The replacement of a single codon in the human prion gene, causing the substitution of glycine with valine at position 127 (G127V) of the prion protein (PrP), prevents development of prion disease. We set out to explore if prion disease survival extension manifests in mice if the V127 mutant is delivered through a recombinant adeno-associated virus (rAAV) packaged as a self-complementary DNA. The notorious delivery limitations of rAAV vectors were designed to be overcome using a cross-correction approach that relied on the expression of the mutation in the context of glycosylphosphatidylinositoI-anchorless (ΔGPI) PrP. In this proof-of-concept study, we inoculated Rocky Mountain Laboratory (RML) prions into knock-in mice, in which the endogenous murine prion protein gene (Prnp) was replaced with the bank vole prion protein gene (BvPrnp). Prion-inoculated mice that were retro-orbitally transduced with a protective rAAV vector encoding BvPrnpV127ΔGPI survived ~50 days longer than control mice that were unprotected. A deep proteomic analysis revealed that BvPrnpV127ΔGPI was protective by slowing perturbations to the proteome observed in late-stage RML prion disease. In addition to capturing details of synaptic decay and depletion of proteins in proximity to PrP, the proteomic dataset revealed the identity of proteins of potential diagnostic value that may be central to the brain's attempt to fight prion disease by contributing to astrocytosis or microgliosis, by coping with calcium influx, or by enhancing the endoplasmic reticulum processing of essential proteins. Taken together, our results demonstrate that a gene therapy based on a GPI-anchorless PrP containing the G127V mutation can delay the onset of prion disease in mice, providing a framework for development of a corresponding therapy in humans.

Toxoplasma gondii (T. gondii) is a prevalent zoonotic parasite that has been implicated in influencing human psychiatric disorders and risk-taking behaviors. Using genome-wide association study (GWAS) data, we selected 25 and 76 single-nucleotide polymorphisms as instrumental variables for anti-T. gondii IgG seropositivity and anti-T. gondii IgG levels, respectively, and conducted two-sample Mendelian randomization (MR) analyses across 18 GWAS datasets to investigate potential causal effects on addiction, bipolar disorder, obsessive-compulsive disorder, schizophrenia, and risk-taking behavior in European populations. Contrary to previous epidemiological evidence, our MR analyses do not support a significant causal association between T. gondii infection and any of the studied psychiatric disorders or risk-taking behavior. Collectively, these results establish that any true causal effect of genetic liability to T. gondii infection is likely to be small (OR < 1.18 for schizophrenia, < 1.29 for bipolar disorder) and below the effect sizes typically reported in observational seroepidemiological studies, although small or infection-phase-specific effects cannot be excluded.

Understanding the potential for resistance to antibodies targeting conserved epitopes on the coronavirus spike protein is essential for developing broad-spectrum antivirals. The S2'-helix and stem helix represent two key conserved epitopes across multiple coronaviruses, with the S2'-helix being broadly conserved throughout the coronavirus subfamily and the stem helix primarily conserved among betacoronaviruses. Here, we demonstrate that the S2'-helix in SARS-CoV-2 possesses a higher genetic barrier to antibody resistance than the stem helix. Potent escape mutations in the stem helix, including D1153G, Y1155S, F1156L and F1156V, were selected under antibody pressure, and variants such as S1147L, E1151D, D1153G, D1153H, D1153Y and Y1155H were frequently identified in naturally circulating strains. These mutations completely abolished neutralization by some stem helix-targeting antibodies. In contrast, under pressure from S2'-helix-targeting antibodies, we did not detect clear viral escape. Only one naturally occurring mutation in the S2'-helix, L822F, was observed at considerable frequency, it weakly or mildly reduced but did not abolish neutralizing activity. Furthermore, combination therapy with S2'-helix-targeting antibodies synergistically suppressed the emergence of escape mutants selected by stem helix-directed antibodies. Our findings underscore the S2'-helix as a promising target for the design of broadly protective coronavirus therapeutics with a reduced risk of viral escape.

