https://orcid.org/0000-0002-8225-1254
Figures & data
Figure 1. Experimental design. Six pigs were inoculated with the MPXV hMPXV/USA/MA001/2022 (Lineage B.1, Clade IIb) isolate acquired from BEI Resources. A 3 ml dose of 3 × 107 TCID50 per animal was administered intranasal (IN), intradermal (ID), and intravenous (IV). At 2 days post-challenge (DPC), two contact sentinel control pigs were co-mingled with the six principally challenged animals. Daily clinical observations and body temperatures were recorded. Nasal, oropharyngeal, and rectal swabs as well as whole blood were collected at −1, 1, 3, 5, 7, 10, 14, 17, and 21 DPC. Oral fluids and serum were collected at −1, 7, 14, and 21 DPC. Post-mortem examinations were performed at 7, 14, and 21 DPC and results compared to baseline post-mortem examinations conducted on 2 additional negative control pigs at -1 DPC. BioRender.com was used to create the figure illustrations.
Figure 2. Histological alterations in the skin and immunohistochemical detection of MPXV antigen in one of the infected pigs. (A) Histologic alterations are characterized by epidermal pustule formation (*), hyperplasia of the adjacent epidermis (arrowheads) and superficial perivascular lymphohistiocytic and eosinophilic dermatitis (arrows). The corneal layer of the epidermis was occasionally colonized by coccoid bacteria (dashed arrows). (B) Within this single pustule, sporadic degenerate inflammatory cells contain minimal intracytoplasmic viral antigen (red). No viral antigen was detected within keratinocytes or other cell types. H&E, 200X total magnification; Fast Red, 400X total magnification.
Figure 3. Viral DNA shedding of MPXV-infected pigs. qPCR was performed on nasal (A, C) and oral (B, D) swabs collected from principal-infected and sentinel contact control pigs at indicated timepoints. Mean (n = 2) viral DNA copy number per swab is reported based on the detection of the MPXV TNF receptor gene (crmB). The dotted line indicates the limit of detection for the assay. (A, B) Numbers indicate the ID code of the individual pig. An open symbol indicates samples with only one of two qPCR reactions positive. (C, D) Line graphs display the mean and error bars indicate the standard deviation.
Figure 4. Detection of binding antibodies targeting MPXV. Indirect multiple ELISAs were conducted using purified recombinant MPXV A35 (A) and H3 (B) antigens were used to detect MPXV-specific binding antibodies in pig sera. Serum was diluted in 3x serial dilutions. An ELISA titer was considered positive when the OD450 was greater than 3 standard deviations above the mean of naïve animal sera run concurrently. (C) Indirect ELISA using crude whole VACV. Plates were coated with either VACV or BSC-40 cell lysate. Sera was diluted 1:100, 1:200, and 1:400. An ELISA titer was considered positive when the average OD450 among two replicates was greater than 2 standard deviations above the mean of corresponding samples in the cell lysate plate.
Figure 5. Immunofluorescent detection of MPXV-specific total IgG. Representative images of stained cells. Naïve Vero E6 cells were infected with MPXV at a MOI of 1 and incubated for 48 hours before fixation with 80% acetone. Pig sera were diluted in PBS with 1% BSA and added to fixed wells. Goat anti-pig IgG secondary antibody conjugated to Alexa-Fluor 488 was used to stain wells, and wells were visualized at 10x magnification using an EVOS fluorescence microscope. Naïve serum collected at -1 DPC was used as a negative control, and a rabbit anti-vaccinia polyclonal antibody was used as a positive control. Scale bars represent 400 µm.
Table 1. Summary of indirect immunofluorescence staining of infected Vero cells to detect MPXV-reactive IgG in pig serum. Detection of IgG was performed with goat anti-pig IgG polyclonal antibodies.
Figure 6. Detection of MPXV-specific antibodies using Protein A. Vero E6 cells were infected with MPXV at a MOI of 1 and incubated for 48 hours before fixation with 80% acetone. Pig sera were diluted in PBS with 1% BSA and added to fixed wells. Biotinylated Protein A conjugated to streptavidin-DyLight488 was used to stain wells, and wells were visualized at 10x magnification using an EVOS fluorescence microscope. Scale bars represent 400 µm. (A) Representative images of stained cells. Naïve serum collected at -1 DPC was used as a negative control, and a rabbit anti-vaccinia polyclonal antibody was used as a positive control. (B) Images from sentinel pig #18, collected at each indicated timepoint.
Table 2. Summary of indirect immunofluorescence staining of infected Vero cells to detect MPXV-reactive antibodies in pig serum. Detection of antibodies was performed with Protein A.