PRRS Resistant Pigs - Dr. Randall Prather, University of Missouri-Columbia, from the 2016 Missouri Pork Expo, February 9-10, 2016, Columbia, Missouri, USA. More presentations at http://www.swinecast.com/2016-missouri-pork-expo

2. • Genetic Engineering
• Porcine Reproductive &
Respiratory Syndrome Virus
–Traditional Genetic Engineering
• SIGLEC1
–CRISPRs
• CD163

3. – Bioengineering
– Cancer
– Cardiovascular Disease/Atherosclerosis
– Cognitive Behavior
– Cutaneous Pharmacology/Wound Repair/Dermatology
– Diabetes
– Immunology
– Lipoprotein Metabolism
– Nutrition
– Organ Transplantation
– Orthopedics
– Ophthalmology
– Physiology
– Regenerative Medicine

4. Adapted from Prather et al 2013
Genetic Engineering
(CRISPRs, Zinc Fingers, TALENS,
Homologous Recombination)
Transgene Injection
Nuclease Injection
Oocyte Transduction
Sperm-Mediated Gene Transfer
Design & Build
Constructs or
Guides

5. • Cancer (CAG-Floxed-Stop, KRASG12D, p53R167H; caCSN2-miBRCA1-RASSF1)
• Cardiovascular Disease (hFAT-1+; TEK-NOS3+; TEK-CAT+)
• Cystic Fibrosis (CFTR-/-; CFTRΔF508/ΔF508; CFTRΔF508/ΔF508, Tet-on CFTR+; CFTRΔF508/ΔF508,
FABP2-CFTR+; shRNA CFTR; MUC5AC-/-,MUC5B-/- )
• Diabetes (ssGIP-hINS+)
• Disease Resistance (SIGLEC1-/-; CD163-/-; CD163-/Δdomain 5; CD163-/-, SIGLEC1-/-;
TMPRSS2-/-; CD1D-/-)
• Enhancing Meat Quality (hFAT1+)
• Muscular Dystrophy (DMD+/Δexon 46)
• Pharmaceuticals (FIX+, VWF+, SERPINA1+; FVIII+, SERPINA1+, FURIN+)
• Regenerative Medicine (IL2RG+/-; RAG2-/-)
• Retinitis Pigmentosa (P23H RHO +)
• Spinal Muscular Atrophy (SMN+/-; SMN+/-, hSMN2+)
• Suicide Prodrug for Liver Transplantation (ALB-TK1+; AFP-CDA+)
• Tracking/Tools (CAG-eGFP+; CMV-eGFP+; ZP3-CRE; UBC-Tomato+; CAG-Tomato+;
UBC-nls eGFP+; PSMA1-eGFP fusion protein +)
• Xenotransplantation (GGTA1-/-; GGTA1-/-, CD55+; GGTA1CD55/+; GGTA1-/-, CD55+,
CD59+, ENTPD1+, THBD+; SIGLEC1-/-)

6. • The PRRS virus (PRRSv) was first detected in
the U.S. in 1987 (Keffaber et al ‘89) and in Europe in
1990 (Wensvoort et al ’91)
• PRRSv replicates in macrophages
– induce prolonged viremia and can cause
persistent infections that last for months
• It also predisposes infected pigs to other
bacterial and viral pathogens

7. • Vaccinations to date have been ineffective
– Non- neutralizing antibodies actually enhance viral
replication in alveolar macrophages
– Antibody dependent enhancement (ADE)
• Sows/Gilts
– severe reproductive failure and a high rate of late abortion
– Early farrowing
– Decreased litter size, Increased number of mummies
• Boars
– Low libido
– Fever, low sperm count
• Young and growing pigs
– Pneumonia, Diarrhea, Mortality of 12-15%
http://www.thepigsite.com/diseaseinfo/97/porcine-reproductive-respiratory-syndrome-prrs

8. • Costs $660,000,000 annually in North
America (Holtkamp et al ‘13)
• Costs €1,500,000,000 annually in
Europe (European PRRSpecive ‘15)
• Translates to ~$6,000,000 each day!
• Doesn’t include Asia.