Respiratory syncytial virus (RSV) is an enveloped, negative-sense, single-stranded RNA virus whose ribonucleoproteins (vRNPs) must be transported from cytoplasmic viral factories to the plasma membrane for efficient virion assembly. Viral vRNPs comprise genomic RNA encapsidated by nucleoprotein N and associated with the polymerase complex (L, P, and M2-1). It was previously demonstrated that newly synthesized vRNPs are transported along microtubules by hijacking Rab11a, a small GTPase involved in the regulation of recycling endosomes. In our previous study, we showed an interaction between Rab11a and vRNPs in infected cells by immunoprecipitation assays, nevertheless the molecular mechanisms underlying Rab11a viral hijacking remained unknown. Here, we provide the first comprehensive characterization of the interaction between RSV vRNPs and Rab11a using immunoprecipitation, immunofluorescence colocalization, GST pull-down assays, and biolayer interferometry. We demonstrate that the viral polymerase L is the sole vRNPs component responsible for Rab11a recognition: immunoprecipitation of L specifically co-precipitates HA-tagged Rab11a, whereas other vRNPs proteins show no interaction. In vitro binding studies confirm that L interacts directly and specifically with the active, GTP-bound form of Rab11a with sub-micromolar affinity. Domain mapping using truncated constructs reveals that this interaction requires the C-terminal methyltransferase and CTD domains of L (residues 1756-2165) and depends on Rab11a's Switch I region, known to mediate interactions with cellular Rab11a partners. Mutagenesis further highlights leucine 1860 in the L polymerase as critical for Rab11a binding. Competitive inhibition of the interaction between Rab11a and L using the minimal Rab11a-binding domain significantly impairs vRNP dynamics during infection, indicating that Rab11a-L binding is involved in the transport of vRNPs. Together, these findings establish RSV polymerase L as the key mediator of Rab11a engagement, define the molecular interface of their interaction, and reveal a potentially conserved viral strategy for genome transport. Targeting the L-Rab11a interaction could therefore be a promising strategy for the development of RSV-specific or broad-spectrum antiviral therapies.

Epstein-Barr Virus (EBV) infection and reactivation in B-lymphocytes are tightly regulated by host antiviral response genes. In the present study, we identify Interferon Stimulated Genes (ISGs) RSAD2 (radical S-adenosyl methionine domain-containing 2) and CMPK2 (Cytidine/Uridine Monophosphate Kinase 2) as key modulators of EBV expression and cellular response during EBV infection and reactivation. EBV primary infection and reactivation lead to a coordinated upregulation of RSAD2 and CMPK2. Depletion of RSAD2 reduced cell viability and limited EBV reactivation, while depletion of CMPK2 led to reactivation of EBV lytic gene expression during latency. Despite distinct subcellular localizations, RSAD2 at the endoplasmic reticulum (ER) and CMPK2 in the mitochondria, transcriptomic analysis revealed that both genes functionally converge and exhibit overlapping roles in driving shared immunometabolic pathways, specifically Interferon (IFN) signaling, MAPK signaling, oxidative phosphorylation, mitochondrial function, eukaryotic translation, and ATF-4-associated unfolded protein response (UPR). We show that RSAD2 and CMPK2 knockdown affects IRAK1-TRAF6-TAK1 expression levels, and RSAD2 overexpression downregulates NF-κB signaling by EBV membrane associated oncoprotein LMP1. Depletion of RSAD2 and CMPK2 had significant effects on global metabolites consistent with a remodeling of nucleotide metabolism, glycolysis, fatty acid biosynthesis and degradation of superoxides. EBV reactivation induced formation of antiviral ribonucleotide ddhCTP during lytic EBV reactivation which was strictly dependent on RSAD2. These observations demonstrate that RSAD2 and CMPK2 function in a coordinated ER-Mitochondria-Interferon signaling axis that shapes EBV reactivation and host immune control, including a novel layer of immunometabolic regulation modulating viral latency and reactivation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone extensive evolution since its emergence in 2019, underscoring the continuous need for vaccines and therapeutics effective against multiple variants of concern (VOCs). The S2 subunit of the viral spike (S) glycoprotein is highly conserved among sarbecoviruses, making it an attractive target for broadly protective countermeasures. To elucidate the S2 antigenic landscape, we employed yeast surface display to isolate S2-targeted antibodies from COVID-19 convalescent donors. Biophysical characterization revealed that these S2 apex-directed antibodies preferentially bind to open spike conformations and a stabilized S2 construct but not to the closed, trimeric prefusion spike. Cryo-electron microscopy structures defined a cryptic epitope encompassing the upper helix and fusion peptide proximal region on S2. This epitope is conserved among sarbecoviruses but remains largely occluded in the closed prefusion conformation of the spikes. As a result, the antibodies exhibited weak neutralization activity against SARS-CoV-2 pseudoviruses, failed to neutralize authentic viruses, and did not provide protection in a lethal mouse challenge model using a mouse-adapted SARS-CoV-2 strain. These findings highlight a non-neutralizing epitope on S2 capable of eliciting antibodies during SARS-CoV-2 infection in humans and provide valuable reagents for probing S2 conformational dynamics and optimizing S2-based vaccine antigens.

