Cell lines

Human hepatocellular carcinoma-derived Huh7, Huh7.5 cells (both kindly provided by Charles Rice, The Rockefeller University) and lung carcinoma-derived A549 (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals), 100 units ml⁻¹ of penicillin and 100 μg ml⁻¹ streptomycin (Thermo Scientific). Huh7 was authenticated by short tandem repeat profiling. There was no further authentication for A549 and Huh7.5 cells.

Generation of YF-VAX stocks

YF-VAX (Sanofi Pasteur), a US-licenced yellow fever vaccine and a 17D-204 strain, was obtained from Princeton University Health Services. The vaccine was amplified by passaging once in Huh7.5 cells, aliquoted and cryopreserved at −80 °C.

Biocontainment

All procedures involving virulent YFV generation and infection were carried out in biosafety level 3 (BSL3) facilities. All in vitro experiments were performed in the International Center for Public Health (ICPH) of Rutgers University in Newark and in accordance with protocols reviewed and approved by the Institutional Biosafety Committee (IBC) of Rutgers University (protocol no. 15-005) and Princeton University (protocol no. 3063). All animal experiments were performed in accordance with protocols reviewed and approved by the Institution Animal Care and Use Committees (IACUCs) of Princeton University (protocol no. 3063) and the National Emerging Infectious Disease Laboratories (PROTO2020000075). All personnel involved in working with YFV received the YFV vaccine.

Antibodies and inhibitors

Monoclonal anti-flavivirus group antigen antibodies (D1-4G2-4-15, 4G2, NBP2-52709 or NBP2-52709APC) were obtained from Novus biologicals, polyclonal anti-YFV NS4B antibodies (GTX134030) from GeneTex, polyclonal anti-human fumarylacetoacetase (FAH) antibodies (PA5-42049) from Invitrogen, polyclonal anti-human ISG15 antibodies (15981-1-AP) from Proteintech and ruxolitinib (also known as Jakavi, INCB-18424 phosphate) from InvivoGen.

Complete viral genome sequencing

Viral stocks (kindly provided by Kenneth Plante and Robert Tesh at the World Reference Center for Emerging Viruses and Arboviruses of UTMB) were amplified for one passage in Huh7.5 cells. Viral RNA was extracted using a Zymo Quick-RNA viral kit (Zymo Research). Purified vRNA was sequenced as described previously⁵⁸ with minor modifications. Viral RNA was DNase treated, underwent human ribosomal RNA depletion^58,59 and then subjected to reverse transcription using SuperScript IV Reverse Transcriptase (Thermo Scientific) with random primers. Following second-strand synthesis, dsDNA was then tagmented using a Nextera XT kit (Illumina). Libraries were amplified and sequenced on an Illumina MiSeq Micro flowcell with 2 × 151 cycle reads. Raw sequencing reads were demultiplexed using Picard v.2.25.6. Consensus viral genomes were generated by reference-guided de novo assembly using viral-pipelines (v.2.1.33.14)⁶⁰ (originally viral-ngs⁶¹) with Asibi (GenBank accession AY640589.1) as the reference. Briefly, reads are depleted of common contaminants and human sequences, deduplicated, filtered to yellow fever virus-like sequences, and assembled into contigs and refined with Spades (v.3.14.1)⁶². In addition, to determine the sequences of the 5′ and 3′ UTRs, the vRNA was decapped using Tobacco Decapping Enzyme (Enzymax) and circularized using T4 RNA ligase (New England Biolabs, NEB). Circularized vRNA was then purified and subjected to reverse transcription using SuperScript IV Reverse Transcriptase (Thermo Scientific) with a reverse primer that targets the 5′ UTR. A PCR amplicon of the 3′–5′ UTR junction was generated by Cloneamp (Takara) using the following PCR cycles: 98 °C for 30 s, followed by 35 cycles of 10 s at 98 °C and 15 s at 72 °C, and a final extension at 72 °C for 2.5 min. The targeted amplicon was gel purified and Sanger sequenced. The contigs of the UTRs sequence and consensus sequence from NGS were assembled using SnapGene v.7.0.2 (GSL Biotech). All sequences have been deposited in the NCBI GenBank database (Supplementary Table 1).

