Surveillance and disease control approaches for pigs and their

application to ASF

Raymond (Bob) RowlandCollege of Veterinary Medicine,

Kansas State University, Manhattan, KansasJuly 21, 2011, Nairobi, Kenya

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

CDRicht

AD:Rowland

Theme 1: Vaccines C: Richt AC:

Garcia-SastreTheme 2: Detection

C: LipkinAC: R. Hesse

Theme 3: Modeling/Epidemiology

C: GrayACs: HabteMariam/Webby/Scoglio

Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)

RVFV: (Richt/Young/Ksiazek/Flick)

FMDV: (Estes/Rodriguez/ Golde)

AIV: (Garcia-Sastre/ Richt)

Vaccine Platform: (Garcia-Sastre/Rowland/Ma/

Rodriguez)

Translational Partners:(LAH/BI-Vetmedica/Merial/ GenVec Inc./Bioprotection

Systems)

RVFV/FMDV/AIV: (Lipkin/Palacios/Wilson)

Novel Pathogens:(Lipkin/Briese)Field Devices:

(Higgins/Culbertson/Anderson/Hesse)

Translational Partners:(Orion Biosciences

Inc./INT/BI-Vetmedica/Akonni Inc,

Synbiotics)

RVFV: (Linthicum/Habtemariam)

FMDV: (Perez/HabteMariam/Scott)

AIV: (Stallknecht/Gray/Webby)

Others (Data collection, GIS, etc.):

(Scoglio/Erickson/Anderson/Schroeder/Damon)

CDRicht

AD:Rowland

Admin. Core with Admin. Core Dir. (ACD)/Emergency Response

Coordinator and Internal/External Committees

Theme 1: Vaccines C: Richt AC:

Garcia-SastreTheme 2: Detection

C: LipkinAC: R. Hesse

Theme 3: Modeling/Epidemiology

C: GrayACs: HabteMariam/Webby/Scoglio

Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)

RVFV: (Richt/Young/Ksiazek/Flick)

FMDV: (Estes/Rodriguez/ Golde)

AIV: (Garcia-Sastre/ Richt)

Vaccine Platform: (Garcia-Sastre/Rowland/Ma/

Rodriguez)

Translational Partners:(LAH/BI-Vetmedica/Merial/ GenVec Inc./Bioprotection

Systems)

RVFV/FMDV/AIV: (Lipkin/Palacios/Wilson)

Novel Pathogens:(Lipkin/Briese)Field Devices:

(Higgins/Culbertson/Anderson/Hesse)

Translational Partners:(Orion Biosciences

Inc./INT/BI-Vetmedica/Akonni Inc,

Synbiotics)

International Collaborations(Africa, Asia, Europe)

RVFV: (Linthicum/Habtemariam)

FMDV: (Perez/HabteMariam/Scott)

AIV: (Stallknecht/Gray/Webby)

Others (Data collection, GIS, etc.):

(Scoglio/Erickson/Anderson/Schroeder/Damon)

National Collaborations with:Agriculture Industry

Federal, State, and Local AgenciesGovernmental Groups

Non-governmental groups

Overlay: Outreach/ Training/EducationC: Roth; AC: Montelone

Outreach/Training/Education: (Roth/Stewart/ Montelone)

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.

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

PRRS“Reproductive Failure of Unknown Etiology”,Kerry K. Keffaber, 1989, AASP1. Anorexia during finishing2. Transient increase in respiration rates3. Temperature increase (102-106)4. Mid- to late-term abortions5. Delay in return to heat and infertility6. Pre-weaning mortality7. Respiratory distress

Blue ear disease-Kansas 2006

PRRS is a swine disease cofactorReston ebolavirus (REBOV)

Philippines 2008

Co-Infection of pigs with REBOV and PHFD PRRSV

PRRSV as a disease cofactor

Days after weaning Mortality in groups of 200 experimentally challenged pigs

MGP5

N2b

GP2GP3

GP4

heterotrimer

viralenvelope

Glycosylation sitesnucleocapsid homopolymer

heterodimer

5a

Complex surface topology & compositionSites for neutralization remain largely unknown

Dockland-Vet Res (2010)154: 86

Minor Proteins• GP2• 2b (E)•GP3•GP4•5a

N

M

GP5

Complex surface topology & composition

Genetic diversity (poor heterologous protection)

Pep

tide

seq

uenc

e va

riabi

lity

Mutations in a single isolate over time

nsp2

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

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)

PRRSV Ecology/EpidemiologyNeumann-2003

• 60% herds infected• $600 million/yr

FAD Threats•Feral pigs (8 million)•Backyard farms

Endemic infection Acute outbreaks

Vaccines as biosecurity tools• Block introduction (antibody)• Break endemicity (T cells and innate responses)•Prevent or lower shedding

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

Large Populations

Loss of immunity- appearance of an escape mutant-introduction of a new virus

Continuous Infection

Natural Termination/Elimination/Extinction

Susceptible Infectious Resistant

Susceptible Infectious ResistantSIRS

SIRSmall Populations

Population size matters

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

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!)

