Noncytopathic, cell culture-adapted HM175/p16 HAV9 (link) was propagated in Huh-7.5 cells. Reverse molecular genetics studies were carried out with pHM175/18f, a molecular clone of a related cytopathic variant28 (link). Buoyant density was assessed in 8–40% iodixanol (Opti-Prep) gradients centrifuged at 141,000 × g for 48 hrs at 4 °C. Viral RNA was measured by qRT-PCR with primers targeting the 5’-untranslated region. Infectivity was quantified by infrared fluorescence immunofocus assay (IR-FIFA)10 (link). For RNAi studies, cells were transfected with SmartPool siRNAs (Dharmacon) and samples collected 48–72 hrs later for viral RNA quantification. To analyze VP2-ALIX interactions, cell lysates were prepared 48 hrs after electroporation of mutant and wild-type viral RNAs, treated with RNase, and immunoprecipitated. RNA extracted from immunoprecipitates was assayed by HAV-specific qRT-PCR. For intracellular neutralization, cells were incubated with eHAV for 1 hr at 37 °C, then washed extensively. Antibodies were added at intervals, and intra- and extracellular HAV RNA quantified at 48–72 hrs. For standard neutralization assays, virus was incubated with antibodies for 1 hr at 37 °C, then inoculated onto cells.