2.1 Resource availability

All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

2.2 NHEK

NHEK-Ad (Normal Human Epidermal Kerationocytoes-Adult; Cat# 00192627; Lonza) were cultured with KGM-Gold Keratinocyte Growth Medium BulletKit (Cat# 00192060; Lonza). NHEK-Ad were seeded on 24-well cell culture plates (2.0 × 10⁴ cells/2 cm²) and cultured for 7 days.

2.3 Human iPS cell-derived colon organoids

The iPS cell line, 1383D6, was maintained on 0.5 μg/cm² recombinant human laminin 511 E8 fragments (iMatrix-511; Cat# 892 012; Nippi) with StemFit AK02N medium (Cat# RCAK02N; Ajinomoto) containing 10 μM Y-27632 (Cat# 034-24024; FUJIFILM Wako Pure Chemical). To passage the cells, iPS cell colonies were treated with TrypLE Select Enzyme (Cat# 12563029; Thermo Fisher Scientific) for 10 min at 37°C. After centrifugation, cells were seeded on Matrigel® Growth Factor Reduced Basement Membrane (Cat# 354230; Corning)-coated cell culture plates (2.0 × 10⁵ cells/4 cm²) and cultured for 2 days. To perform definitive endoderm differentiation, human iPS cells were treated with 100 ng/mL Activin A (Cat# 338-AC-01M; R&D Systems) and 10 μM Y-27632 in RPMI1640 medium (Cat# R8758-500; Sigma-Aldrich) supplemented with 1× B-27 Supplement Minus Vitamin A (Cat# 12587001; Thermo Fisher Scientific), 1× GlutaMAX (Cat# 35050-079; Thermo Fisher Scientific), and 1× penicillin−streptomycin for 3 days. To perform hindgut differentiation, the cells were treated with 200 ng/uL FGF2 (Cat# 160-0010-3; Katayama Chemical Industries) in RPMI1640 medium supplemented with 1× B-27 Supplement Minus Vitamin A, 1× GlutaMAX, and 1× penicillin−streptomycin for 4 days. To generate colon organoids, the cells were dissociated and embedded in Matrigel Growth Factor Reduced Basement Membrane to generate organoids. To perform colonic differentiation, the cells were treated with 3 μM CHIR99021 (Cat# 034-23103; FUJIFILM Wako Pure Chemical), 0.5 μM A-83-01 (Cat# 035-24113; FUJIFILM Wako Pure Chemical), 50 ng/mL Noggin (Cat# 120-10C; PeproTech), 30 ng/uL Forskolin (Cat# 063-02193; FUJIFILM Wako Pure Chemical), and 50 ng/mL EGF (Cat# AF-100-15; PeproTech) in advanced DMEM/F12 supplemented with 1× N2 (Cat# 141-08941; FUJIFILM Wako Pure Chemical), 1× B-27 Supplement Minus Vitamin A, 1× GlutaMAX, 0.05% bovine serum albumin, and 1× penicillin−streptomycin for 13 days. To perform the infection experiments, the colon organoids were recovered from Matrigel and the suspension of colon organoids (small free-floating clumps) was seeded onto Matrigel-coated 24-well cell culture plates (1.0 × 10⁵ cells/2 cm²) and cultured for 3 days.

2.4 MPXV preparation

MPXV infection experiments were performed in a biosafety level 3 (BSL3) facility. Three MPXV strains, Zr-599 (Congo Basin strain, clade I), Liberia (West African strain, clade IIa), and the 2022 outbreak strain (MPXV/human/Japan/Tokyo/TKY220091/2022, clade IIb, Genbank accession no. LC722946.1), were propagated using VeroE6 cells (kindly gifted by Tokyo Metropolitan Institute of Public Health). Zr-599 and Liberia strains were kindly gifted by National Institute of Infectious Diseases. The 2022 outbreak strain was kindly gifted by Tokyo Metropolitan Institute of Public Health.

