Animal Infections
Animal studies were described in10. Briefly, 3 rhesus macaques (Macaca mulatta) - two female, one male – were inoculated intravenously with mpox (MPXV/USA/MA001/2022; lineage B.1, clade 2b; BEI NR-58622; 106 TCID50 (108 plaque-forming unit [PFU]); intravenous). On day 10 post-infection, a full necropsy with description of gross lesions and collection of major organs was performed. All animal studies were approved by the institutional animal care and use committee of Bioqual, Inc. in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals; The Guide for the Care and Use of Laboratory Animals; the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training; and ARRIVE guidelines.
Histopathology
Tissues were fixed in 4% paraformaldehyde for 24 hours, transferred to 70% ethanol, and paraffin embedded and blocks sectioned at 5 mm for routine hematoxylin and eosin staining (H&E) and for IHC and ISH. Tissue pathology was independently assessed by three veterinary pathologists (AJM, GMR, CEL).
Immunohistochemistry
Immunohistochemical staining was performed on selected formalin-fixed, paraffin-embedded sections of reproductive tissues using standard techniques. The slides were baked for 30 minutes at 60ºC, deparaffinized through xylene, 100% ethanol, 95% ethanol, and 1x tris-buffered saline (TBS). Heat-induced epitope retrieval was performed with a 10% citrate buffer (Sigma-Aldrich, C9999) for all antibodies. To detect mpox, primary mouse anti-Vaccinia antibody (Santa Cruz, SC-58210) was applied at 1:100 followed by mouse Mach-2 HRP-Polymer (Biocare MHRP520) for 30 min, then Nova-Red (Vector, SK4800) for 10 min, and counterstained with hematoxylin followed by bluing using 0.25% ammonia water. For CD10 (1:4000; SinoBiological, 90177-C07H), primary antibody was applied for 60 min by rabbit Mach-2 HRP-Polymer (Biocare, RHRP520) for 30 min, then 3,3’-diaminobenzidine (DAB,Cell Marque 957D-30). The slides were counterstained using 50% hematoxylin solution (BioCare CATHE-MM). Vaccinia and CD10 IHC was performed using a Biocare intelliPATH autostainer. For cytokeratin (1:140, clone AE1/AE3, Dako M3515), CD68 (1:410, clone KP1, Dako M0814), alpha smooth muscle actin (1:1000; clone 1A4, Dako M0851), and vimentin (1:162; clone 3B4, ProGen, 61013) primary antibodies were diluted in Da Vinci Green Diluent (BioCare PD900M) and incubated for 30 min at room temperature, slides were then washed and treated with biotinylated horse anti-mouse secondary antibody (Vector Labs BA-2001, diluted 1:200 in Da Vinci Green) for 30 minutes at room temperature. Elite Avidin-Biotin Complex solution (Vector Labs, PK-6100) was applied and incubated for 30 minutes, followed by DAB solution. Matched negative control slides were incubated with Universal Negative Control Serum (BioCare, NC498L). All slides were counterstained using 50% hematoxylin solution (BioCare, CATHE-MM) and were scanned at 20x using a Midi II Scanner (3DHistotech) on default brightfield settings.
In situ Hybridization
Duplex chromogenic in situ hybridization was performed using the RNAscope 2.5 HD Duplex Detection Kit (ACD Bio 322500) with customized and general probes (1260381-C1, Mmu-KRT10-C1), 1226271-C2 (V-MPXV-OPG124-C2), 461341 (Mmu-POLR2a), 457711-C2 (Mmu-PPIB-C2), 2-plex negative control (320751) following recommended guidelines (Protocol 322500-QCK Rev B) as previously described10. Slides were then scanned at 20x using a Midi II Panoramic Scanner (3D Histotech) on default brightfield settings.
Cyclic ISH and Fluorescence Microscopy
Cyclic dual RNAscope ISH and immunofluorescence staining was also performed on selected cervical tissues from Animal 1 (T451F). Baking, deparaffinization, and rehydration were performed in the same manner as with IHC, with the substitution of 1x phosphate-buffered saline with 0.2% fish skin gelatin (FSG, Aurion 900.033) in place of 1x TBS. Heat-induced epitope retrieval was achieved using the same method as above. The slides were then washed with 1x PBS/FSG twice for 5 minutes. A protein block was performed using Intercept Blocking Buffer (LiCor 927-70001) for 30 minutes. A photochemical bleaching solution (3% H2O2 in 1xPBS with 20mM NaOH) 22 was used prior to antibody application to decrease autofluorescence and between cycles of fluorescence staining. For each application slides were photo bleached for 60 minutes at room temp while illuminated from above and below with LED light panels (Miroco MI-CL008) on full power. Slides were washed twice with 1x PBS/FSG for 5 minutes. In a lightproof humidity chamber, the slides were protein blocked with Intercept for 30 minutes. For cycle 1, primary antibodies cytokeratin AE1/AE3 (1:140) and SMA (1:100) with Hoechst 1:10,000). For cycle 2, primary antibody vimentin 1:50 with Hoechst 1:10,000 incubated overnight at 4ºC. For both cycles, secondary antibodies against mouse IgG1 conjugated with AlexaFluor 647 (1:1,000, Jackson Immunoresearch 115-605-205) and mouse IgG2a conjugated with AlexaFlour 488 (1:1,000, JIR 115-545-206) were applied and incubated sequentially at room temperature for 30 minutes each. All primary and secondary antibodies were diluted in Intercept. After each cycle slides were coverslipped with 80% glycerol solution and scanned at 20x using a Midi II Scanner in fluorescence mode. Between cycles coverslips were removed by soaking in 1xPBS/FSG for approximately 10 minutes. For cycle 3, dual-plex RNAscope was performed as listed above followed by detection with the Multiplex-Fluorescent reagent kit V2 (323100) as recommended.
Slide Analysis
HALO (v3.6, Indica Labs) was used to fuse serially obtained fluorescence scans with the HALO registration module within the HiPlex FL Module (v4.2). The fused image underwent thresholding for each fluorescent color based on isotype control slides and according to chromogenic IHC identification of staining pattern and intensity.