood is not yet fully elucidated. Methods: We conducted single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) obtained from patients acutely infected with CHIKV and from healthy control subjects. Cellular interactions were inferred, and the transcriptomic results were orthogonally validated through quantitative real-time PCR (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) to assess systemic interferon-stimulated responses. Furthermore, a comparative analysis was performed using publicly available single-cell data from Dengue virus (DENV) infections. Results: CHIKV infection significantly altered the immune system, increasing monocytes and dendritic cells while reducing T and B lymphocytes. Monocytes and NK cells showed strong activation of interferon-stimulated genes (ISGs). Monocytes were identified as key in driving inflammatory and immune responses. In adaptive immunity, CHIKV led B cells to become plasmablasts with antiviral immunoglobulins and caused T cells and NK-like T cells to show signs of cytotoxicity and exhaustion. Validation showed increased levels of IFN-{gamma}, IFN-{beta}1, MX1, and ISG15. CHIKV triggered a more intense, monocyte-driven interferon response than DENV. Conclusions: Acute CHIKV infection induces a systemic interferon response predominantly centered on monocytes, accompanied by significant alterations in adaptive immunity. Circulating ISG products, including MX1 and ISG15, reflect the transcriptomic activation and may serve as potential biomarkers for assessing the early intensity of innate antiviral responses.

Orthologs of the herpes simplex virus (HSV) pUL16 tegument protein are conserved throughout the Orthoherpesviridae family. During HSV infection, pUL16 functions in the nuclear egress of nascent nucleocapsids from the nucleus to the cytoplasm, prevents the docking of nascent cytoplasmic nucleocapsids to nuclear pore complexes, promotes the final envelopment of cytoplasmic nucleocapsids, and enhances cell-to-cell spread of virus infection. How pUL16 performs these diverse functions is poorly understood. To gain further insight into the mechanisms by which pUL16 mediates its activities, we utilized a BioID approach to identify cellular and viral proteins in proximity to pUL16 during the infection of human keratinocytes. By comparing proteins in proximity to pUL16 during infection with proteins in proximity to its well-known virus-encoded binding partner, pUL21, we provide new insight into the activities of pUL16 that likely occur in complex with pUL21 and those that are independent of pUL21. A key function of pUL21 is to deliver protein phosphatase 1 (PP1) to viral and cellular substrates to mediate their dephosphorylation. Intriguingly, the findings presented suggest that pUL16 interactions with pUL21 may regulate the isoform of PP1 that is bound to pUL21 and thereby regulate the specificity of substrate dephosphorylation. ImportanceHSV-1 and HSV-2 are important human pathogens that currently infect roughly 3.8 billion and 520 million people, respectively. These viruses cause lifelong, recurrent, infections and cause a variety of diseases including vesicular lesions of the oral and genital mucosa, corneal blindness, meningitis, encephalitis and devastating neonatal infections. The HSV pUL16 tegument protein performs a number of critical functions for the virus that influence virion assembly and the spread of infection between cells. In this study we have identified cellular and viral proteins that are proximal to pUL16 during infection of human keratinocytes, providing new insight into the mechanisms used by pUL16 to perform its activities.

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.

Background: RSV is a major cause of severe lung infections in young children, with over 95% of deaths occurring in poorer countries. Bangladesh has high rates of RSV illness in children but lacks genetic data from after the COVID-19 pandemic. New vaccines and antibody treatments are now available, making local genetic information essential. Objectives: We sequenced complete RSV genomes from Bangladeshi patients to study virus types, genetic changes, and protein mutations, and shared our data openly. Methods: From August 2024 to December 2025, we took 59 RSV-positive samples with high virus levels from hospital patients and sequenced their full genomes using Oxford Nanopore technology. Results: Among 11,874 patients, 1,390 (11.7%) had RSV, mostly RSV-A (94.6%). We obtained 49 good-quality full genomes from the 59 samples (83% success): 43 RSV-A (ON1 type, five sub-lineages) and 6 RSV-B (BA9 type). We found S276N in 35% of RSV-A and S389P in all RSV-B, but neither stops current antibody treatments. All RSV-A viruses gained a new sugar attachment site on their F protein, and most RSV-B viruses gained one too. We uploaded all 49 genomes to GISAID for public use. Conclusion: This work shows we can do full RSV genome sequencing in Bangladesh. The viruses here still match the targets of new vaccines and antibodies, which is reassuring. Our findings provide a foundation for planning RSV prevention in Bangladesh and South Asia.