Sow Herd Filtration StudyScott Dee, University of Minnesota

Pipetsone, MN

Attic installation of filter boxes

Advances in biosecurity

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

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

Minnesota(Bob Morrison, Montse Torremorell)

164,000 pigs-83 sites

InfectedUnknownFreeNurserySowsFinish

2004

2006

2010

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

pA2pA3pA4pA5pAM

2a

2b3

4 56

7

5’ UTR

pA1a1b

pAN

pAGFP GFP

PRRSV as dual vaccine vector

N

M

GP5

GFP

Nsp2-fusion protein

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

Analysis of virus and antibodies in Oral Fluids Jeff Zimmerman, Iowa State

Non-invasive and easy to collectCollect dailyPopulation sampleModification of existing tests (PCR and Ab)Sensitivity

Circulation of three infectious agentsPRRSV, SIV, PCV2

Oral fluid testing for routine surveillance of infectious diseases in swine populations

Jeff Zimmerman, IA State

Measure SurfaceTag Florescence Green laser

Red laserMeasure Internal Dye Florescence

AntigenHost antibody (analyte)

Luminex- Microsphere Immunoassay

Each sphere is coated with a different antigen“multiplexing to assess quantity and quality of immunity”

Microimmunoassay (MIA) Luminex• Substitute for standard ELISA• Can detect multiple analytes (antigens)

including native and denatured proteins, peptides

• Interrogate host antibody responseDIVA

Targets of neutralizing antibodyImmunopathogenic responses

• Sensitivity/Specificity• Multiple variations

• 2010 MAGPIX instrument (magnetic beads)LED detectionLower cost-instrument and sample prep96 well formatSimple sample prep

Serum IgG and IgM responses (Luminex)PRRSV N protein

n=16 pigs

0 5 10 15 20 250

1

2

3

4

5

6

7

2000

4000

6000

8000

10000

12000

0

MFI LogPRRSV

Day after infection

RNAIgG

IgM

Oral fluid PRRSV IgG and IgM responses(Luminex, PRRSV protein)

0

2000

4000

6000

8000

10000

12000

14000

1 3 5 7 9 11 13 15 17 19 21 23 25

IgM- anti-PRRSV N mean for 12 pensIgG- anti-PRRSV N mean for 12 pens

Pig movementPoint-of-care tests

Transportwithin a region

Transportout of a region

TransportInto a region

Host geneticsPRRS 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 markersthat predict outcomes following infection

3. Conduct “ultra-deep” phenotyping to identify gene pathways and novel biomarkers related to virus replication and disease

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

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)

“Deep” phenotyping

Weight gain over time Log PRRSV RNA over time

Viral Load(AUG)

Quantified as area under the curve from day 0 to 21

Subpopulations with unique phenotypic properties

PRRS tolerant

J.P. SteibelMich. St. U.

GWAS-Illumina Porcine 60k Beadchip

Genomic Association Model

y = Xb + ziα ii=1

k

∑ δ i +ε

Where y = phenotypic observations for VL or WGX = incidence matrix relating fixed effects to phenotypesb = vector of fixed effects of experiment(pen) and

experiment*parityzi = vector of genotypes at SNP i , coded 0/1/2αi = substitution effect of SNP iδi = 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

Bayes B analysispi = 0.99

Prop

ortio

n of

Gen

etic

Var

ianc

e

Prop

ortio

n of

Gen

etic

Var

ianc

e

5-SNP window ordered by chromosome5-SNP window ordered by chromosome

Viral Load (AUG) Weight Gain (ADWG)

SSC 4SSC 4

SSC X

SSC 17

Jack Dekkers and Nick Boddicker

Integrated approach (no single solution)

• Good vaccines• Good diagnostics• Good understanding of ecology epidemiology• Good surveillance approaches• Good understanding of social psychology • Good pig

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