Presented by Raymond (Bob) Rowland at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011

1. Surveillance and disease control
approaches for pigs and their
application to ASF
Raymond (Bob) Rowland
College of Veterinary Medicine,
Kansas State University, Manhattan, Kansas
July 21, 2011, Nairobi, Kenya

3. USDA Coordinated Agricultural Project
(PRRS CAP)
• Stakeholder driven (scientists, producers,
veterinarians)
• “Out-of-the-box” approaches to infectious
disease problem solving
• Conduct activities that are unique and not
routinely supported by existing mechanisms
• Activities converge at the control and elimination
of virus in the field
• Leverage

4. Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)
CD
Richt
AD:
Rowland
Theme 1: Vaccines
C: Richt AC: Theme 2: Detection
Garcia-Sastre C: Lipkin Theme 3: Modeling/
AC: R. Hesse Epidemiology
RVFV:
(Richt/Young/Ksiazek/Flick) RVFV/FMDV/AIV: C: Gray
(Lipkin/Palacios/Wilson) ACs: HabteMariam/Webby/Scoglio
FMDV:
(Estes/Rodriguez/ Golde) Novel Pathogens:
(Lipkin/Briese) RVFV:
AIV: (Linthicum/Habtemariam)
Field Devices:
(Garcia-Sastre/ Richt) FMDV:
(Higgins/Culbertson/
(Perez/HabteMariam/Scott)
Vaccine Platform: Anderson/Hesse)
AIV:
(Garcia-Sastre/Rowland/Ma/ Translational Partners: (Stallknecht/Gray/Webby)
Rodriguez) (Orion Biosciences Others (Data collection, GIS,
Inc./INT/ etc.):
Translational Partners: BI-Vetmedica/Akonni Inc, (Scoglio/Erickson/Anderson/
(LAH/BI-Vetmedica/Merial/ Synbiotics) Schroeder/Damon)
GenVec Inc./Bioprotection
Systems)

5. Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)
Admin. Core with Admin. Core Dir.
CD (ACD)/Emergency Response
Richt Coordinator and Internal/External
International Collaborations Committees
AD:
(Africa, Asia, Europe) National Collaborations with:
Rowland
Agriculture Industry
Federal, State, and Local Agencies
Theme 1: Vaccines Governmental Groups
C: Richt AC: Theme 2: Detection Non-governmental groups
Garcia-Sastre C: Lipkin Theme 3: Modeling/
AC: R. Hesse Epidemiology
RVFV:
(Richt/Young/Ksiazek/Flick) RVFV/FMDV/AIV: C: Gray
(Lipkin/Palacios/Wilson) ACs: HabteMariam/Webby/Scoglio
FMDV:
(Estes/Rodriguez/ Golde) Novel Pathogens:
(Lipkin/Briese) RVFV:
AIV: (Linthicum/Habtemariam)
Field Devices:
(Garcia-Sastre/ Richt) FMDV:
(Higgins/Culbertson/
(Perez/HabteMariam/Scott)
Vaccine Platform: Anderson/Hesse)
AIV:
(Garcia-Sastre/Rowland/Ma/ Translational Partners: (Stallknecht/Gray/Webby)
Rodriguez) (Orion Biosciences Others (Data collection, GIS,
Inc./INT/ etc.):
Translational Partners: BI-Vetmedica/Akonni Inc, (Scoglio/Erickson/Anderson/
(LAH/BI-Vetmedica/Merial/ Synbiotics) Schroeder/Damon)
GenVec Inc./Bioprotection
Systems)
Overlay: Outreach/
Training/Education Outreach/Training/Education:
C: Roth; AC: Montelone (Roth/Stewart/ Montelone)

6. Elimination vs. Eradication
Eradication
• Based on laws- legislated
• Government supported. Indemnify against losses.
• Draconian. Non-palatable to producers.
Elimination
• Stakeholder driven- Incentive to maintain profitability.
• Varied in scope (herd, region).
• Incorporates education and organization (sociology).
• Must be “voluntary”, can be chaotic and divisive.

