RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.

: Disruption of HIV-1 Integrase (IN) interactions with the host-factor Lens Epithelium-Derived Growth Factor (LEDGF)/p75 leads to decreased, random integration, increased latent infection, and described here, accumulation of HIV-1 antisense RNA (asRNA). asRNA increase was observed following interruptions of IN-LEDGF/p75 interactions either through pharmacologic perturbations of IN-LEDGF/p75 by treatment with allosteric HIV-1 integrase inhibitors (ALLINIs) or in cell lines with LEDGF genetic knockout. Additionally, by impairing Tat-dependent HIV transcription, asRNA abundance markedly increases. Illumina sequencing characterization of asRNA transcripts in primary T cells infected in the presence of ALLINIs showed that most initiate from within the HIV-1. Overall, loss of IN-LEDGF/p75 interactions increase asRNA abundance. Understanding the relationship between ALLINIs, integration sites, asRNA, and latency could aid in future therapeutic strategies.

A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

Background Inborn errors of immunity (IEIs) include more than 500 genetic disorders that cause recurrent infections, autoimmunity, and inflammatory complications. Although classified as rare diseases, their prevalence is increasing, making IEI an emerging public health problem. However, reliable national epidemiological data and coordinated follow-up data of IEI patients are lacking, which potentially affects up to 20,000 people in Peru. Objective In this study, we aim to describe the protocol of a three-phase mixed-method study that will design, build, and evaluate the usability and feasibility of the first Peruvian National IEI Registry. Methods Guided by the Exploration-Preparation-Implementation-Sustainment (EPIS) framework, Phase I (qualitative) will map stakeholder needs through in-depth interviews and focus groups; the data will undergo hybrid thematic analysis in ATLAS.ti. Phase II (pre-experimental) will enroll ≥20 clinical immunology consultants and residents to complete standardized tasks on the REDCap-based prototype; efficiency (task time), effectiveness (error rate), and satisfaction will be compared pre/post-iteration. Phase III (observational) will assess feasibility by enrolling ≥126 IEI cases across six national reference hospitals; the primary outcomes are clinical profile, data completeness, and accuracy. Results The study protocol received institutional review board approval in February 2025 from the UNMSM-Research Ethics Committee. Phase I enrollment started in March 2025. We expect our REDCap-based prototype to incorporate key needs and preferences gathered from focus groups and interviews to develop the platform. By the completion of the study phase II, we will have assessed the usability of the prototype platform. Study phase III will begin in April 2026. The final results are expected by November 2026 and will be published thereafter. Conclusions By evaluating usability and feasibility in real-world conditions, this protocol lays the groundwork for a national IEI registry. The resulting evidence will inform scale-up decisions and facilitate its incorporation into Peruvian health policy. Contributions to the literature - Shows how the EPIS framework can steer real-world adoption of a REDCap-based registry and prospectively measure usability and feasibility across six public hospitals involved in rare-disease surveillance. - Provides a mixed-methods protocol that designs, iterates and field-tests Peru’s first national registry for inborn errors of immunity, tackling persistent barriers to rare-disease data capture in low- and middle-income countries and offering a scalable blueprint for other resource-constrained settings. - Outlines a FAIR-aligned governance and data-sharing strategy that can be adapted by future programmes planning registries for rare and orphan diseases in similar contexts.

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.