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Background  The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface.    Results  Fluorescence activated cell sorting (FACS) of 1.5 × 10 7  clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in  E. coli  shake flask culture, with post-purification yields exceeding 10 mg/L.    Conclusions  FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.    Electronic supplementary material  The online version of this article (10.1186/s13036-019-0203-2) contains supplementary material, which is available to authorized users.

De Novo Isolation & Affinity Maturation of yeast-displayed Virion-binding human fibronectin domains by flow cytometric screening against Virions