Viral variants calling

Variants were filtered on standard metagenomic criteria: frequency (>0.5%), strand bias, detection in replicate libraries, based on vphaser2 (v.2.0)⁶³, called from within viral-phylo v.2.1.19.1 (ref. ⁶⁰), followed by additional manual removal of any variants in homopolymers of length ≥4.

Virus sequence conservation analysis

To identify and quantify sequence variations within a multiple sequence alignment of YFV vaccine and virulent strains, we utilized the Biopython library’s AlignIO module to read the alignment from a FASTA-formatted file generated using MUSCLE aligner⁶⁴. The reference sequence, ‘YF-17D’, was identified within the alignment and used as the baseline for comparison. A tab-separated output file was generated to record the following: reference position, reference residue, total sequence coverage, alternate residues with their counts, total count of variations and specific residue variations across all sequences. For each alignment position, residues differing from the reference sequence were identified, tallied and counted. Only positions where 17D differed from Asibi were then manually picked for conservation analysis.

Highlighter plots were generated with the sequence alignment file as input using https://www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter_top.html.

Generation of the virulent YFV infectious clones

pACNR-FLYF-17D (pANCR-17D), constructed on the basis of the sequence of the 17D-204 strain, was kindly gifted by Charles Rice from Rockefeller University. To generate pACNR-Asibi, plasmid A and plasmid B encoding for 2 parts of the Asibi genome with digestion sites NotI-XhoI and XhoI-ClaI, respectively, were synthesized (GenScript). These plasmids were digested with NotI-XhoI and XhoI-ClaI to generate Asibi fragments A and B, and cloned into NotI-ClaI-digested pACNR-17D to generate pACNR-Asibi.

To generate pACNR-Dakar, randomly primed complementary (c)DNAs from vRNA isolated from a serum sample of an infected rhesus monkey were used as templates to generate 6 PCR fragments by Cloneamp HiFi premix (Takara) using CPER primer pairs that have complementary ends with at least a 20-nucleotide overlap (Supplementary Table 6). The PCR fragments were cloned into a TOPO vector (Thermo Fisher) to generate plasmids with fragments that match the viral genome sequence. Sequence-confirmed TOPO plasmids were then used as templates to generate PCR fragments using the corresponding CPER primer pairs. A seventh PCR fragment was amplified using pACNR-17D as a template, using a forward primer annealing to the SP6 promoter region with an overhang of the 5′ UTR of the viral genome and a reverse primer annealing immediately downstream of the viral 3′ UTR with an overhang of the 3′ UTR of the viral genome and a SnaBI restriction site. The resulting PCR products were purified using DNA Clean and Concentrator (Zymo Research) and mixed in equimolar concentrations (0.1 pmol each) to generate circular DNA by CPER using PrimeSTAR GXL DNA polymerase (Takara). The following PCR cycling was used: 98 °C for 2 min, followed by 18 cycles of 10 s at 98 °C, 15 s at 55 °C, 12 min at 68 °C, and a final extension at 68 °C for 12 min. The PCR product was diluted 1:50 and transformed into JM109 cells (Promega) and propagated at 30 °C.

Generation of the YFV mScarlet reporter genomes

The generation of YF-17D-mScarlet was described previously¹². Briefly, the first 27 nucleotides of the sequence encoding 17D NS1, the Gaussia luciferase (GLuc) gene and a dengue virus E linker coding sequence (E stem and transmembrane domain, TMD) were amplified by PCR from pBSC-YFV-GLuc (provided by Laura Gil, Oswaldo Cruz Foundation). The amplified gene cassette was then inserted between the E and NS1 coding sequences of pACNR-FLYF-17D-RLuc through In-Fusion-based molecular cloning (Takara), yielding pACNR-FLYF-17D-GLuc. The pACNR-FLYF-17D-GLuc construct was then digested with NarI as two NarI sites flank the GLuc-coding sequence. The mScarlet-I gene was amplified by PCR and subcloned in place of the GLuc-coding sequence into NarI-digested pACNR-FLYF17D-GLuc using the In-Fusion cloning kit. To generate pACNR-Asibi or -Dakar mScarlet plasmids, gene cassettes of Asibi/Dakar partial E, the mScarlet-DENV-linker from pACNR-17D-mScarlet and Asibi/Dakar partial NS1 were generated by overlapping extension PCR and inserted into NsiI-MluI-digested pACNR-Asibi or -Dakar using the In-Fusion cloning kit.