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.

Aedes albopictus is a major arboviral vector whose global expansion, driven by human activities and climate change, poses a growing public health concern for a number of neglected tropical diseases in both tropical and temperate regions. As a poikilotherm, its biology and population dynamics are strongly influenced by temperature, thereby shaping disease transmission. To thwart and control its geographic expansion, effective vector control strategies are increasingly critical. Densoviruses (DVs), such as AalDV2, are being explored as mosquito viral biocontrol agents due to their restricted host range and ability to disseminate through oviposition sites. However, the influence of environmental parameters on the interactions between Ae. albopictus and AalDV2 remains poorly understood. This makes their performance under realistic, fluctuating thermal regimes difficult to estimate. In this study, we investigated the combined effects of temperature and AalDV2 exposure on Ae. albopictus survival and development across its full life cycle. Mosquitoes were reared under fluctuating temperature regimes (26-28 {degrees}C and 32-34 {degrees}C, 12:12 day[ndash]night cycles) and exposed to AalDV2 or a control treatment. Chronic exposure to 32-34 {degrees}C significantly reduced overall survival, decreasing median lifespan by approximately 10 days (HR=2.21, p=0.0018), with a deleterious effect increasing over time. It extended aquatic lifespan and increased pupal mortality. It also reduced adult lifespan in both sexes with a stronger effect in females. AalDV2 exposure had no significant effect on overall survival, stage-specific mortality, or adult lifespan. However, a significant interaction between viral exposure and thermal stress was detected on aquatic lifespan: AalDV2-exposed females showed further extended larval and pupal development specifically under the 32-34 {degrees}C regime, without any effect on survival. These results indicate that the biocontrol potential of AalDV2 cannot be assessed independently of thermal context: while lethal effects were absent under both fluctuating regimes, the prolongation of aquatic development by the virus under thermal stress may have indirect consequences for mosquito population dynamics that warrant further investigation.

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.

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.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer that frequently carries integrated Merkel cell polyomavirus DNA and expresses oncogenic viral small T antigen (sTAg) and truncated large T antigen (tLTAg). We previously reported a mouse model of MCC with skin-targeted expression of sTAg, tLTAg, and the Merkel cell transcription factor ATOH1, combined with deletion of Trp53. Here, we optimized this model to achieve 100% tumor penetrance with lymph node metastases, established four mouse MCC cell lines, and selected one line, mMCC2, for pilot preclinical trials. In immunocompetent C57BL/6J mice, mMCC2 cells reliably produce MCCs and lymph node metastases following subcutaneous or intradermal (orthotopic) injection, and liver and lung metastases after tail vein injection. Mouse MCC allografts resemble parental tumors histologically and express a full complement of MCC differentiation markers. Treatment of allografted mice with anti-PD-1 resulted in variable inhibition of tumor growth. In contrast, treatment with lysine-specific histone Wdemethylase 1 (LSD1) inhibitors, with or without anti-PD-1, led to consistently lower tumor volumes by 5.7-fold in both groups (P < 0.0001) and smaller or undetectable lymph node metastases. Growth-inhibited tumors in all groups showed a marked reduction in proliferating tumor cells and increased infiltration by F4/80+ macrophages and CD8+ T cells. These findings support a role for immune-cell recruitment in treatment response and underscore the importance of immunocompetent preclinical models, even in studies using targeted therapies. This unique virus-positive MCC allograft model, which produces local tumors as well as regional and distant metastases in immunocompetent hosts, provides a critical platform for preclinical evaluation of new therapeutic strategies and sets the stage for much-needed translational studies to inform future clinical trials.