7. Arteriviruses (order Nidovirales)
Porcine reproductive and respiratory syndrome virus (PRRSV)
(Type 1 and Type 2 genotypes)
Lactate dehydrogenase elevating virus (LDV)
Equine arteritis virus (EAV)
Simian hemorrhagic fever virus (SHFV)
• Enveloped
• Positive single-stranded RNA genome (10-15kb)
• During replication, produce a nested set of subgenomic mRNAs
with a common leader and poly A tail
• Macrophage-tropic
• Persistent infection and severe and fatal disease

8. PRRS
“Reproductive Failure of Unknown
Etiology”,Kerry K. Keffaber, 1989, AASP
1. Anorexia during finishing
2. Transient increase in respiration rates
3. Temperature increase (102-106)
4. Mid- to late-term abortions
5. Delay in return to heat and infertility
6. Pre-weaning mortality
7. Respiratory distress

9. Blue ear disease-Kansas 2006

10. PRRS is a swine disease cofactor
Reston ebolavirus (REBOV)
Philippines 2008
Co-Infection of pigs with
REBOV and PHFD PRRSV

11. PRRSV as a disease cofactor
Days after weaning Mortality in groups of 200
experimentally challenged pigs

12. Complex surface topology & composition
Sites for neutralization remain largely unknown
heterodimer heterotrimer
GP5 GP3
M GP2 GP4
viral
envelope
2b 5a
N
Glycosylation sites
nucleocapsid
homopolymer

13. Complex surface topology & composition
M
Minor
N GP Proteins
5 • GP2
• 2b (E)
•GP3
•GP4
•5a
Dockland-Vet Res (2010)154: 86

14. Genetic diversity
(poor heterologous protection)
Mutations in a single isolate over time
Peptide sequence variability
nsp2

15. Subversion of innate immunity by NSP1
(inhibition of the Type 1 IFN response)
• Degradation of CREB-binding protein (CBP) in the nucleus, making
it unable to form a complex with p300 and interferon regulatory
factor 3 (IRF3; Kim et al., 2010)
• Inhibition of dsRNA-induced IRF3 and IFN promoter activities (Kim
et al., 2010)
• Interaction with PIAS1 (Yoo unpublished)
• Inhibition NF-kB function (Song et al., 2010)
• Interference of RIG-I signaling (Yoo et al., 2010)
• Inhibit nuclear translocation of STAT1 (Chen et al., 2010)
nsp1α nsp1β

16. Virus properties that relate
to control and eradication
• Complex virion composition and surface topology
• Subversion of innate and adaptive immunity
• Capacity to generate a large degree of genetic diversity
in structural and nonstructural proteins
• Macrophage tropism (lymphotropism during subclinical
infection)
• Capacity to exist as a subclinical infection and cause
severe disease
• Delayed and reduced Ab neutralizing activity (no
heterologous protection)
• Co-factor with other infections (ASFV and CSFV)

17. PRRSV Ecology/Epidemiology • 60% herds infected
Neumann-2003 • $600 million/yr
Endemic infection Acute outbreaks
FAD Threats
•Feral pigs (8 million)
•Backyard farms
Vaccines as biosecurity tools
• Block introduction (antibody)
• Break endemicity (T cells and innate
responses)
•Prevent or lower shedding

18. PRRS vaccines
• Modified live virus (MLV) vaccine introduced in 1994-
suitable for infected herds
• MLV limitations-virus shedding, persistent infection,
incomplete immune protection, inability to differentiate
infected from vaccinated animals (DIVA), potential for
reversion to virulence
• Killed vaccines are not effective
• Acclimation with wild-type virus as an alternative to
vaccination

19. Population size matters
Natural Termination/Elimination/Extinction
Small SIR
Populations Susceptible Infectious Resistant
Continuous Infection
SIRS
Susceptible Infectious Resistant
Large
Populations
Loss of immunity- appearance of an escape mutant-introduction
of a new virus