Generation of YFV or reporter YFV by in vitro RNA transcription

Midi-prepped (QIAGEN) plasmids of YFV infectious clones or mScarlet reporter YFVs were linearized with AflII (17D and Asibi; NEB) or SnaBI (Dakar; NEB) and in vitro transcribed using the mMESSAGE mMACHINE SP6 Transcription kit (Thermo Scientific) at 37 °C for 2 h. The DNA template was eliminated by adding 1 µl Turbo DNAse (Thermo Scientific) and incubating for 15 min at 37 °C. Viral RNA was purified using the MEGAclear Transcription Clean-Up kit (Thermo Scientific) following manufacturer instructions, and quality control was performed by gel electrophoresis to ensure that there was no notable RNA degradation. Viral RNA was transfected into Huh-7.5 cells via the TransIT-mRNA Transfection kit (Mirus) for viral production. Upon observation of cytopathic effects (CPE) around 2 days post transfection, culture supernatant was collected to recover newly generated viruses, and supernatants were replenished with fresh medium containing 2% FBS. This process was repeated daily until the cultures reached complete CPE. Virus-containing supernatants were passed through a 0.45 μm syringe filter to remove cell debris, thoroughly mixed by vortex, aliquoted and cryopreserved at −80 °C.

Generation of recombinant YFV and chimaeric viruses by CPER

Stocks of infectious YFV were generated by CPER as described previously¹⁵. Briefly, for wildtype 17D, Asibi and Dakar, genetically defined infectious clone or reporter virus plasmids were used as templates (10 ng) to generate 6 PCR fragments using Cloneamp HiFi premix (Takara) with 21 PCR cycles and CPER primer pairs that have complementary ends with at least a 20-nucleotide overlap (Supplementary Table 6). For chimaeric viruses with mutations in individual viral genes, overlapping extension PCR was performed to generate chimaeric fragments, which were then cloned into the TOPO vector. Sequence-confirmed TOPO plasmids were then used as templates to generate PCR fragments using the corresponding CPER primer pairs. A linker fragment that contains the cytomegalovirus (CMV) promoter and hepatitis delta virus ribozyme (HDVr) site was amplified from the Linker-TOPO-plasmid with corresponding primers to create 25-nucleotide overlaps with the 5′ and 3′ UTR of the viral genome. The resulting PCR products were then DpnI- treated for 1 h at 37 °C and purified using the DNA Clean and Concentrator kit (Zymo Research). These seven DNA fragments were then mixed in equimolar concentrations (0.1 pmol each) to generate circular DNA by CPER using PrimeSTAR GXL DNA polymerase (Takara). The following PCR cycling was used: 98 °C for 2 min, followed by 18 cycles of 10 s at 98 °C, 15 s at 55 °C, 12 min at 68 °C, and a final extension at 68 °C for 12 min. The CPER products were transfected into two wells of Huh7.5 cells in 6-well plates with ~80% density using the 10 μl X-tremeGENE HP DNA Transfection Reagent (Roche). At 24 h post transfection, the culture medium was replaced by fresh medium to avoid transfection reagent-induced cytotoxicity. At 48 h post transfection, cells were expanded into a 10 cm dish. Upon observation of the CPE at 2 days post transfection, culture supernatant was collected to recover newly generated viral strains and cells were replenished with 2% FBS-containing fresh medium. This process was repeated daily until the cells were completely dead due to CPE. Virus-containing supernatant was passed through a 0.45 μm syringe filter to remove cell debris, thoroughly mixed by vortexing, aliquoted and cryopreserved at −80 °C.

