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Summary: Glial-vascular interactions are critical for the formation and maintenance of brain blood vessels and the blood-brain barrier (BBB) in mammals, but their role in the zebrafish BBB remains unclear. Using three glial gene promoters—gfap, glast, and glastini (a truncated glast)—we explored glial-vascular development in zebrafish. Sparse labeling showed fewer glial-vascular interactions at early stages, with glial coverage and contact area increasing with age. Stable transgenic lines for glast and glastini revealed similar developmental increases, starting at ∼30% coverage at 3 days post-fertilization (dpf) and peaking at ∼60% by 10 dpf, and consistently higher glial coverage in the forebrain and midbrain than in the hindbrain. Electron microscopy analyses showed similar progressive increases in glial-vascular interactions, with maximal coverage of ∼70% in adults—significantly lower than the ∼100% seen in mammals. These findings define the temporal and regional maturation of glial-vascular interactions in zebrafish and highlight differences from mammalian systems.

Background: Over 296 million people worldwide are living with chronic Hepatitis B (CHB) infection who require monitoring of viral activity and disease progression. Serum HBV RNA is a promising new biomarker in CHB management. No standardized method for serum HBV RNA quantification has been established. Objectives: To develop and validate, both analytically and clinically, a 3′ RACE RT-qPCR assay for quantification of serum HBV RNA. Study design: The 3′ RACE RT-qPCR method was developed using published primers. The analytical limit of detection and quantification, linearity, inter- and intra-assay repeatability, and clinical specificity and sensitivity were evaluated using synthetic pre-genomic RNA and specimens from various patient populations. Intra- and inter-laboratory ring trials involving three laboratories were completed. Results: The 3′ RACE RT-qPCR assay dynamic range is 25 to 108 copies/µL of synthetic RNA. Theoretical and measured serum HBV RNA quantities from a serially diluted sample showed excellent linearity (R2=0.9795). The inter- and intra-assay repeatability were 94.91% and 95.12%, respectively. Clinical specificity and sensitivity were 100% and 90%, respectively, in treated patients. Inter-laboratory analysis demonstrated moderate to high agreement among participating laboratories (κ = 0.581 to 0.867). High agreement was also observed between both operators participating in the intra-laboratory evaluation (κ = 0.867). Conclusions: Our methodology for the quantification of serum HBV RNA is specific and repeatable and employs a biologically relevant RNA standard suitable for medium throughput laboratories. Method standardization is required to facilitate the comparison of studies and better understand the clinical role of this novel biomarker.