Article Figures & Data
Figures
- FIG 1
Generation of ΔSRCR5 pigs and experimental setup. (A) Genome editing to generate ΔSRCR5 pigs. Genome-edited founder animals were generated by zygote injection of CRISPR/Cas9 editing reagents using Cas9 mRNA and two guide RNAs, sgSL26 and sgSL28, in combination to generate a deletion of exon 7 in CD163. Animals were bred to generate F1 and F2 generations, focusing on one genotype showing clean religation at the cutting sites of both guide RNAs. Homozygous F2 animals carry this genotype in both alleles (bottom). (B) Structure prediction and expression of ΔSRCR5 in pulmonary alveolar macrophages of F2 animals. Protein structure prediction using RaptorX points toward an intact protein product upon the deletion of SRCR5. (C) Experimental design of the challenge study. Four homozygous (green) and 4 wild-type (orange) siblings from heterozygous/heterozygous mating of the F1 generation animals were cohoused from weaning. Genotypes were confirmed by PCR amplification across exon 7 (see panel A) and by Sanger sequencing. Piglets were cohoused after weaning and after acclimation to the specific-pathogen-free unit for 1 week and throughout the 14-day challenge experiment that was initiated by inoculating each pig intranasally with 5E6 TCID50 of PRRSV-1 subtype 2 strain BOR-57 at day 0 and day 1 of the challenge. The piglets were 7 to 8 weeks of age at the start of the acclimation period. (D) Piglets 1 day before the start of the challenge.
- FIG 2
Serum levels of soluble C163. Serum samples collected 2 weeks prior to and on day 0 of the challenge were assessed for the level of sCD163 using a commercial ELISA (n = 4 pigs per genotype, serum collected at 2 different time points, assessed in duplicate in 3 replicates). Minima/maxima and 90th percentiles are displayed. Statistical analysis using an unpaired t test showed no significant difference.
- FIG 3
ΔSRCR5 pigs show no clinical signs or pathology of PRRSV-1 infection. (A) Rectal temperatures of ΔSRCR5 (green) and wild-type (orange) piglets during challenge with BOR-57. Rectal temperatures were measured daily during feeding. Error bars represent standard errors of the means (SEM) (n = 4). (B) Average daily weight gain based on weight measurements at day 0, 7, and 14 of the challenge. For panels A and B, statistical analysis was performed using two-way ANOVA and Sidak's multiple-comparison test. ns, not significant. (C) Viremia during challenge with BOR-57. Serum samples were collected at days 0, 3, 7, 10, and 14 from the jugular vein using vacuum tubes, and viral RNA was isolated and quantified using RT-qPCR with primers specific to ORF5 of BOR-57. (D) Antibody response to PRRSV-1 during the challenge. Serum samples were analyzed for the presence of PRRSV antibodies using the Idexx PRRSV X3 ELISA, where a value of <0.40 is negative and a value of ≧0.4 is positive. Each data point/line represents data for a single animal, with 4 animals per genotype group. (E) Lung and lymph node pathology, histopathology, and immunohistochemistry (IHC) scores. Lung pathology was assessed in a blind fashion, and a subjective score for the severity of gross lung lesions using an established scoring system was applied (scale, 0 to 100). Lung histopathology sections were scored for the presence and severity of interstitial pneumonia, ranging from 0 to 6 (0, normal; 1, mild multifocal; 2, mild diffuse; 3, moderate multifocal; 4, moderate diffuse; 5, severe multifocal; 6, severe diffuse). Immunohistochemistry staining against PRRSV-N of lung and lymph node sections was scored, ranging from 0 to 3 (0, no signal; 1, low numbers of positive cells; 2, moderate numbers of positive cells; 3, abundant). Numbers represent averages (n = 4) ± SEM. (F) Lung histology and immunohistochemistry. (Top) Formalin-fixed, paraffin-embedded, hematoxylin-and-eosin-stained lung sections from necropsy on day 14 postchallenge. (Left) ΔSRCR5 piglets; (right) wild-type piglets. Bar, 100 μm. (Bottom) Formalin-fixed, paraffin-embedded immunohistochemical staining against PRRSV antigen (brown) and hematoxylin counterstain. (Left) ΔSRCR5 piglets; (right) wild-type piglets. The scale bar represents 50 μm. (G) Lung pathology. Shown are lungs from pigs at necropsy at 14 days postchallenge. (Left) Lungs from two ΔSRCR5 pigs; (right) lungs from two wild-type pigs.