Virus titration

YFV titres were determined using a focus-forming unit (f.f.u.) assay. Briefly, Huh7.5 cells were seeded into a collage-coated 48-well plate at a density of 1 × 10⁴ cells per well and infected the next day with 10-fold serial dilutions of the virus stock for 2 h at 37 °C. Following virus adsorption, 0.5 ml of overlay media (culture medium supplemented with 1% methylcellulose and 10% FBS) was added to each well. The overlay medium was removed when the plaques became visible under the microscope; the cell monolayer was washed with PBS three times and fixed for 30 min at room temperature with 4% paraformaldehyde in PBS (PFA, Millipore Sigma). Fixed cells were then permeabilized with 0.2% Triton X-100 (Thermo Scientific) in PBS for 15 min and subsequently with blocking buffer (0.2% BSA in PBS) for 30 min. Following blocking, cells were incubated with the flavivirus group antigen antibody 4G2 (1:1,000, 1 μg ml⁻¹, Novus) in blocking buffer at room temperature for 2 h or 4 °C overnight, and then with HRP-conjugated secondary antibody (1:500, 2 μg ml⁻¹, Thermo Scientific) diluted in blocking buffer at room temperature for 30 min. F.f.u.s were visualized using a DAB Substrate kit (Vector Laboratories). Each experiment was performed in duplicate. Wells with foci numbers from 10–100 were counted, averaged and expressed as f.f.u.s ml⁻¹.

In vitro virus infection

Before virus infection, Huh7 or A549 cells were seeded in a 24-well or 6-well plate format at densities of 1.2 × 10⁵ or 8 × 10⁵ cells per well. Infections were conducted with different MOIs as indicated on the basis of viral titres previously determined in the Huh7.5 cells. The viral inoculum was removed after 2 h incubation at 37 °C and replaced with the complete medium. For the ruxolitinib treatment experiments, the viral inoculum was replaced with 2 μM ruxolitinib (10 mM stock, Invivogen) or a corresponding amount of dimethylsulfoxide (DMSO) as solvent control in complete medium. The medium containing DMSO or ruxolitinib was exchanged every 24 h. At indicated timepoints, cells were trypsinized and pelleted for fixation with 4% PFA in PBS for flow cytometry or for RNA extraction using a Biobasic Total RNA Miniprep Super kit (Biobasic).

RT–qPCR

Viral RNA was quantified using a Luna Universal Probe One-Step RT–qPCR kit (NEB) with primers and TaqMan probes (Supplementary Table 6) targeting a conserved region of the 5′ UTR of the YFV genome. Host mRNAs were quantified using the Luna Universal One-Step RT–qPCR kit (NEB). One-step RT–qPCR was accomplished in a Quantstudio 7 pro PCR system (Applied Biosystems, Thermo Scientific) using the following thermal cycling procedure: 55 °C for 10 min, 95 °C for 2 min, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Relative expression level of host mRNA was calculated as the fold change over mock-infected cells, with human HPRT1 as an internal control. Absolute viral RNA copy numbers were quantified using standard curves and normalized to total RNA in μg.

Flow cytometry

For mScarlet reporter viruses, fixed cells were washed twice with FACS buffer (2% (v/v) FBS in PBS). For non-reporter viruses, fixed cells were permeabilized in PBS supplemented with 1% (w/v) saponin (Sigma Aldrich), followed by incubation with the flavivirus group antigen antibody 4G2 conjugated with APC (1:100, 10 μg ml⁻¹, Novus) and washed 3 times with FACS buffer. Flow cytometry was performed using an LSRII flow cytometer (BD Bioscience).

IFN-beta ELISA

Supernatants from the virus or mock-infected A549 cells were collected at indicated timepoints and stored at −20 °C before use. The ELISA assay was performed using the Human IFN-beta Quantikine ELISA kit (Bio-Techne) following manufacturer instructions without modification.

