HSV-1 anterior uveitis is a relapsing inflammatory disease driven by recurrent episodes of viral reactivation. Repeated inflammation can lead to corneal endothelial dysfunction, iris atrophy, ocular hypertension, secondary glaucoma and irreversible visual impairment. Diagnosis is based largely on the clinical phenotype, while polymerase chain reaction testing of aqueous humor can confirm viral DNA in selected cases. The review cites a reported sensitivity of 91.3% and specificity of 98.8% for HSV PCR, but emphasizes that sensitivity depends on factors such as intraocular viral load, sample timing, limited specimen volume, rapid clearance of viral DNA and inhibitory compounds. Sampling during active inflammation may therefore improve the chance of detecting intracellular viral DNA, while a negative result cannot by itself exclude a viral cause.

Even when positive, aqueous-humor PCR establishes the presence of HSV-1 DNA but provides little information about the host immune state, inflammatory activity or molecular mechanisms associated with recurrence. The review argues that extracellular vesicles may complement PCR by providing a broader view of the anterior chamber. These vesicles are released by ocular cells and infiltrating leukocytes and can carry proteins, lipids, messenger RNAs and microRNAs. Their lipid bilayers offer relative protection against extracellular degradation, although the in vivo lifetime of individual vesicle-associated microRNAs in human aqueous humor has not been established and may vary with vesicle origin, cargo and the local inflammatory environment.

Aquous humor is normally relatively acellular, making extracellular vesicles a major potential source of molecular information in the anterior chamber. Human aqueous-humor vesicle preparations have been shown to contain small RNAs and mature microRNAs, providing a methodological precedent for sequencing low-input samples. In neovascular age-related macular degeneration, vesicle fractions from affected eyes contained candidate disease-associated proteins, including cathepsin D and cytokeratin 8. Label-free proteomic analysis of aqueous-humor small EVs from 84 patients with Vogt-Koyanagi-Harada disease or Behçet disease identified 65 and 40 differentially expressed proteins, respectively, with complement-related pathways prominent in the functional analysis.

MicroRNA profiling has also demonstrated biomarker potential in uveitis, although most of that work has used peripheral blood, plasma vesicles or blood mononuclear cells rather than intraocular samples. In an intraocular tuberculosis study, ultra-low-input small RNA sequencing of aqueous humor identified 56 differentially expressed microRNAs despite the limited sample volume. Quantitative reverse-transcription PCR subsequently validated several candidates, including miR-423-5p, miR-328-3p, miR-21-5p and miR-16-5p. That study analyzed total aqueous-humor microRNAs rather than the EV-associated fraction, so it establishes analytical proof of principle without directly demonstrating that vesicle-enriched profiling is feasible in HSV-1 anterior uveitis.

Experimental evidence provides a biological rationale for studying microRNAs in HSV-1 infection. Both host- and virus-derived microRNAs regulate viral replication, latency, reactivation and inflammatory responses. Productive HSV-1 infection can restrict nuclear export of host precursor microRNAs while allowing viral precursors to escape that restriction. Several viral microRNAs have been linked to different stages of infection: miR-H1 is abundant during productive infection, whereas miR-H3 and miR-H4 are prominent during latency. Other viral microRNAs target immediate-early proteins involved in lytic gene expression, helping tune the balance between latency and reactivation. Host microRNAs also affect ocular immunopathology; for example, experimental deletion or inhibition of miR-155 reduced inflammation, Th1 and Th17 responses, neovascularization and disease severity in models of herpetic stromal keratitis.

Vesicle-associated microRNAs are not simply copies of the total microRNA pool. Regulated sorting can preferentially place particular molecules into extracellular vesicles, making their cargo biologically distinct from unfractionated aqueous humor. In Behçet uveitis, miR-19b-3p was increased in plasma EV fractions and experimentally promoted T-helper 17 differentiation while suppressing regulatory T-cell differentiation through reduced CD46 expression. However, this systemic evidence cannot be assumed to apply to the immune-privileged anterior chamber. The local mixture of resident ocular cells, infiltrating leukocytes and virus-infected tissues, together with barriers that may limit equilibration with the circulation, could make aqueous-humor vesicles more closely tied to intraocular events.

Translating this hypothesis will require control of several technical and biological variables. Anterior chamber paracentesis typically yields only about 100 to 200 microliters, potentially limiting vesicle and RNA recovery. Isolation methods—including ultracentrifugation, polymer precipitation, size-exclusion chromatography, ultrafiltration and immunoaffinity capture—differ in recovery, purity and reproducibility, and may co-isolate protein or ribonucleoprotein complexes. Sample timing, inflammatory activity, first versus recurrent episodes, concurrent keratitis, intraocular pressure, previous ocular procedures and exposure to antiviral or corticosteroid treatment could also alter vesicle release and cargo. Prospective studies should therefore record these variables prospectively, standardize workflows and include comparator groups such as varicella-zoster virus, cytomegalovirus and non-infectious anterior uveitis.

Normalization is especially difficult for low-input vesicle studies. Exogenous spike-in RNAs can track extraction and reverse-transcription variability but do not correct for biological differences in vesicle abundance. Particle-number normalization depends on accurate quantification and can be distorted by co-isolated material, while endogenous reference microRNAs may themselves change with inflammation. Total-RNA normalization can be unreliable when RNA concentrations approach the detection limit. The review therefore calls for predefined, justified and consistent normalization strategies, together with adherence to Minimal Information for Studies of Extracellular Vesicles guidance. Future work should also compare EV-associated and total aqueous-humor microRNAs in the same specimens rather than assume vesicle enrichment is analytically superior.

The most informative design may be longitudinal, sampling eyes during active disease, early treatment and, where clinically possible, quiescent or recurrent episodes. Host- and virus-derived microRNAs could be analyzed alongside HSV-1 PCR, quantitative viral load when available, inflammatory cell counts, intraocular pressure, corneal involvement and treatment exposure. Such within-patient comparisons could reveal whether candidate signatures track viral replication, reactivation or clinical remission while reducing inter-individual variation. Before any clinical use, candidate assays would require standardized collection and processing, analytical validation across laboratories, independent prospective testing in multicentre cohorts, meaningful diagnostic thresholds and evidence that they add value beyond established aqueous-humor PCR. The present evidence therefore supports a carefully defined research program, not a ready diagnostic test or a clinical recommendation.

Subject of Research: Evaluation of extracellular-vesicle-associated microRNAs as candidate biomarkers in HSV-1 anterior uveitis

Article Title: Aqueous humor exosomal microRNAs as emerging biomarkers in HSV-1 anterior uveitis

Article References: Saturno, M. C., Kalogeropoulos, D., Carbone, E., Piscopo, P., Gagliardi, O. M., La Cava, M., Lambiase, A., & Iannetta, D. (2026). Aqueous humor exosomal microRNAs as emerging biomarkers in HSV-1 anterior uveitis. Frontiers in Cellular and Infection Microbiology, 16, Article 1986644. https://doi.org/10.3389/fcimb.2026.1986644

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1986644

Keywords: HSV-1, anterior uveitis, aqueous humor, extracellular vesicles, microRNAs, ocular biomarkers, viral reactivation, intraocular inflammation, Aqueous, humor, exosomal, emerging