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The Role of CD163 in the Sensitivity to the Porcine
Reproductive and Respiratory Syndrome Virus
Outline• Genetic Engineering • Porcine Reproductive &
Respiratory Syndrome Virus–Traditional Genetic Engineering
• SIGLEC1–CRISPRs
• CD163
Swine are being used extensively as biomedical models of human health
– Bioengineering– Cancer– Cardiovascular Disease/Atherosclerosis– Cognitive Behavior– Cutaneous Pharmacology/Wound Repair/Dermatology – Diabetes– Immunology– Lipoprotein Metabolism– Nutrition– Organ Transplantation– Orthopedics– Ophthalmology – Physiology– Regenerative Medicine
Adapted from Prather et al 2013
Genetic Engineering(CRISPRs, Zinc Fingers, TALENS, Homologous Recombination)
Transgene InjectionNuclease Injection
Oocyte TransductionSperm-Mediated Gene Transfer
Design & Build Constructs or
Guides
Genetic Engineering of Pigs at Missouri
• 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-/-)
• 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
Porcine Reproductive and Respiratory Syndrome (PRRS)
Animal Wellbeing Cost• 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
Economic Cost• 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.
Other Costs• Sustainability costs• Threat to food security• Psychological & emotional costs
PRRSv Infectivity Was Thought to Result from 3 Specific Entry Mediators
– Initial binding with heparan sulfate– Binding/Internalization by Sialoadhesin– Internalization/uncoating of the virus by CD163
Van Breedam et al., 2010
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
Organization of the Sialoadhesin Gene and Targeting Vector Design
Southern Probe
Prather et al ‘13 J Virology
Enucleation
Fusion or Injection and Activation
Development
Transfect or Transduce and Select
Synchronize?Nuclear TransferUS Patent #6,211,429
SIGLEC1+/- pigsBorn 1/16-18/2011 Patent Pending
Prather et al ‘13 J Virology
First SIGLEC1 -/- Pigs (F2 pigs) BornOctober 18, 2012
4 SIGLEC1 -/-
4 SIGLEC1 +/-
4 SIGLEC1 +/+ Wild Type
Prather et al ‘13 J Virology
PRRSv Challenge• 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
• Viral RNA/nuclei acidPrather et al ‘13 J Virology
PRRSV RT-PCR
SIGLEC1 Conclusions• SIGLEC1-/- is not embryonic lethal.• SIGLEC1-/- did not affect susceptibility
to PRRSv.
Prather et al ‘13 J Virology
CRISPRsClustered Regularly Interspaced Short Palindromic Repeats
• 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.
Zygote Injection
Gene Editing• 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.
Weaned wildtype and CD163 edited piglets prior to transport
to Kansas State University.
Patent Pending
Whitworth et al, ‘15Nature Biotechnology
Pigs Included in the Study
• 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 genotypesPiglet ID Predicted
TranslationMaternal 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, ‘15Nature Biotechnology
Experimental Design• 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, ‘15Nature Biotechnology
From the Beach in Florida!
http://www.scuba-diving-smiles.com/palm-tree-clipart.html
Whitworth et al, ‘15Nature Biotechnology
Clinical Signs• 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, ‘15Nature Biotechnology
Clinical Signs
Whitworth et al, ‘15Nature Biotechnology
Data provided by Maureen Kerrigan
Typical Viremia Response
Over 3000 pigs challenged at Kansas State with a response like these.
Viremia
Whitworth et al, ‘15Nature Biotechnology
Day After Infection
Antibody
Whitworth et al, ‘15Nature Biotechnology
Day After Infection
Lung Pathology
• 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, ‘15Nature Biotechnology
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 043 CD163-/- No changes 055 CD163-/- No changes 0*Lung lesion scores calculated according to Halbur et al. 8 Vet Pathol. 1995 Nov;32(6):648-60
Lung Pathology
Whitworth et al, ‘15Nature Biotechnology
Overall- Conclusions
• 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).
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
From Here to Where?• 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
Zygote InjectionsKiho LeeLee Spate
CRISPR and Targeting Vector DesignKevin WellsKristin Whitworth
Transfection and GenotypingMykel AndersonMariah ThomasJoshua Benne
Nuclear Transfer and Embryo TransferJoshua BenneStephanie MurphyJennifer TesonJiude MaoClifton Murphy
Surrogate and Piglet CareMelissa SamuelJason DowellTricia MeyerEntire Prather Lab
Funding SourcesGenus plcThe Christopher Columbus Fellowship FoundationFood for the 21st CenturyUSDA ARS
Genus plcAlan MilehamDave McLarenJon Lightner
SIGLEC1-/-
ProjectJon GreenTina Egen
Kansas State UniversityBob RowlandBenjamin TribleMaureen KerriganCatherine EwenAda Cino-OzunaBhupinder Bawa
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