FACS enrichment of YFV antigen-bearing cells for RNA-seq

Huh7 cells were infected with YFVs or chimaeras at an MOI of 0.5. After 48 h, cells were collected, washed and fixed with 4% PFA supplemented with SUPERase•In RNase Inhibitor (Thermo Scientific) for 30 min at 4 °C. The cells were centrifuged, resuspended in sort buffer (PBS containing 0.2% (w/v) BSA and SUPERase•In RNase Inhibitor) for FACS. High mScarlet-expressing cells were sorted by a FACSAria sorter (BD Biosciences,). Sorted cells were then centrifuged and subjected to RNA extraction using the RecoverAll Total Nucleic Acid Isolation kit (Thermo Scientific) according to manufacturer instructions with slight modifications. Briefly, cell pellets were resuspended in digestion buffer and incubated at 50 °C for 3 h with constant shaking to reverse PFA cross-links. During column purification, in-column DNase treatment was omitted. Instead, eluted RNA was treated with TURBO DNase (Thermo Scientific) for 30 min at 37 °C and cleaned up with 0.8× RNAClean XP Beads (Beckman Coulter). RNA integrity was assessed using an Agilent TapeStation 4200 system (Agilent) with High Sensitivity RNA ScreenTape (Agilent).

3′ mRNA sequencing library preparation and sequencing

Total RNA (300 ng) was used as input for reverse transcription using barcoded oligo-dT primers to label each sample in the 96-well plate following the drop-seq method⁶⁵. The pooled barcoded cDNA samples were amplified by PCR and purified, then turned into sequencing libraries using the Tn5 transpose-based tagmentation method to include only the poly-A tail adjacent 3′ ends of transcripts. These libraries were sequenced on an Illumina NovaSeq 6000 SP 100-cycle flowcell.

RNA-seq analysis

Raw Illumina sequence data were demultiplexed using fastq-multx. Reads were then mapped to the human genome hg38 with hisat2 (2.1.0)⁶⁶, and reads mapping to the comprehensive gene annotation on the primary assembly were counted with htseq-count (v.1.99.2)⁶⁷ using snakemake wrapper RASflow⁶⁸. Raw read counts were processed and pairwise differential expression analysis was performed using DESeq2 (v.1.38.3)⁶⁹. Differentially expressed genes (DEGs) were defined with the cut-off value |log₂ fold change| >2 and P_adj < 0.05. Gene set enrichment analysis (GSEA) was performed using clusterProfiler (v.4.6.2)⁷⁰ on hallmark gene sets from the Molecular Signatures Database (MSigDB)⁷¹. Heat maps were generated using the pheatmap package (v.1.0.12), with z-score normalization performed across rows and hierarchical clustering applied to both rows and columns. Z-score normalization was performed gene-by-gene (row-wise) on vst-transformed read counts across samples by subtracting the mean and dividing by the standard deviation. Compressed expression of gene clusters were performed as follows: vst-transformed gene counts were first normalized as z-scores. The per-gene average z-score was then calculated from across replicates. Data were then plotted as mean ± s.d. or s.e.m. across the indicated gene clusters. Spearman’s correlation coefficients were calculated using the cor() function in RStudio (2024.04.2) with the argument method = “spearman”.

Chemical synthesis of 2A3 for SHAPE-MaP analysis

The SHAPE-chemical 2A3 was synthesized as described previously²² with slight modifications. Briefly, 2-aminopyridine-3-carboxylic acid (138.12 mg, 1 mmol, Sigma Aldrich) was dissolved in anhydrous acetonitrile (Sigma Aldrich). 1,1′-carbonyldiimidazole (CDI, 162 mg, 1 mmol, Sigma Aldrich) was added in portions. The reaction was stirred for 3 h at room temperature, then diluted into dichloromethane (Sigma Aldrich) and washed with saturated sodium bicarbonate solution (Sigma Aldrich) three times. The organic layer was dried over MgSO₄ and concentrated under reduced pressure. The product was confirmed by nuclear magnetic resonance (NMR core in the Department of Chemistry of Princeton University).