- FIG 4
Cytokine response to BOR-57 PRRSV infection. Cytokine levels in serum samples collected prior to challenge on day 0 and on challenge days 3, 7, 10, and 14 were measured using cytokine antibody arrays. (A) Alpha interferon (IFN-α); (B) interleukin-17A (IL-17A); (C) interleukin-1 receptor antagonist (IL-1ra); (D) IL-4; (E) IL-6; (F) IL-8; (G) monokine induced by gamma interferon (MIG/CXCL9); (H) macrophage inflammatory protein 1β (MIP-1β/CCL4); (I) chemokine ligand 3-like 1 (CCL3L1); (J) granulocyte-macrophage colony-stimulating factor (GM-CSF); (K) tumor necrosis factor alpha (TNF-α); (L) IL-12; (M) IL-1β; (N) IL-10; (O) transforming growth factor β1 (TGFβ1); (P) IFN-γ; (Q) IL-18; (R) platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31); (S) IL-1α; (T) IL-13. Error bars represent SEM (duplicates of 4 replicates). Statistical analysis was performed using two-way ANOVA and Sidak's multiple-comparison test. *, P ≤ 0.5; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Tables
- TABLE 1
Whole-blood-count results for ΔSRCR5 (animals 4 to 7) and wild-type (animals 8 to 11) piglets at week 6a
Indicator Value for animal Reference value (range) 4 5 6 7 8 9 10 11 WBC count (10E9/liter) 22.5 24 14 15.1 12.4 19.6 26.1 14.4 11–22 Neutrophil count (segmented) (10E9/liter) 5.85 4.8 4.62 5.889 4.34 7.252 7.83 4.32 2–15 % neutrophils (segmented) 26 20 33 39 35 37 30 30 20–70 Neutrophil count (nonsegmented) (10E9/liter) 0 0 0 0 0 0 0 0 0–0.8 % neutrophils (nonsegmented) 0 0 0 0 0 0 0 0 0–4 Lymphocyte count (10E9/liter) 15.3 18.72 8.82 8.305 7.564 11.76 16.182 9.36 3.8–16.5 % lymphocytes 68 78 63 55 61 60 62 65 35–75 Monocyte count (10E9/liter) 0.675 0.48 0.42 0.755 0.496 0.588 1.044 0.576 0–1 % monocytes 3 2 3 5 4 3 4 4 0–10 Eosinophil count (10E9/liter) 0.675 0 0 0.151 0 0 1.044 0.144 0–1.5 % eosinophils 3 0 0 1 0 0 4 1 0–15 Basophil count 0 0 0.14 0 0 0 0 0 0–0.5 % basophils 0 0 1 0 0 0 0 0 0–3 RBC count (10E12/liter) 6.03 6.64 6.99 6.58 6.3 6.53 7.52 6.97 5–9 PCV/hematocrit 0.384 0.391 0.383 0.388 0.382 0.39 0.429 0.421 0.36–0.43 Hb level (g/dl) 11.5 11.9 10.9 11.8 11.6 12 13.8 12.3 10–16 MCV (fl) 63.7 58.9 54.8 58.9 60.7 59.8 57.1 60.5 50–62 MCHC (g/dl) 29.9 30.4 28.3 30.5 30.3 30.9 32.1 29.1 30–36 No. of platelets 219 230 605 397 483 519 219 606 120–720 RDW 20.9 23.1 28.9 20.6 21 18 17 22.6 ↵a WBC, white blood cell; RBC, red blood cell; PCV, packed cell volume; Hb, hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width.