Microbial keratitis caused by Exotoxin U (ExoU)-producing strains of Pseudomonas aeruginosa is associated with poor clinical outcomes and reduced responsiveness to antimicrobial therapy. ExoU, a phospholipase, is secreted directly into host cells, causing their lysis. A screen of 3,034 FDA-approved compounds identified zinc pyrithione (Zp), bismuth subcitrate (Bis), and polymyxin B (Pol) as lead inhibitors of ExoU selectively inhibiting it without affecting human PLA2s or bacterial viability. The compounds have distinct inhibitory mechanisms: Zp disrupted ExoU oligomerization and protein stability; Bis impaired phosphatidylinositol 4,5-bisphosphate-dependent membrane association; Pol directly inhibited catalysis via its lipid-peptide architecture. Compound combinations enhanced ExoU inhibition in vitro to nanomolar concentrations. In mammalian cells, Bis and Pol promoted lysosomal trafficking and degradation of ExoU. A high content screen of 52 P. aeruginosa keratitis isolates demonstrated the broad efficacy of these inhibitors in exoU[+] strains. Therapeutic efficacy was evaluated in ex vivo porcine corneas, Galleria mellonella, and in vivo mouse keratitis models. Topical delivery of ExoU inhibitors, particularly in combination, significantly reduced corneal opacity, ulceration, and stromal damage in porcine corneas without affecting bacterial load. In Galleria, compound combinations significantly maintained larval health, improved larval survival and delayed mortality. In a mouse eye infection model, combinatorial treatment reduced disease severity and preserved tissue viability without altering bacterial burden. These findings validate ExoU as a druggable virulence factor and support the repurposing of these compounds as an antivirulence strategy for the treatment of P. aeruginosa infections in human beings and in veterinary medicine.

Nelson Bay orthoreovirus (NBV) was initially discovered in a bat sample but has since been isolated from patients with acute respiratory tract diseases. Accumulating reports of NBV isolation from patients with respiratory tract viral infections suggest that NBV is able to transmit and cause disease in humans. However, the underlying molecular mechanisms remain unclear. We previously established a reverse genetics system for NBV Miyazaki-Bali/2007 (MB) strain isolated from a patient with an acute respiratory tract disease. We found that the fusion-associated small transmembrane protein (FAST)-which is necessary for syncytium formation-and cell attachment protein σC play crucial roles in MB virulence; however, whether these gene products determine the strain-specific difference in NBV virulence remains unclear. Therefore, here, we compared the virulence of the MB strain with that of the NBV strain isolated from a bat sample (NelB strain). We found that the NelB strain did not cause a virulent phenotype in the mouse model. Using reverse genetics, we found that the S1 gene segment correlates with the virulent phenotypes of NBV strains. Moreover, among the three proteins encoded by the S1 gene segment, structural protein σC, but not nonstructural proteins FAST or p17, contributed to the difference in virulence in vivo. Further analysis using a panel of σC mutant viruses showed that the middle body domain in σC was involved in the different virulent phenotypes, rather than the C-terminal head domain, which contains a putative receptor-binding domain. These results provide new insights into the mechanisms underlying NBV transmission and pathogenesis.

Epidemic preparedness depends on tracking microbial evolution that drives shifts in ecological behaviors such as disease emergence. However, the genetic constraints mediating microbial emergence for generalist and specialist behaviors remain poorly described. Here, we addressed this question by combining comparative and functional genomics with phylogeny-based evolutionary analyses of the cereal pathogen Xanthomonas translucens. We show that a generalist X. translucens subgroup arose from a specialist ancestor, and the loss of a single effector gene, xopAL1, contributed to the generalist host expansion by promoting host jump from barley to wheat. Deleting barley-specialist X. translucens xopAL1 recapitulated the host jump to wheat and demonstrates risk across each globally distributed genetic lineage. However, this niche expansion via XopAL1 loss incurs a significant fitness cost to colonize barley. Moreover, the specialist lineage gained an additional effector gene, xopAJ, which enhanced virulence on barley while restricting oat infection, thereby reinforcing niche specialization. We further conducted transcriptomic analysis of wheat and determined that XopAL1 triggers a defense response that involves the reduction of photosynthetic processes. Our work provides an experimentally validated evolutionary framework to understand mechanisms of intergenera host jump. Overall, we demonstrate that single events of gene loss and gain shape ecological behaviors by creating a dynamic trade-off between niche breadth and specialization.