SHAPE-MaP

Viral whole-genome RNA structures were determined using SHAPE-MaP as previously described²⁰. Briefly, Huh7 cells were infected at an MOI of 0.05; at 2 (17D and Asibi) or 5 (Dakar) days post infection (dpi), cells were washed with PBS twice, trypsinized and resuspended in PBS. 2A3 from a 500 mM stock in DMSO was added at a final concentration of 100 mM. A corresponding amount of DMSO was added to the control sample. Samples were then incubated at 37 °C in a heat block with constant shaking for 15 min, followed by quenching of 2A3 by adding dithiothreitol at a final concentration of 0.5 M. Following centrifugation at 10,000 × g for 1 min, the supernatant was discarded and cell pellets were lysed in 1 ml Trizol (Thermo Scientific), followed by the addition of 0.2 ml of chloroform. The aqueous phase was transferred to a new tube, mixed with one volume of 70% ethanol, transferred to an RNeasy mini column (QIAGEN), then washed and eluted according to manufacturer instructions. An amount of 1.5 μg of total RNA was first subjected to human ribosomal RNA depletion. Ribo-depleted RNA was purified using 0.8× RNAClean XP Beads (Beckman Coulter) and fragmented in RNA Fragmentation Buffer (NEB) at 94 °C for 105 s. After addition of the stop solution (NEB), fragmented RNA was purified with 2× RNAClean XP Beads (Beckman Coulter) and eluted in 9 μl nuclease-free H₂O. Eluted RNA was then supplemented with 1 μl 50 μM random hexamers (Thermo Scientific) and 1 μl dNTPs (10 mM, NEB), then incubated at 70 °C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 20 μl with 120 mM MnCl₂, SHAPE-RT buffer (50 mM Tris-HCl pH 8.0, 55 mM KCl), 0.1 M dithiothreitol, 2 U of SuperScript II (SSII, Thermo Scientific) and 2 U of SUPERase•In (Thermo Scientific). Reactions were incubated at 25 °C for 10 min to allow partial primer extension, followed by 3 h at 42 °C and heat inactivation of SSII at 75 °C for 15 min. EDTA (0.1 M) was added to a final concentration of 6 mM, and reactions were incubated at room temperature for 5 min to chelate Mn²⁺ ions. After addition of MgCl₂ to a final concentration of 6 mM, the resulting RNA:cDNA duplex then underwent second-strand synthesis using NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB) according to manufacturer instructions. cDNA were then ligated with the xGen Dual Index UMI Adapter (IDT) and sequenced on an Illumina NovaSeq 6000 SP 100-cycle flowcell.

RNA structure modelling

Raw sequencing reads were demultiplexed using Picard v.2.25.6 and merged using samtools (v.1.9)⁷². Analysis of SHAPE-MaP data was performed using RNAframework (v.2.8.8)²³ as described previously²⁰. Reads preprocessing and mapping was performed using the rf-map module (parameters: -ctn -cmn 0 -cqo -cq5 20 -b2 -mp ‘–very-sensitive-local’). The SHAPE mutational signal was then derived using the rf-count module (parameters: -m -rd). Data normalization was performed using the rf-norm module (parameters: -sm <3). The rf-fold module of RNAframework was used for SHAPE-MaP-informed full-length YFV genome structure modelling using the following parameter (-sl 2.0 -in -0.6 -w -fw 3000 -fo 300 -wt 200 -pw 1500 -po 250 -dp -sh -nlp -md 200). RNA structures were visualized using VARNA (3.93)⁷³.

In vivo experiments

All animal experiments were performed in accordance with protocols reviewed and approved by the IACUCs of Princeton University (protocol no. 3063) and the National Emerging Infectious Disease Laboratories (PROTO2020000075). All mice were bred and generated in the Laboratory Animal Resource (LAR) Center of Princeton University and transferred to the National Emerging Infectious Disease Laboratory (NEIDL) for infection experiments.