9. • Sustainability costs
• Threat to food security
• Psychological & emotional costs

10. – Initial binding with heparan sulfate
– Binding/Internalization by Sialoadhesin
– Internalization/uncoating of the virus by CD163
Van Breedam et al.,
2010

11. Heparan Sulphate
Binding
Binding and
Internalization
by
Sialoadhesin
(SIGLEC1)
Uncoating of
the virus by
CD163
Genome Release
in endosomes
dependent on low
pH
Van Breedam et al., 2010

12. Southern Probe
Prather et al ‘13 J Virology

13. Enucleation
Fusion or
Injection and
Activation
Development
Transfect or
Transduce and
Select
Synchronize?
Nuclear
Transfer
US Patent #6,211,429

14. SIGLEC1+/- pigs
Born 1/16-18/2011
Patent Pending
Prather et al ‘13 J Virology

15. Prather et al ‘13 J Virology

16. • Piglets were shipped to Kansas State
University
• After 1 week acclimation
– Low passage PRSSV isolate (KS-06: North
American isolate collected in 2006)
– 105 TCID50 of virus diluted in 3 mL
• ½ intramuscularly
• ½ intranasally
– Blood sampled on days 0, 4, 7, 14, 21, 28 &
35.
Prather et al ‘13 J Virology

17. • Viral RNA/nuclei acid
Prather et al ‘13 J Virology

18. • SIGLEC1-/- is not embryonic lethal.
• SIGLEC1-/- did not affect susceptibility
to PRRSv.
Prather et al ‘13 J Virology

19. • Bacterical immune system that
degrades foreign genetic elements
(plasmids, phages).
• Can edit the genome at a very high
efficiency.
• Uses two main components: a guide
sequence and a Cas9 protein.

21. • Non-Homologous End Joining
• Can result in changing a handful of
base pairs
• Thus can remove exons/domains or
knockout a gene.
• Swine genome is 2,700,000,000+ base
pairs.

22. Weaned
wildtype
and CD163
edited
piglets prior
to transport
to Kansas
State
University.
Patent Pending
Whitworth et al, ‘15
Nature Biotechnology

23. • 3 piglets predicted to be null (CD163-/- )
• 8 WT piglets
– One WT piglet humanely euthanized on day 1, due to poor body
condition and not included in the study
• K-State veterinarians and staff blinded to
genotypes
Piglet ID Predicted
Translation
Maternal Allele Paternal Allele
#43 CD163-/- 7 bp addition in exon 7 2 bp deletion in exon 7 + 377
bp intron deletion in the
preceding intron
#55 CD163-/- 7 bp addition in exon 7 2 bp deletion in exon 7 + 377
bp intron deletion in the
preceding intron
#40 CD163-/- 7 bp addition in exon 7 11 bp deletion in exon 7
Whitworth et al, ‘15
Nature Biotechnology

24. • Identity blinded to the crew at KSU
• Housed in the K-State BL-2 LARC facility
• Allowed to acclimate for 3 days after arrival at facility
• Challenge IM and IN with 105 TCID50 of NVSL 97-7985.
(Standard lab isolate from 1997, relatively “hot” in terms of
replication and pathogenesis)
• Pigs were maintained in the same pen; therefore, all pigs
constantly exposed to virus
• Monitored daily for clinical signs
• Blood collected on days 0,4,7,11,14,21,28,35 and weights
collected at least weekly
• Study terminated 35 days after infection
• Lung lavage for PAMs
• Lungs and tissues removed for histopathology
Whitworth et al, ‘15
Nature Biotechnology

25. http://www.scuba-diving-
smiles.com/palm-tree-clipart.html
Whitworth et al, ‘15
Nature Biotechnology