To prepare working MPXV stock, VeroE6 cells (5 million cells) were seeded in a T-75 flask. Next day, the culture medium was refreshed with DMEM (low glucose) (Cat# D6046-500ML; Sigma-Aldrich) containing 2% fetal bovine serum (FBS; Cat# 172012-500ML; Sigma-Aldrich), and 1× penicillin−streptomycin (Cat# P4333-100ML; Sigma-Aldrich), and seed virus was inoculated. At 3 or 4 dpi, the infected flask including culture supernatant and cells were frozen at –80°C. Before use, the flask was placed at room temperature to facilitate thawing. The thawed medium in the flask was harvested and centrifuged, and the supernatant was collected as a working MPXV stock.

2.5 MPXV titration

Virus infectious titers were determined by plaque assay.³¹ One day before infection, VeroE6 cells (0.2 million cells) were seeded into a 12-well plate and infected with MPXV. Before infection, the culture medium was changed to DMEM (low glucose) containing 2% FBS, and 1% penicillin−streptomycin. The cells were incubated at 37°C in a fully-humidified atmosphere of 5% CO₂. At 3 dpi, the culture medium was removed, and the cells were washed with PBS once and fixed with 4% paraformaldehyde phosphate (Cat# 09154-85; Nacalai Tesque). The fixed cells were washed with tap water, dried, and stained with a staining solution (0.1% methylene blue [Cat# 22412-14; Nacalai Tesque] in water) for 30 min. The stained cells were washed with tap water and dried, and the number of plaques was measured to calculate plaque forming unit.

2.6 Quantification of viral DNA copy number of the cell culture supernatant

The cell culture supernatant was mixed with an equal volume of 2× DNA lysis buffer (distilled water containing 2% Triton X-100, 50 mM KCl, 100 mM Tris-HCl [pH 7.4], and 40% glycerol) and incubated at room temperature for 10 min. The mixture was diluted 10 times with distilled water. Viral DNA was quantified using a Probe qPCR Mix (Cat# RR391A; Takara Bio) with 5’-GGA AAA TGT AAA GAC AAC GAA TAC AG-3’ as the forward primer and 5’-GCT ATC ACA TAA TCT GGA AGC GTA-3’ as the reverse primer, and 5’-FAM-AAG CCG TAA TCT ATG TTG TCT ATC GTG TCC-TAMRA-3’ as the probe. To quantify viral DNA copy number, a plasmid encoding MPXV G2R gene was used as the standard.

2.7 Quantification of viral DNA copy number in the cell

Total DNA was isolated using DNeasy Blood & Tissue Kit (Cat# 69506; QIAGEN). For quantification of MPXV DNA, Monkeypox virus qPCR Detection Kit (Cat# RC420A; Takara Bio) was used on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Standard curves were prepared using plasmids encoding MPXV G2R gene.

2.8 Quantitative PCR

Total RNA was isolated using ISOGENE (Cat# 319-90211; NIPPON GENE). cDNA was synthesized using 500 ng of total RNA with the Superscript VILO cDNA Synthesis Kit (Cat# 11754250; Thermo Fisher Scientific). Real-time RT-PCR was performed with SYBR Green PCR Master Mix (Cat# 4385614; Thermo Fisher Scientific) using a StepOnePlus real-time PCR system, QuantStudio 1, or QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The relative quantification of target mRNA levels was performed using the $${2}^{ \mbox{-} \unicode{x02206}\unicode{x02206}{C}_{t}}$$ method. The values were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences are summarized in Supporting Information: Table S1.