IFN alpha beta receptor knockout mice

Mice lacking type I IFN receptor (Ifnar −/−) on a C57BL/6 genetic background were kindly provided by Sergei Kotenko (Rutgers University) and generated as described previously⁷⁴. Mice were 11–18 weeks of age when used for experiments. For each condition, female and male mice were randomly assigned to the study in a 3:5 ratio. The number of mice used is described in the figure legends.

Generation of human liver chimaeric mice

The generation of Fah^−/− NOD.Cg-Rag1^tm1MomIL2rg^tmlWjl/SzJ IL2Rg^null (FNRG) mice has been previously described⁷⁵. Briefly, female FNRG mice older than 6 weeks of age were transplanted with ~1.0 × 10⁶ cryopreserved adult human hepatocytes (BioReclamation). FNRG mice were cycled on water supplemented with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC, Yecuris) to block the build-up of metabolites to toxic concentrations. Approximately 3 months after transplantation, blood was collected by submandibular puncture and serum was separated by centrifuging the coagulated blood at 1,100 × g for 15 min. Mouse sera were analysed via human albumin ELISA. HLCM were 35–50 weeks old when used for experiments and were randomly assigned for the study. The number of mice used is described in the figure legends.

Human albumin ELISA for assessment of human hepatocyte engraftment of chimaeric mice

Levels of human albumin in mouse serum were quantified by ELISA as described previously⁷⁶. Briefly, 96-well flat-bottomed plates (Nunc, Thermo Fisher) were coated with goat anti-human albumin antibody (1:1,000, 2 μg ml⁻¹, Bethel) in coating buffer (1.59 g Na₂CO₃, 2.93 g NaHCO₃, 1 l dH₂O, pH 9.6) for 1 h at 37 °C. Plates were washed four times with wash buffer (0.05% (v/v) Tween 20 (Sigma Aldrich) in 1× PBS), incubated with superblock buffer (Fisher Scientific) for 1 h at 37 °C and subsequently washed twice with wash buffer. Human serum albumin (Sigma Aldrich) was diluted to 1 µg ml⁻¹ in sample diluent (10% (v/v) Superblock, 90% (v/v) wash buffer), then serially diluted 1:2 in 135 µl sample diluent to establish an albumin standard. Mouse serum (5 µl) was used for a 1:10 serial dilution in 135 µl sample diluent. The coated plates were incubated for 1 h at 37 °C, then washed three times. A volume of 50 µl mouse anti-human albumin (1:2,000, 1 μg ml⁻¹, Abcam) in sample diluent was added and plates were incubated for 2 h at 37 °C. Plates were washed four times and 50 µl of goat anti-mouse HRP (1:1,0000, 0.1 μg ml⁻¹, Life Technologies) in sample diluent were added and plates incubated for 1 h at 37 °C. Plates were then washed six times. TMB (100 µl) substrate (Sigma Aldrich) was added and the reaction was stopped after 1 min incubation with 25 µl 2 N H₂SO₄. Absorbance was read at 450 nm on the BertholdTech TriStar microplate reader.

Infection of mice with yellow fever virus

Infections were carried out as previously described³⁴. Briefly, human liver chimaeric mice with a human liver chimaerism of >3 mg ml⁻¹ albumin in the serum (Supplementary Table 4) were injected intravenously³⁴ with 2 × 10⁵ f.f.u.s of the viruses, and tissues were collected at 3 dpi.

Ifnar^−/− mice were inoculated intravenously with 1 × 10⁶ f.f.u.s via the retroorbital route with the YFV strains indicated in the main text. Disease progression was monitored using an IACUC-approved clinical scoring system. Categories evaluated included body weight (1 = >10% weight loss), general appearance (1 = moderate hunched posture, ruffled fur) and neurological signs including tremors (1 = shaky/trembling posture) and paralysis (1 = severe hunched posture and/or paralysis), for a maximum score of 4. Animals were considered moribund and humanely euthanized in the event of the following: a score of 4 for 2 consecutive observation periods, weight loss greater than or equal to 25%, severe respiratory distress, or lack of responsiveness. Clinical signs were recorded once per day for the duration of the study.

