Dr. Randall Prather - PRRS Resistant Pigs
-
Upload
john-blue -
Category
Health & Medicine
-
view
449 -
download
0
Transcript of Dr. Randall Prather - PRRS Resistant Pigs
![Page 1: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/1.jpg)
The Role of CD163 in the Sensitivity to the Porcine
Reproductive and Respiratory Syndrome Virus
![Page 2: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/2.jpg)
Outline• Genetic Engineering • Porcine Reproductive &
Respiratory Syndrome Virus–Traditional Genetic Engineering
• SIGLEC1–CRISPRs
• CD163
![Page 3: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/3.jpg)
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
![Page 4: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/4.jpg)
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
![Page 5: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/5.jpg)
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-/-)
![Page 6: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/6.jpg)
• 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)
![Page 7: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/7.jpg)
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
![Page 8: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/8.jpg)
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.
![Page 9: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/9.jpg)
Other Costs• Sustainability costs• Threat to food security• Psychological & emotional costs
![Page 10: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/10.jpg)
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
![Page 11: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/11.jpg)
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
![Page 12: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/12.jpg)
Organization of the Sialoadhesin Gene and Targeting Vector Design
Southern Probe
Prather et al ‘13 J Virology
![Page 13: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/13.jpg)
Enucleation
Fusion or Injection and Activation
Development
Transfect or Transduce and Select
Synchronize?Nuclear TransferUS Patent #6,211,429
![Page 14: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/14.jpg)
SIGLEC1+/- pigsBorn 1/16-18/2011 Patent Pending
Prather et al ‘13 J Virology
![Page 15: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/15.jpg)
First SIGLEC1 -/- Pigs (F2 pigs) BornOctober 18, 2012
4 SIGLEC1 -/-
4 SIGLEC1 +/-
4 SIGLEC1 +/+ Wild Type
Prather et al ‘13 J Virology
![Page 16: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/16.jpg)
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
![Page 17: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/17.jpg)
• Viral RNA/nuclei acidPrather et al ‘13 J Virology
PRRSV RT-PCR
![Page 18: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/18.jpg)
SIGLEC1 Conclusions• SIGLEC1-/- is not embryonic lethal.• SIGLEC1-/- did not affect susceptibility
to PRRSv.
Prather et al ‘13 J Virology
![Page 19: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/19.jpg)
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.
![Page 20: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/20.jpg)
Zygote Injection
![Page 21: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/21.jpg)
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.
![Page 22: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/22.jpg)
Weaned wildtype and CD163 edited piglets prior to transport
to Kansas State University.
Patent Pending
Whitworth et al, ‘15Nature Biotechnology
![Page 23: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/23.jpg)
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
![Page 24: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/24.jpg)
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
![Page 25: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/25.jpg)
From the Beach in Florida!
http://www.scuba-diving-smiles.com/palm-tree-clipart.html
Whitworth et al, ‘15Nature Biotechnology
![Page 26: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/26.jpg)
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
![Page 27: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/27.jpg)
Clinical Signs
Whitworth et al, ‘15Nature Biotechnology
![Page 28: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/28.jpg)
Data provided by Maureen Kerrigan
Typical Viremia Response
Over 3000 pigs challenged at Kansas State with a response like these.
![Page 29: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/29.jpg)
Viremia
Whitworth et al, ‘15Nature Biotechnology
Day After Infection
![Page 30: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/30.jpg)
Antibody
Whitworth et al, ‘15Nature Biotechnology
Day After Infection
![Page 31: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/31.jpg)
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
![Page 32: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/32.jpg)
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
![Page 33: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/33.jpg)
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).
![Page 34: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/34.jpg)
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
![Page 35: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/35.jpg)
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
![Page 36: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/36.jpg)
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
![Page 37: Dr. Randall Prather - PRRS Resistant Pigs](https://reader034.fdocuments.in/reader034/viewer/2022042611/58f2d9581a28abb4538b4579/html5/thumbnails/37.jpg)