2.9 Immunofluorescence staining

For immunofluorescence staining of human iPS cell-derived colon organoids, the cells were fixed with 4% paraformaldehyde in PBS at 4°C. Human iPS cell-derived colon organoids were harvested and used for the preparation of paraffin Section (5 μm). The sections were permeabilized using Tris Buffered Saline with 0.1%-Tween 20 Detergent (Cat# 34874-91; Nacalai Tesque) for 10 min and blocked using Blocking One Histo (Cat# 06349-64; Nacalai Tesque) for 45 min. The sections were incubated in Tris Buffered Saline with 0.1%-Tween 20 Detergent with or without primary antibodies overnight at 4°C. The sections were washed with PBS and incubated in Tris Buffered Saline with 0.1%-Tween 20 Detergent containing Alexa Fluor 488- or 594-conjugated secondary antibodies for 45 min. The sections were finally washed and mounted with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Cat# 12593-64; Nacalai Tesque) and analyzed using the BZ-X700 (Keyence Corporation). The antibodies are summarized in Supporting Information: Table S2.

2.10 RNA sequencing

Total RNA was isolated from cells using ISOGENE. RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies). Library preparation was performed using the TruSeq Stranded mRNA Sample Prep Kit (Cat# 20020594; Illumina) according to the manufacturer's instructions. Sequencing was performed on an Illumina NextSeq. 550. The fastq files were generated using bcl2fastq-2.20. Adapter sequences and low-quality bases were trimmed from the raw reads using Cutadapt ver v3.4.³² The trimmed reads were mapped to human reference genome sequences (hg38) using STAR ver 2.7.9a³³ with the GENCODE (release 36, GRCh38.p13)³⁴ gtf file. The raw counts were calculated using htseq-count ver. 0.13.5³⁵ with the GENCODE gtf file. Gene expression levels were determined as transcripts per kilobase million (TPM) values with DESeq. 2 v1.30.1.³⁶ Raw data generated from this study were submitted under Gene Expression Omnibus accession number GSE219036.

2.11 Phylogenetics analysis of MPXV

A phylogenetic analysis of MPXV was performed based on core SNP alignment (906 variant positions) retrieved from a rapid alignment (parsnp version 1.2) of the available MPXV genome sequences, with reference genomes, Zaire-96-I-16 (Genbank accession no. NC_003310.1, MPXV clade I) and Liberia_1970_184 (Genbank accession no. DQ011156.1, MPXV clade IIa). Phylogenetic data visualization was performed with Microreact (https://microreact.org/).

2.12 Statistical analyses

Statistical significance was evaluated using unpaired two-tailed Student's t-test, one-way analysis of variance (ANOVA) followed by Dunnett or Tukey post hoc tests, or two-way ANOVA followed by Tukey post hoc tests. Statistical analyses were performed using GraphPad Prism 9. Details are described in the figure legends.

