Ethical use of virus and animals in research
The use of animals and viruses was in accordance with the Federation of Animal Science Societies (FASS) Guide for the Care and Use of Agricultural Animals in Research and Teaching, the USDA Animal Welfare Act, and Animal Welfare Regulations, Biosafety in Microbiological and Biomedical Laboratories, 5th edition. Pigs were humanely euthanized by intravenous pentobarbital injection according to American Veterinary Medical Association Guidelines on Euthanasia. Prior to initiating the study, all activities involving the use of virus and animals were approved by Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee at Kansas State University (#1079), the IACUC at University of Missouri (#8813), and by the IACUC and IBC committees of Midwest Veterinary Services/Central States Research Centre (#AC21009P). The study was carried out in compliance with the ARRIVE guidelines.
Generation of ANTXR1 KO pigs
Generation of the ANTXR1 KO pigs by using CRISPR/Cas9 is previously described by Whitworth et al.21. Single guide RNAs (sgRNAs) were designed in exon 1 of the ANTXR1 gene (GenBank accession: NC_010445.4). Four 20 bp guides were designed adjacent to a Streptococcus pyogenes (Spy) protospacer adjacent motif (PAM) according to the Zhang Lab CRISPR Design22. Two pairs of guide sequences were located downstream from the start codon in exon 1 (Table S1).
In vitro transcription of sgRNAs
Template guide DNA was synthesized by Integrated DNA Technologies (idtdna.com, Skokie, IL, USA) as a gBlock. For in vitro transcription, a T7 promoter sequence was added upstream of the guide. Each gBlock was diluted to a final concentration 0.1 ng/µL and amplified with gBlock Forward (5ʹ-ACTGGCACCTATGCGGGACGAC) and gBlock Reverse (5ʹ-AAAAGCACCGACTCGGTGCCAC) primers by using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) following standard procedures. PCR amplification included an initial denaturation of 98 °C for 1 min followed by 35 cycles of 98 °C (10 s), 68 °C (30 s), and 72 °C (30 s). Each PCR-amplified gBlock was purified by using a QIAGEN (Valencia, CA, USA) PCR purification kit following standard procedures. The purified gBlock amplicons were used as templates for in vitro transcription with the MEGAshortscript T7 Transcription Kit (Ambion, ThermoFisher, Grand Island, NY, USA). Cas9 mRNA with the 5ʹ cap and polyadenylation signal was purchased from Sigma (St. Louis, MO, USA). Total single guide RNA (sgRNA) and Cas9 mRNA were diluted in nuclease-free water and combined to final concentrations of 20 ng/µL and 20 ng/µL, respectively. Mix 1 contained sgRNA guides 58 and 99 with Cas9 mRNA. Mix 2 contained sgRNA guides 64 and 101 with Cas9 mRNA.
Preparation of embryos and injection of guide pairs were as described by Whitworth et al.21. Guide pairs Mix 1 or Mix 2 were co-injected as individual treatments into the cytoplasm of presumptive zygotes at 14 h post-fertilization. Injected zygotes were placed into MU2 for culture until day 5 or 623. To assess blastocyst development and editing efficiency, three replicates of injections were performed for Mix 1 and Mix 2. Development to the blastocyst stage on day 6 for non-injected controls, Mix 1-injected, and Mix 2-injected groups were 33.3 ± 3.3%, 20.7 ± 3.4%, and 18.5 ± 5.4%, respectively. Embryo transfers were performed at the University of Missouri Swine Research Complex. Fifty blastocysts and morulae were loaded into a tomcat catheter and surgically transferred into the ampullary-isthmic junction of a cycling gilt on days 3 or 4 after standing estrus. Pregnancy was determined by heat checking and monitoring by ultrasound after day 25. Surrogates were checked weekly until farrowing. The sexes and birth weights of the piglets were recorded.
