Protocols and procedures were conducted in the enhanced laboratory Biosafety Level 2 facility of the Adolfo Lutz Institute. All formalin-fixed, paraffin-embedded liver tissue samples were processed and stained with hematoxylin and eosin for histopathologic examination. IHC was performed according to our laboratory protocols. Liver tissue sections were subjected to antigen retrieval in a pressure cooker in citrate buffer for 3 min (120°C, pH 6.0) and then incubated overnight with polyclonal anti-YF (mouse hyperimmune antiserum against wild strain; Núcleo de Doença de Transmissão Vetorial, Virology Center, Adolfo Lutz Institute) (3,12). Signal amplification was performed by using the Horseradish Peroxidase–Conjugated Polymer Detection System (REVEAL Biotin-Free Polyvalent; Spring Bioscience Corp., https://www.cmocro.com) and visualized by using diaminobenzidine (D-5637; Sigma-Aldrich, https://www.sigmaaldrich.com) and counterstaining with Harris hematoxylin. In selected instances, amplification was performed by using AP conjugated polymer (MACH4 Universal AP Polymer Kit; Biocare Medical, https://biocare.net) and visualized by using fast red chromogen (WARP RED chromogen kit; Biocare Medical). Known NWPs and human positive and negative control tissues with omitted first-layer antibody were included.

NWP cases were classified according to the distribution, extent, and nature of microscopic findings after staining with hematoxylin and eosin as described (Appendix Table 1). For IHC, cases were classified as positive, negative, or inadequate on the basis of YFV antigen detection and varying degrees of typical YF-associated lesions. Inadequate classification refers to highly autolyzed/decomposed cases with lack of immunolabeling. Human cases were classified as full spectrum of YF-associated hepatic lesions or other histologic patterns. We provide a detailed description of other histologic patterns.

*IHC, immunohistochemical analysis; NWPs, New Word primates; qRT-PCR, quantitative reverse transcription PCR.

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