3.1 MPXV is more likely to infect keratinocytes than colon organoids

Three MPXV strains were used in this study to characterize MPXV infections: Zr-599 MPXV (Congo Basin monkeypox virus, clade I), Liberia MPXV (West African monkeypox virus, clade IIa), and an isolate from the 2022 outbreak (strain TKY220091, clade IIb; referred to as “2022 MPXV” hereafter). 2022 MPXV is classified as a clade IIb strain (lineage B.1) and phylogenetically distinct from prior endemic MPXV strains (Figure 1). In this study, normal human epidermal keratinocytes (NHEK) and colon organoids were infected separately with the above MPXV strains and cultured for 3 days (Figure 2A). MPXV genomes were detected in the cell culture supernatants of infected keratinocytes and colon organoids at 1, 2, and 3 day(s) postinfection (dpi) (Figure 2B). In the cell culture supernatant of 2022 MPXV-infected keratinocytes, the virus copy number increased 16.7-fold from 1 dpi to 3 dpi (Figure 2B, left), while the increase observed during the same period was only 1.4-fold for infected colon organoids (Figure 2B, right). At 3 dpi, viral copy numbers in the cell culture supernatant of infected keratinocytes were 41.6, 7.0, and 8.2 times higher for Zr-599 MPXV, Liberia MPXV, and 2022 MPXV than those of infected colon organoids, respectively. We did not observe any considerable variation between the MPXV clades in the quantity of viral genome in the culture supernatant of infected colon organoids. On the other hand, the viral copy number in the culture supernatant of Zr-599 MPXV-infected keratinocytes was higher than that of Liberia MPXV-infected keratinocytes. In addition, MPXV DNA and mRNA were detected in infected keratinocytes and colon organoids (Figure 2C,D). The intracellular MPXV mRNA expression in infected keratinocytes was higher than that in infected colon organoids (Figure 2D). Consistent with this, a Circos plot showing the distribution of reads along the MPXV genome indicated that keratinocytes were more efficiently infected than colon organoids (Figure 2E). Interestingly, the amounts of Zr-599 MPXV DNA (Figure 2C) and mRNA (Figure 2D) were slightly higher than those detected from Liberia MPXV- and 2022 MPXV-infected colon organoids (Zr-599 MPXV/Liberia MPXV = 1.3 and 1.6, Zr-599 MPXV/2022 MPXV = 3.1 and 4.8) suggesting that Zr-599 MPXV had a higher infection efficiency in colon organoids than Liberia MPXV and 2022 MPXV. These results suggest that 2022 MPXV shares similar infection tropism with prior endemic MPXV strains.

In addition, infection experiments with the three MPXV clades were performed using VeroE6 cells, which are widely used for MPXV research. Although there was almost no viral genome present in the cell culture supernatant of infected VeroE6 cells, both intracellular MPXV DNA and mRNA were detected (Supporting Information: Figures S1A, S1B). MPXV mRNA expression detected in infected VeroE6 cells was similar to infected colon organoids, suggesting that the infection efficiency was similar between VeroE6 cells and colon organoids, but much lower than in keratinocytes.

3.2 MPXV infection causes cellular damage in keratinocytes

As shown in Figure 3A, cytopathic effects of Zr-599 MPXV, Liberia MPXV, or 2022 MPXV infections were observed in keratinocytes. In addition, most keratinocytes became KRT10 (a keratinocyte marker)-negative following Zr-599 MPXV, Liberia MPXV, and 2022 MPXV infections (Figure 3B). We next performed RNA-seq analysis to examine the host response by keratinocytes caused by MPXV infection. Volcano plots in Figure 3C show that infections with Zr-599 MPXV, Liberia MPXV, or 2022 MPXV significantly altered the expression levels of 938, 1529, and 968 genes in keratinocytes, respectively. Cluster analysis suggested that the gene expression profile of keratinocytes infected with 2022 MPXV was different from those of keratinocytes infected with Zr-599 MPXV or Liberia MPXV (Figure 3D). In Figure 4, dot plots of the top 10 significantly enhanced or decreased gene sets from Gene Set Enrichment Analysis (GESA) of Zr-599 MPXV- (Figure 4A), Liberia MPXV- (Figure 4B), or 2022 MPXV- (Figure 4C) infected keratinocytes compared with uninfected keratinocytes (mock) are shown. The expression levels of genes in the “keratinization” and “mitochondrial respiratory chain complex assembly” categories were decreased in Zr-599 MPXV-, Liberia MPXV-, and 2022 MPXV-infected keratinocytes (Figure 4). Consistently, previous histologic and immunohistochemical analyzes in human mpox patients have also demonstrated keratinocyte dysfunction, including necrosis of keratinocytes.¹⁵ These results suggest that efficient proliferation of MPXV in keratinocytes disrupts their normal functions. Moreover, genes in the “nucleosome assembly” category were increased in Zr-599 MPXV-, Liberia MPXV-, and 2022 MPXV-infected keratinocytes (Figure 4). A previous study, which performed microarray analysis in MPXV-infected MK2, showed that the majority of modulated histone-encoding genes were significantly elevated in expression.¹⁶ Taken together, these results suggest that MPXV infections may alter the regulation of chromatin structure in host cells.