Histology and multiplex fluorescence immunohistochemistry

Tissue samples were fixed for 72 h in 10% neutral buffered formalin (Sigma Aldrich), processed in a Tissue-Tek VIP-5 automated vacuum infiltration processor (Sakura Finetek), followed by paraffin embedding using a HistoCore Arcadia paraffin embedding station (Leica). Generated formalin-fixed, paraffin-embedded (FFPE) blocks were sectioned to 5 μm using an RM2255 rotary microtome (Leica), transferred to positively charged slides, deparaffinized in xylene and dehydrated in graded ethanol. A Ventana Discovery Ultra (Roche) tissue autostainer was used for multiplex fluorescence immunohistochemistry (fmIHC). In brief, tyramide signalling amplification (TSA) was used in an iterative approach to covalently bind opal fluorophores (Akoya Bioscience) to tyrosine residues in tissue, with subsequent heat stripping of primary–secondary antibody complexes until all antibodies were developed. A 7-colour IHC panel was employed and specific details of the assay are outlined in Supplementary Table 5, with a more concise overview provided below.

Fluorescence immunohistochemistry

Antigen retrieval was conducted using a Tris-based buffer-CC1 (Roche). All primary antibodies were of rabbit origin (Supplementary Table 5) and thus developed with a secondary goat anti-rabbit HRP-polymer antibody (Vector Laboratories) for 20 min at 37 °C. All Opal TSA-conjugated fluorophore reactions took place for 20 min. Fluorescent slides were counterstained with spectral DAPI (Akoya Biosciences) for 16 min before being mounted with ProLong gold antifade mountant (Thermo Scientific).

Multispectral microscopy and unmixing of multiplex fluorescence immunohistochemistry

Fluorescently labelled slides were imaged using a PhenoImager Quantitative Pathology Imaging System (Akoya Biosciences). Exposures for all opal dyes on the imaging system were set based on regions of interest with strong signal intensities to minimize exposure times and maximize the specificity of signal detected. Images were unmixed using spectral libraries affiliated with each respective opal fluorophore including removal of autofluorescence.

Quantitative analysis of multiplex fluorescence immunohistochemistry

View settings were adjusted to allow for optimal visibility of immunomarkers and to reduce background signal by setting threshold gates on minimum signal intensities. Tissue was annotated using the flood tool in HALO v.3.6.4134.263 (Indica Labs) and edge effect artefacts were removed from the analysis area. An AI-based nuclear segmentation algorithm was developed by annotating examples of nuclei for the module to train with. For quantifying the absolute number and overall percentage of cells expressing various biomarkers, we utilized the HALO (Indica Labs) HighPlex (HP) phenotyping module (v.4.2.3) with the nuclear segmentation algorithm. In brief, the nuclear segmentation algorithm was used to first segment all cells within the annotated liver sections using DAPI counterstain. Detection threshold and nucleus geometry were defined until segmentation appeared accurate. Next, minimum nucleus and cytoplasm thresholds were set for each fluorophore to detect low and high expression within each of the segmented cells. Parameters were set using the real-time tuning mechanism that was tailored for each individual sample on the basis of signal intensity. Phenotypes of NS4B⁻/ISG15⁺, NS4B⁺/ISG15⁺ and NS4B⁺/ISG15⁻ were determined by selecting inclusion and exclusion parameters appropriate to phenotype. The algorithm was run across all tissue outputting counts of FAH⁺, NS4B⁺, ISG15⁺ and all defined phenotypes mentioned before. The quantitative output for the automated quantitation and HP was exported as a .csv file.

Statistics and reproducibility

Sample sizes, number of replicates and statistical methods are described in the figure legends. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications^{33,77,78,79,80}. Data distribution was assumed to be normal, but this was not formally tested. For RNA-seq, only genes with greater than 10 counts for more than 2 samples were included in the analysis as a standard prefiltering step. No other data were excluded from the analyses. For in vivo study, mice were randomly allocated to the experiments. No randomization was performed for in vitro experiments. Histopathology assessment was performed in a blinded fashion. All other experiments were not blinded.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.