Genotyping
Zona pellucidae of Day 6 blastocyst-stage embryos were removed, and genomic DNA was extracted as described by Whitworth et al.21. Briefly, genomic DNA was extracted from blastocyst-stage embryos by adding 6 μL of embryo lysis buffer (ELB) (40 mM Tris, pH 8.9, 0.9% Triton X-100, 0.9% Nonidet P-40, 40 μg/mL Proteinase K) and incubating at 60 °C for 45 min. Genomic DNA was isolated from Day 1 piglets by incubating a piece of ear skin in 300 μL of ELB at 56 °C for 4–5 h. Proteinase K was inactivated by incubating lysates at 85 °C for 10 min. Primers for the genotyping assay were ANTXR1 Forward (5ʹ-GGTCCTGGCGACTTAGA) and ANTXR1 Reverse (5ʹ-TGTATGCGGGACAACTTCT) which were located upstream of the start codon and within the first intron, respectively. The PCR amplicon was predicted to be 446 bp for the WT gene. PCR conditions consisted of 95 °C for 2 min, 38 cycles of 95 °C (30 s), 56 °C (40 s), 72 °C (30 s), and 72 °C for 1 min, and products were separated on a 2% agarose gel. PCR products were also subjected to Sanger sequencing with the ANTXR1 Forward primer or cloned into pCR™4-TOPO vector (ThermoFisher) following standard procedures.
Histology
One ANTXR1 KO pig and one WT pig were humanely euthanized at one year of age for post-mortem examination at the University of Missouri Veterinary Medical Diagnostic Laboratory. Tissue samples were collected and fixed in 10% neutral buffered formalin. Fixed tissues were trimmed, processed by using a Sakura Tissue-Tek VIP 6 tissue processor (Torrance, CA, USA), embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin for routine histopathology. In addition, Masson’s Trichrome stain and Alcian blue-PAS stain were used for the identification of connective tissue.
Immunohistochemistry
For detection of ANTXR1 in lung tissue of WT and KO pigs, paraffin-embedded tissue sections were sectioned, deparaffinized, and enzymatically digested with Proteinase K. Sections were blocked with Dual Endogenous-Enzyme blocking reagent, avidin/biotin (Agilent, Santa Clara, CA, USA), in 1% blocking reagent (Roche, Basel, Switzerland) in TBS (100 mM Tris pH 7.5, 150 mM NaCl) with 1% Triton-X for 30 min at room temperature. Incubation was with rabbit anti-human TEM8 (clone 37, Abcam, Cambridge, United Kingdom) or IgG (Abcam) at 4 °C overnight, and detection was performed with Vectastain rabbit-IgG ABC HRP Kit (Vector Labs, Burlingame, CA, USA). Images were captured on Zeiss upright microscope and processed in Zen Blue software (Carl Zeiss, Thornwood, NY, USA).
Cells and virus
Primary cultures of porcine fibroblast cells were prepared from tail snips of newborn pigs and placed at 4 °C in PBS with 10 μg/mL gentamicin. Fibroblast cells were isolated by incubating the tail snips at 37 °C in a humidified incubator with an atmosphere of 5% CO2 in air in DMEM supplemented with 15% fetal bovine serum (FBS), 10 μg/mL gentamicin, 400 U/mL collagenase (Sigma), and 4 Kunitz/μL DNase I (Sigma) for 3 h. Cells were grown until 90% confluent in a T75 flask, trypsinized, and frozen in FBS with 10% DMSO. To isolate individual colonies for confirming genotypes, cells were plated at clonal density (30 cells in a 100 mm dish) and grown for 7 days at 37 °C. Individual colonies were collected, genotyped, and frozen in liquid nitrogen.