Getah virus (GETV), a mosquito-borne alphavirus, poses an emerging threat to public health with its increasingly broad host spectrum. While glycosaminoglycans (GAGs) serve as critical attachment factors for many alphaviruses and the low-density lipoprotein receptor (LDLR) facilitates the cellular entry of several members, the precise viral determinants governing these interactions and their implications for viral virulence remain poorly defined. Here, we introduced an H86Y substitution, a potential adaptive mutation site, within the E2 glycoprotein of GETV using reverse genetics. The H86Y mutant replicated more efficiently in mosquito C6/36 cells but was consistently attenuated across several mammalian cell lines. In susceptible mouse models, H86Y infection led to reduced viral loads, milder histopathology, and lower inflammatory responses compared with the parental virus, yet still elicited robust protective immunity in adult mice. Mechanistically, a series of functional assays, including infection in GAG-deficient cells, decoy inhibition, co-immunoprecipitation, receptor overexpression and knockdown, and biolayer interferometry, demonstrated that the residue 86 in E2 glycoprotein is a critical determinant for GETV binding to both GAGs and LDLR. The H86Y mutation concurrently reduces these interactions, contributing the impairment of virus attachment and entry into mammalian cells. Furthermore, the GAG-binding site functionally overlaps with the LDLR interaction interface. In LDLR-deficient suckling mice, the impaired replication of H86Y persisted in examined tissues. However, pre-treatment with heparinase nearly completely eliminated this attenuation phenotype, further confirming that LDLR and GAG are the key host factors mediating attenuation phenotype for H86Y. In summary, the residue 86 of the GETV E2 glycoprotein represents a determinant of viral virulence, and an H86Y mutation attenuates GAGs and LDLR-dependent infection, providing mechanistic insights into alphavirus-host interactions and a potential target for antiviral and vaccine development.

CD4+ T cells that produce IFN-γ (T helper 1 [Th1] cells) chiefly mediate vaccine acquired resistance to fungal infections. However, the key regulators of the development of Th1 cells and acquired resistance to fungi are incompletely understood. Here, we report that Natural Killer Cell Granule Protein (NKG7) acts as an unappreciated regulator inducing anti-fungal Th1 cells to produce IFN-γ and converting plastic Th17 cells into polyfunctional memory Th1 cells.

Successful viruses including SARS-CoV-2 antagonize and evade immune detection to establish infection. As population immunity to the ancestral strain has risen, new variants-particularly Omicron-have evolved to avoid detection by both circulating antibodies and the innate immune system. Screening innate immune agonists, we previously identified STING agonists as potent inhibitors of the ancestral variant of SARS-CoV-2. When we explored the sensitivity of Omicron BA.1 to diverse innate stimuli, we found that Omicron is also sensitive to interferons and STING agonists; however, Omicron variants are uniquely restricted by small DNA oligonucleotide ligands known to activate TLR9. Mechanistic studies showed that short, but not long, DNA oligonucleotides of diverse sequences, including those that do not engage TLR9, block Omicron infection at an entry step downstream of TMPRSS2-mediated activation. Using genetic and pharmacological assays we confirmed that activity was independent of TLR9. Furthermore, these oligodeoxynucleotides (ODNs) are active in vivo, reducing SARS-CoV-2 Omicron titers. Together, these findings identify a previously unrecognized antiviral activity of short DNA oligonucleotides and establish them as selective inhibitors of SARS-CoV-2 Omicron entry.

Chronic hepatitis B virus infection persists as a global health crisis, driving life-threatening liver pathologies such as hepatocellular carcinoma. Central to HBV's resilience is the covalently closed circular DNA (cccDNA), a viral minichromosome that orchestrates viral transcription and sustains infection. Dissecting cccDNA-driven transcription remains a formidable challenge due to the dense overlap of viral open reading frames, which generates RNA transcripts with shared sequences, complicating precise quantification and hindering efforts to unravel HBV's transcriptional regulation. To address this bottleneck, we developed a multiplexed assay named ddPCR for Overlapping Transcripts (ddOTs) capable of simultaneously quantifying all major HBV RNA species, including splice variants, with unprecedented specificity and sensitivity. This method overcomes current limitations by leveraging the high-resolution power of ddPCR to deconvolute overlapping transcripts, enabling direct interrogation of individual promoter/enhancer activities and RNA stability. By offering a cost-effective, scalable solution for precise RNA profiling in rare biological samples, this breakthrough tool unlocks new avenues for exploring cccDNA biology and accelerating antiviral drug development.