Disruption of anthrax toxin receptor 1 in pigs leads to a rare disease phenotype and protection from senecavirus A infection

Figures

• 

  Figure 1. Anatomical and histological features of ANTXR1 KO pigs. (A) The photographs show examples of anatomical abnormalities in ANTXR1 KO pigs at two weeks of age. The upper left photograph shows an example of frontal bossing and shortened snout in the KO pig compared to a WT pig (left). The lower left photograph shows an example of a broadened stance (white arrow). The top right picture shows the post-legged rear legs of the KO pig. The bottom right picture shows an example of a KO pig with cataract (black arrow) in the left eye. (B) Presence of partial anodontia in the KO pig at one year of age. (C) Presence of excessive tissue development in the orbital socket with exophthalmos in the KO pig at one year of age. Representative images of hematoxylin and eosin (H&E) staining are shown for (D) lung, (E) skeletal muscle from the leg, and (G) cardiac muscle of WT and KO pigs at one year of age. (F) Periodic acid-Schiff staining was performed for skeletal muscle to show peripheral nerve degeneration with dilated fascicles filled with mucin (black arrow). (H) Masson’s trichrome staining was also performed for the cardiac muscle to show collagen accumulation (blue staining). Excessive…

• 

  Figure 2. Infection of WT and KO cells with SVA-EGFP. Photomicrographs taken (A) 8 h and (B) 24 h after infection of fibroblast cells with SVA isolate, KS15-01-EGFP. The KO cells were generated from Pig 1–3 (52) (Fig. S1 and Table S2).

• 

  Figure 3. Circulating SVA nucleic acid and SVA-specific antibody responses in WT and KO pigs during the SVA challenge. (A) The concentration of nucleic acid is shown as the Ct value of the sample subtracted from 40, the Ct value cutoff. An asterisk identifies a serum sample that was positive for virus isolation on PK15 cells. (B) Total antibody response as measured by IFA. Serum, beginning with an initial dilution of 1:5, was serially diluted and incubated with SVA-infected PK-15 cells. (C) Virus neutralizing results for serum serially diluted 1:2. Dilutions less than 1:8 showed cytotoxicity on PK-15 cells. Results are shown for individual pigs.

• 

  Figure 4. ANTXR1 detection in lung tissue from WT and KO pigs after the SVA challenge. Immunohistochemistry for ANTXR1 in lung sections for WT (54 and 59) and KO (52 and 53) at 10 × and 20 ×. Negative control sections incubated with rabbit IgG are shown to the right. Scale bars are 200 μm for 10 × and 100 μm for 20 ×.

• 

  Figure 5. Gross anatomical features of ANTXR1 in-frame (IF) pigs for the second SVA challenge. (A) The founder boar who was used for breeding was mosaic for edits in ANTXR1 with two knockout alleles and one in-frame (IF) allele (Figure S3), and he exhibited a strong GAPO phenotype. After breeding to propagate his alleles, only the IF mutation was detected in progeny, and piglets were produced that were homozygous for this mutation. As shown, these piglets exhibited (B) frontal bossing and shorter snouts and (C) a post-legged stance as compared to WT piglets from the same litter. (D) Two homozygous IF piglets also had cataracts at birth (black arrow).

• 

  Figure 6. Infection of WT, KO, and IF cells with SVA-EGFP. Photomicrographs taken 8 h after infection of fibroblast cells with SVA isolate, KS15-01-EGFP, at the same exposure. KO cells were from the two pigs of the first SVA challenge, and IF cells were from two pigs of the second SVA challenge.