The KS15-01 virus was originally isolated from a pig nasal swab sample submitted to the Kansas Veterinary Diagnostic Laboratory10. The virus was propagated on PK-15 cells with Minimal Essential Medium (MEM; ThermoFisher) supplemented with 2% horse serum (Sigma), antibiotics (100 units/mL of penicillin, 100 µg/mL of streptomycin, 0.25 µg/mL fungizone; ThermoFisher), and incubated at 37 °C and 5% CO2. For titration, the virus was serially diluted 1:10 in quadruplicate on confluent PK-15 cells in a 96-well tissue culture plate (BD Biosciences, San Jose, CA, USA). After two days, the wells were examined for the presence of cytopathic effect (CPE). The last well showing CPE was used as the titration endpoint, and the 50% tissue culture infectious dose (TCID50) per mL was calculated by using the method of Reed and Muench24. Modification of KS15-01 included the insertion of an EGFP gene fused with a Teschovirus 2A element between SVA genes 2A and 2B of the KS15-01 infectious clone and propagated as described by Chen et al.10. The EGFP-expressing virus, KS15-01-EGFP (GenBank accession No. KX349734), was used to infect fibroblast cells from WT and KO pigs. All images were captured at the same exposure. The growth kinetics in culture for the KS15-01 and the EGFP variant are the same10.
Animal infection study
Pigs were infected with SVA isolate, KS15-01, according to the protocol described by Chen et al.10. Animal experiments were performed at the BSL-2 facility operated by Midwest Veterinary Services (Manhattan, Kansas, USA). WT and ANTXR1 KO pigs (P generation) at 4 weeks of age or WT and ANTXR1 IF pigs (F2 generation) at 8 weeks of age were maintained in the same room and allowed to co-mingle throughout the challenge period. After a one-week acclimation period, the pigs were infected with KS15-01, which was delivered intranasally as a 5 mL dose containing 108 TCID50 of virus. Animals were examined daily for the presence of clinical signs. Serum, fecal swabs, and nasal swabs were collected at 0, 3, 7, and 10 days after infection. The experiment was terminated 10 days after infection, and the pigs were euthanized and necropsied.
qRT-PCR of SVA nucleic acid
Viral RNA was extracted from serum, nasal swabs, or fecal swabs by using a MagMax 96-Viral RNA Isolation kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The qRT-PCR was performed by using the EZ-SVA Real-Time RT-PCR detection kit from Tetracore (Rockville, MD, USA). Briefly, a 25 μL reaction was carried out by using 7 μL of extracted RNA, and all steps were performed according to the manufacturer’s instructions. Reverse transcription and amplification were performed on a CFX96 C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA), under the following conditions: reverse transcription at 48 °C for 15 min, initial denaturation at 95 °C for 2 min, and 45 cycles at 95 °C for 5 s and 60 °C for 40 s. A sample was considered negative a cycle threshold (Ct) ≥ 40.
Total antibody and virus neutralization assays
Assays for the measurement of SVA-specific antibody and neutralizing activity in serum were performed according to Chen et al.10. For the measurement of SVA-specific antibodies, confluent PK-15 cells on a 96-well plate were infected with SVA KS15-01 and fixed with 80% acetone the next day. Beginning with an initial dilution of 1:5 in PBS with 5% goat serum (PBS-GS), serum was diluted 1:2 on a 96-well plate. All samples were assayed on the same plate. After incubation at 37 °C for 1 h, the wells were washed with PBS, and bound antibody was detected with FITC-conjugated goat anti-porcine IgG (Novus Biologicals, Centennial, CO, USA), diluted 1:400 in PBS-GS. Titration results were reported as the last dilution of serum showing fluorescence.
For measurement of SVA neutralizing activity, serum samples were first heated to 56 °C for 30 min to inactivate complement activity. Samples (100 μL) were mixed with 100 μL of medium containing 200 TCID50 of KS15-01. After 1 h incubation at 37 °C, the wells containing the virus-serum mixture were transferred to a 96-well plate of confluent PK-15 cells and incubated at 37 °C. After overnight incubation, the cells were fixed with 80% acetone for 10 min and then stained with anti-SVA VP2-specific monoclonal antibody, mAb30-158, followed by staining with Alexa Fluor® 488 AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Inc., West Grove, PA, USA) as a secondary antibody. Neutralizing titer results were reported as the highest serum dilution showing 90% or greater inhibition of virus growth.
Statistical analysis
Ct values on Day 7 after infection for serum, feces, and nasal swabs of WT and IF pigs were analyzed by a two-tailed Student’s t-test in R (version 3.6.1). Type I error rate was controlled at a level of 0.05.
