Double-stranded RNA (dsRNA) is a universal indicator of viral replication and dysregulated RNA metabolism. Detection of dsRNA triggers some of the most powerful innate immune responses in human cells. Although these molecules differ in origin and structure, viral dsRNAs share the defining geometric and electrostatic features of the A-form helix, enabling their sequence-independent recognition by multiple sensor systems. Cytosolic receptors, like retinoic acid-inducible gene I (RIG-I), melanoma differentiation associated gene 5 (MDA5), and protein kinase R (PKR), as well as the oligoadenylate synthase (OAS)/RNase L pathway, convert dsRNA binding into interferon induction, translational arrest, and widespread RNA decay, while endosomal Toll-like receptor 3 (TLR3) and the inflammasome sensor NLR family pyrin domain containing 1 (NLRP1) expand surveillance to internalised or structurally disruptive RNAs. Counterbalancing these pathways, the RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) marks endogenous dsRNA through A-to-I conversion, preventing inadvertent activation of innate immune response and maintaining self versus non-self discrimination. Although all of these sensors recognise the A-form helix, each extracts distinct structural and chemical information from dsRNA and converts it into a specific response: RIG-I detects short duplexes with 5′-triphosphorylated ends; MDA5 assembles cooperatively along long uninterrupted helices; PKR integrates duplex length with translational control; OAS proteins act as strict reporters of helix regularity; and TLR3 as well as NLRP1 respond to dsRNA in compartment- and context-dependent ways. Epitranscriptomic marks and chemical modifications—including 2′-O-methylation, N6-methyladenosine, pseudouridine, and ADAR1-mediated inosine—further refine sensing by modulating helical stability and end structure, establishing a biochemical ‘self-code’ that shapes RNA immunogenicity. Together, these pathways form an integrated network that distinguishes between viral and endogenous dsRNA and coordinates antiviral defence with immune tolerance.

Recognition of viral double-stranded RNA (dsRNA) is a central trigger of innate antiviral immunity, yet the role of RNA length in shaping immune sensing across species remains poorly defined. Here, we identify RNA length as a key determinant of innate immune activation and demonstrate that this parameter is differentially tuned across vertebrate hosts. Using defined dsRNA molecules, we compared immune responses in human, duck, and chicken cells. Short 5′-triphosphorylated dsRNAs, including influenza-derived mini viral RNAs, robustly activated antiviral responses in human and duck cells but failed to do so in chicken cells. In contrast, longer dsRNAs induced immune activation across all species, revealing a species-specific threshold for dsRNA recognition. Mechanistically, this threshold is defined by differential engagement of RNA sensors. In human cells, sensing of short and intermediate dsRNA is strictly dependent on RIG-I, whereas in chicken cells, which lack RIG-I, MDA5 mediates dsRNA recognition and imposes a higher length requirement for activation. Consistent with this, duck cells, which retain RIG-I, respond to short dsRNA similarly to human cells. These differences in dsRNA sensing are accompanied by differential activation of downstream antiviral pathways, including PKR-dependent translational control and OAS/RNase L activation. Together, our findings identify RNA length as a fundamental parameter governing innate immune recognition and reveal that species-specific usage of RNA sensors constrains antiviral sensing, with implications for host susceptibility and virus-host interactions.

Vectors based on adeno-associated viruses (AAVs) are widely used as a delivery system in gene therapy. However, preexisting immunity against AAV remains a major limitation. This study examines the prevalence of AAV genomes and anti-AAV2 immunity in a Polish cohort of adult patients undergoing planned surgical interventions, which serves as a random sample of the general Polish population. The presence of AAV capsid sequences was analyzed in tissue samples using PCR and endogenous AAVs were detected in 33.95% of samples. Anti-AAV2 IgG antibodies were measured by a single point ELISA and were present in 80.93% of patients. Neutralizing activity was evaluated using an AAV2 transduction inhibition assay in HEK293T cells, revealing a 60% reduction in functional activity by patients’ sera. Tissue AAV PCR-positivity was associated with stronger neutralizing activity although the correlation with seropositivity was not as strong. These results suggest that pre-existing humoral immunity against AAV is common in the Polish adult population, indicating frequent exposure to naturally occurring AAVs. The findings provide important data on pre-existing anti-AAV immunity—a key factor considered in clinical trials and gene therapy development. Furthermore, the identification of naturally occurring AAV variants in human tissues points to the potential use of new sources of capsids with potentially distinct biological properties, which could contribute to the advancement of gene therapies.