Stage-discrimination assay for autophagy-modulating agents in coxsackievirus B3 infection v1

A 48-hour in-vitro assay that separates agents blocking autophagosomal membrane formation from agents blocking lysosomal degradation, using a formation marker (WIPI2 puncta), intracellular viral RNA, LC3 flux and an egress ratio. Five arms are compared: a VPS34 inhibitor, a Beclin-1 pathway inhibitor, bafilomycin A1, rapamycin and vehicle, at MOI 0.01 and 1.0. The protocol includes pre-registered assignment rules and thresholds, so that an agent is classified as acting at the formation step or excluded as acting downstream. It accompanies a preprint describing the rationale and an analysis code deposit implementing the classification and the scaffold-dependence interval estimation.