26. • Fever was considered positive if it was ≥ 104°F
• Respiratory scores ranged from:
– 0: Normal, to
– 1: mild dyspnea and/or tachypnea when stressed
(when handled),
– 2: mild dyspnea and/or tachypnea when at rest,
– 3: moderate dyspnea and/or tachypnea when stressed
(when handled),
– 4: moderate dyspnea and/or tachypnea when at rest,
– 5: severe dyspnea and/or tachypnea when stressed
(when handled),
– 6: severe dyspnea and/or tachypnea when at rest. Is
there evidence of diarrhea (grade) or vomiting?
Whitworth et al, ‘15
Nature Biotechnology

27. Whitworth et al, ‘15
Nature Biotechnology

28. Data provided by Maureen
Kerrigan
Over 3000 pigs challenged at Kansas
State with a response like these.

29. Whitworth et al, ‘15
Nature Biotechnology
Day After Infection

30. Whitworth et al, ‘15
Nature Biotechnology
Day After Infection

31. • CD163+/+ pigs had interstitial edema with the infiltration of
mononuclear cells and the mononuclear infiltrate consisted
mainly of lymphocytes and plasma cells and lesser numbers of
macrophages. In contrast there was no evidence for pulmonary
changes in the CD163-/- pigs.
Whitworth et al, ‘15
Nature Biotechnology

32. Pig Genotype Description Score*
41 Wild Type 100% congestion. Multifocal areas of edema. Infiltration of moderate numbers
of lymphocytes and macrophages.
3
42 Wild Type 100% congestion. Multifocal areas of edema. Infiltration of moderate numbers
of lymphocytes and macrophages.
3
47 Wild Type 75% multifocal infiltration with of mononuclear cells and mild edema. 2
50 Wild Type 75% moderate infiltration of mononuclear cells within alveolar spaces and
around small blood vessels. Perivascular edema.
3
51 Wild Type 25% atelectasis with moderate infiltration of mononuclear cells. 1
52 Wild Type 10% of alveolar spaces collapsed with infiltration of small numbers of
mononuclear cells.
1
56 Wild Type 100% diffuse moderate interstitial infiltration of mononuclear cells.
Interalveolar septae moderately thickened by hemorrhage and edema.
4
40 CD163-/- No changes 0
43 CD163-/- No changes 0
55 CD163-/- No changes 0
*Lung lesion scores calculated according to Halbur et al. 8 Vet Pathol. 1995 Nov;32(6):648-60
Whitworth et al, ‘15
Nature Biotechnology

33. • SIGLEC1-/- pigs are not resistant to
PRRSv.
• CD163 -/- pigs are resistant to PRRSv.
• Working on challenging other
genotypes.
• Working on challenging with other
isolates (multiple Type 1 and Type 2).

34. Heparan Sulphate
Binding
Binding and
Internalization
by
Sialoadhesin
Uncoating of
the virus by
CD163
Genome Release
in endosomes
dependent on low
pH
CD163 is a Gatekeeper!
Van Breedam et al., 2010

35. • Technology Permits Answering Basic
and Applied Questions about Human
Medicine and Domestic Animal
Biology
• Any Genetic Modification that you can
imagine, we can probably build.
– Miniature Pig
– Domestic Pig
• CD163-/- Entry Into Production
– Growth and Performance

36. Zygote
Injections
Kiho Lee
Lee Spate
CRISPR and Targeting Vector
Design
Kevin Wells
Kristin Whitworth
Transfection and
Genotyping
Mykel Anderson
Mariah Thomas
Joshua Benne
Nuclear Transfer and Embryo
Transfer
Joshua Benne
Stephanie Murphy
Jennifer Teson
Jiude Mao
Clifton Murphy
Surrogate and Piglet Care
Melissa Samuel
Jason Dowell
Tricia Meyer
Entire Prather Lab
Funding Sources
Genus plc
The Christopher Columbus Fellowship
Foundation
Food for the 21st Century
USDA ARS
Genus plc
Alan Mileham
Dave McLaren
Jon Lightner
SIGLEC1-/-
Project
Jon Green
Tina Egen
Kansas State University
Bob Rowland
Benjamin Trible
Maureen Kerrigan
Catherine Ewen
Ada Cino-Ozuna
Bhupinder Bawa