Interestingly, both cluster analysis (Figure 3D) and principal component analysis (PCA) (Figure 5A) revealed that infection with 2022 MPXV led to gene expression profile changes that were unique to this strain. To further explore this finding, we extracted the set of genes whose expression levels were specifically altered by 2022 MPXV infection for an in-depth examination. As shown in Figure 5B, we performed DAVID-based gene ontology (GO) enrichment analysis on this gene set. The top five significantly enriched GO terms in 2022 MPXV-infected keratinocytes compared with Zr-599 MPXV- and Liberia MPXV-infected keratinocytes are listed in Figure 5B. Interestingly, the top significantly enriched GO term was “response to hypoxia.” We confirmed by RT-PCR that the expression levels of Egl-9 Family Hypoxia Inducible Factor 1 (EGLN1)¹⁷ and LIM Domain Containing 1 (LIMD1),¹⁸ which are closely related to hypoxia, were increased only by 2022 MPXV infection (Figure 5C). In addition, a hypoxia-induced gene, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2),¹⁹ was also specifically upregulated by 2022 MPXV infection (Figure 5C). These results suggest that skin lesions induced by MPXV may be associated with abnormal hypoxic signaling and collagen production capacity. Altogether, our findings raise the possibility that 2022 MPXV induces skin lesions via mechanisms different from those of prior endemic MPXV strains.

3.3 Zr-599 MPXV induces cellular damage in colon organoids

We next turned our attention to the MPXV-infected colon organoids and characterized how they responded to MPXV infections via RNA-seq analysis. Volcano plots in Figure 6A show that Zr-599 MPXV, Liberia MPXV, and 2022 MPXV infections in colon organoids, in contrast to keratinocytes, did not induce much changes in gene expression profile. Nonetheless, we did identify 85 genes whose expression levels were changed more than twofold in Zr-599 MPXV-infected colon organoids. Cluster analysis suggested that the gene expression profile of colon organoids infected with Zr-599 MPXV differs from those of colon organoids infected with Liberia MPXV or 2022 MPXV (Figure 6B). As such, we performed GESA on the set of genes whose expression was decreased in colon organoids by Zr-599 MPXV infection (Figure 7A). The top two significantly enriched GO terms were “response to zinc ion” and “zinc ion homeostasis.” Barcode plots similarly showed that the gene expression levels of the “response to zinc ion” and “zinc ion homeostasis” categories were decreased in Zr-599 MPXV-infected colon organoids (Figure 7B). It is known that zinc absorption and excretion capacities are deeply involved in intestinal homeostasis.²⁰ Both solute carrier family 30 member 2 (SLC30A2) and metallothionein 1G (MT1G) are essential for maintaining an appropriate concentration of zinc, but their expressions were significantly decreased in Zr-599 MPXV-infected colon organoids (Figure 7C). Moreover, immunostaining analysis showed that the protein level of MT1G was consistently decreased in Zr-599 MPXV-infected colon organoids (Figure 7D). Previous studies have reported SLC30A2 gene depletion^{21, 22} or reduced MT1G expression^{23, 24} to induce intestinal disorders. Importantly, Zr-599 MPXV infection induced gene expression of intestinal inflammation markers, such as trefoil factor 1 (TFF1), fos proto-oncogene, AP-1 transcription factor subunit (FOS), and fatty acid binding protein 1 (FABP1), in colon organoids (Figure 7E). Immunostaining analysis revealed that cleaved Caspase-3 expression was highest in Zr-599 MPXV-infected colon organoids, suggesting that Zr-599 MPXV infection induced apoptosis in colon organoids (Figure 7F). Consistent with this, Zr-599 MPXV, but not Liberia MPXV, has been reported to induce severe intestinal damage in nonhuman primates.⁷ These results suggest that Zr-599 MPXV may induce intestinal disorders by disturbing zinc homeostasis in the colon.