20. Trends (10 year)
• Less government involvement in disease control and
eradication (government may not indemnify producers)
$20 million spent to support market prices during the
pandemic SIV outbreak covered 84 minutes of
production
How much does $20 million buy in research?
• Developing infectious disease models that are predictive
• Implementing regional approaches to PRRSV elimination

21. PRRS herd control methodologies
• Syndromics (1990)
• Test and removal (1992)
• Vaccination (1994)
• Depop-repop
• All in - all out
• Acclimation with known viruses
• Herd closure (200 days-virus extinction)
• Barn filtration (high density areas – expensive!)

22. Sow Herd Filtration Study
Scott Dee, University of Minnesota
Pipetsone, MN
Attic installation of filter boxes
Advances in biosecurity

23. Regional approach to elimination
• Herd-level strategies generally fail (virus re-enters from and
unknown source)
• For herd-level control and elimination to be sustainable, the
effort must be regional
• Sufficient tools, technologies, resources and leadership
(political will) to initiate and conduct regional scale projects
• Regional efforts will identify gaps in knowledge that future
research can fill

24. PRRS CAP regional elimination projects
•Oral fluid analysis for molecular and
immunological sureveillance
•Risk analysis tools (PADRAP)
•Risk-based testing
and surveillance
•Point of care testing
(serology and PCR)
•Sociology
•Economic cost-benefit analysis
•Vaccines
•Host genetics

25. Minnesota
(Bob Morrison, Montse Torremorell)
164,000 pigs-83 sites
2004
Infected
Unknown 2006
Free
Nursery
Sows
Finish
2010

26. Technologies to facilitate elimination
•Oral fluid analysis for molecular and
immunological surveillance
•Risk analysis tools (PADRAP)
•Risk-based testing
and surveillance
•Point of care testing
(serology and PCR)
•Sociology
•Economic cost-benefit analysis
•Vaccines
•Host genetics

27. PRRSV as dual vaccine vector
5’ UTR Nsp2-fusion protein
2b
1b 3 6
1a 2a 4 5 7 pA
GFP GFP pA
2 pA
M
3 GFP pA
4 pA
N GP5 pA
5
M pA
N pA

28. Advantages of dual vaccine
• Targets PRRSV and cofactor infection
• Insertion of Ag attenuates or inactivates wild-type virus
• Compliance and DIVA markers
• In the case of nsp2, antigen expressed as a fusion
protein-incorporated in macromolecular complexes
(replication complexes) or VLP
• PRRSV is resistant to MDA or existing antibody

29. Analysis of virus and antibodies in Oral Fluids
Jeff Zimmerman, Iowa State
Non-invasive and easy to collect
Collect daily
Population sample
Modification of existing tests (PCR and Ab)
Sensitivity

30. Circulation of three infectious agents
PRRSV, SIV, PCV2
Oral fluid testing for routine surveillance
of infectious diseases in swine populations
Jeff Zimmerman, IA State

31. Luminex- Microsphere Immunoassay
Measure Surface
Tag Florescence Green laser
Host antibody (analyte)
Measure Internal Antigen
Dye Florescence
Red laser
Each sphere is coated
with a different antigen
“multiplexing to assess quantity
and quality of immunity”

33. Microimmunoassay (MIA) Luminex
• Substitute for standard ELISA
• Can detect multiple analytes (antigens)
including native and denatured proteins,
peptides
• Interrogate host antibody response
DIVA
Targets of neutralizing antibody
Immunopathogenic responses
• Sensitivity/Specificity
• Multiple variations
• 2010 MAGPIX instrument (magnetic beads)
LED detection
Lower cost-instrument and sample prep
96 well format
Simple sample prep

34. Serum IgG and IgM responses (Luminex)
PRRSV N protein
n=16 pigs
IgG
RNA 7
12000
6
10000
5
MFI 8000 Log
4
6000
PRRSV
3
4000
2
2000 IgM 1
0 0
0 5 10 15 20 25
Day after infection

35. Oral fluid PRRSV IgG and IgM responses
(Luminex, PRRSV protein)
14000
12000
10000
8000
6000
4000
2000
0
1 3 5 7 9 11 13 15 17 19 21 23 25
IgM- anti-PRRSV N mean for 12 pens
IgG- anti-PRRSV N mean for 12 pens

36. Pig movement
Point-of-care tests
Transport
Transport Into a region
out of a region
Transport
within a region

37. Host genetics
PRRS Host Genetics Consortium (PHGC)
(CoPDs, Bob Rowland and Joan Lunney, ARS)
1. Use genotyping and phenotyping tools to determine if
there are host genes that control host response to
infection (resistance vs. susceptibility)
2. Identify relative importance of different protein markers
that predict outcomes following infection
3. Conduct “ultra-deep” phenotyping to identify gene
pathways and novel biomarkers related to virus
replication and disease

38. Nursery Pig Model
• 200 pigs (2-3 weeks of age) from different sources in
Canada and the US
• 5-10 pigs set aside as reference pigs
• 15-16 pigs per pen
• A week after arrival pigs challenged with isolate NVSL
97-7985
• Collect blood at 0, 3, 7, 10, 14, 21, 28, 35, 42 days dpi
(serum, RNA Tempus tubes)
• Weigh weekly
• Collect tonsils and ears at Day 42

39. Phenotypic data (deep phenotype)
• Morbidity and mortality
• Viremia, qRT-PCR (ABI) log PRRSV RNA templates/rxn
• Virus Load, area under curve for the 21 days
• Weight (weekly)
• Total antibody and virus neutralizing activity (42 dpi)
• Circulating cytokine levels
• Transcriptome analysis of whole blood and tonsil
(ultradeep phenotyping)

40. “Deep” phenotyping
Weight gain over time Log PRRSV RNA over time

41. Viral Load
(AUG)
Quantified as area under the curve from day 0 to 21

42. Subpopulations with unique phenotypic properties
PRRS tolerant
J.P. Steibel
Mich. St. U.

43. GWAS-Illumina Porcine 60k Beadchip

44. Genomic Association Model
k
y = Xb + ∑ z iα i δ i + ε
i =1
Where y = phenotypic observations for VL or WG
X = incidence matrix relating fixed effects to phenotypes
b = vector of fixed effects of experiment(pen) and
experiment*parity
zi = vector of genotypes at SNP i , coded 0/1/2
αi = substitution effect of SNP i
δi = indicator for whether SNP i was included (δi=1) or
excluded (δi=0) in the model for a given iteration of the
Monte Carlo Markov Chain
The prior probability of δi= 0 was set equal to pi = 0.99

45. Bayes B analysis
pi = 0.99
Viral Load (AUG) Weight Gain (ADWG)
SSC 4 SSC X
Proportion of Genetic Variance
Proportion of Genetic Variance
SSC 4
SSC 17
5-SNP window ordered by chromosome 5-SNP window ordered by chromosome

46. Jack Dekkers and Nick Boddicker

47. Integrated approach (no single solution)
• Good vaccines
• Good diagnostics
• Good understanding of ecology epidemiology
• Good surveillance approaches
• Good understanding of social psychology
• Good pig

48. Researchable Issues
• Role of PRRSV as a cofactor in ASF pathogenesis and
ecology
• Development of tools for the study of ASF in BSL-2
(pseudotyped virus system)
• Multiplex serological assays (Luminex) for investigating
ASFV immune response and cofactors
• Novel surveillance approaches (oral fluids)
• Risk assessment and biosecurity
• Role of host genes in ASF disease susceptibility,
resistance and persistence
• Transcriptome analysis to investigate virus-host
interactions during acute and subclinical infection
• Education