John Harding, Andrea Ladinig, Lyn Ashley, Joan Lunney, Jamie Wilkinson, Tianfu Yang, Predrag

Novakovic, Susan Detmer, Graham Plastow

PRRSV and the pregnant female Genomics and Swine Health, Banff Pork Seminar

January 13-14, 2016

Reproductive effects of PRRSV • PRRS associated with over $660M annually losses: 45%

due to reproductive disease (Holtkamp, 2013)

• Reproductive failure in late gestation • Late-term abortions, early farrowing, increase of dead and mummified

fetuses and weak-born piglets

• PRRSV replication in endometrium associated with Sialoadhesin and CD163-positive macrophages (Karniychuk et al. 2011)

• Not all fetuses are infected at same time, some may escape infection (Rowland et al. 2003)

• PRRSV replicates in different fetal tissues, proposed thymus primary site of virus replication (Rowland 2010)

Karniychuk et al. 2013

PRRSV attached to maternal macrophages adherent to endothelial wall

PRRSV replicates in maternal macrophages and crosses uterine epithelium fetal allantochorion

Efficient PRRSV replication in fetal macrophages, travels to fetus via umbilical

circulation

Multifocal to complete detachment and degeneration of placenta

Open questions & opportunities • Mechanism of PRRSV-induced reproductive failure and

fetal mortality are poorly understood • Other mechanisms than apoptosis at fetal implantation sites? • Does the dam’s immune response contribute? • Any direct or indirect viral effects on fetal or placental health?

• Can we identify replacement females that are genetically resistant/resilient to PRRSV? • Does WUR10000125 confer resilience to reproductive PRRSV?

• Are there specific phenotypic responses/characteristics that are associated with improved resistance/resilience • Factors/knowledge for management of PRRS outbreaks or

development of new therapeutics/vaccines

Objectives • Improve understanding of the pathophysiology and

immunology of PRRSV infection in 3rd trimester pregnant gilts • Mechanism of transplacental passage • Mechanisms of fetal death and viral load

• Determine genotypic and phenotypic predictors of reproductive PRRSV severity • Characterize phenotypic response following infection • 60K genotyping – sires, dams, fetuses

• Determine associations between phenotypic responses to PRRSV and birth weight of dam and fetuses

PRRSV Pregnant Gilt Model • 133 pregnant purebred Landrace gilts

• blocked by dam’s litter birth weight • 19 sham controls, 114 PRRSV-infected

• Sourced high health: free of PRRSV

• Vaccinated against: Parvo, Lepto, Ery, PCV2

• Subdivided into 12 groups (3-15/group): E2W

• Bred with homospermic semen from 26 Yorkshire boars • 5 litters/boar (target 6)

• All gilts and non-autolysed fetuses genotyped using Illumina SNP60 BeadChip

• PRRSV 105 TCID50 (IM/IN) 97-7895 day 85 gestation

Fetal/conceptus outcomes (GD106)

Fetal development (viable only) • morphometrics • organ/brain ratios

Preservation: • viable mummy • spacial arrangements

PRRS assessments: • qPCR • fetal cytokines • histopathology • PRRS sequencing • transcriptome • DNA (SNPs)

(Gestation) GD80 GD85 GD87Fri GD91 GD106

Day-5Fri

Day2Fri

Day0Wed

Day21Wed

Day6Tue

GD104

Day19Mon

Clinical assessments (abortion, temp, respiratory, lethargy)

PRRS qPCR (dam serum & tissues) Cytokines, Transcriptome, WBC subsets, pathology

PBMC stimulation (PMA-I, PRRS): Cytokines, Transcriptomics

Collection of BALF/cells Kinome analysis (Napper)

Macrophage function (Yates)

PRRS inoculation or

sham CTRL

Termination

Transportation from farm to USask

3-15 gilts (x12 reps)

Selection on BW, gilt development, synchronize, breeding DNA collection

• dam (LR) • sire (Y; 4 gilt per)

Experimental design & outcomes

Analyses & results Phenotypes • Viral load • Fetal location • Immune responses • Birth weight & IUGR • Pathology (uterus, fetus) • Phenotypic predictors

Genotypic predictors • GWAS • Viral load (fetus, endometrium) • Fetal death, viability Transcriptomics • Fetuses (disease progression) • Susceptible / resilient gilts • Susceptible / resilient fetuses

Metabolomics • Fetuses (disease progression) • Susceptible / resilient gilts • IUGR fetuses

Variability in gilt viremia

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 2 6 21

PRRS

RN

A co

ncen

trat

ion

(log 10

per

µL)

days post inoculation

Variation in PRRS RNA concentration in gilt serum following inoculation (dpi 0, 2, 6, 19)

>100

0 fo

ld v

aria

tion

n=113

Variability in body weights of viable fetuses

Weight not associated with RNA concentration: • Fetal thymus and/or serum • Uterus/placenta

Ctrl: 1204 (±36) g Inoc: 999 (±21) g P<0.001

Variability in gilt susceptibility

Percent dead

6.0

Variability in fetal presentation

G57L

L8 L7 L6 L5 L4 L3 L2 L1

1.4

41

010203040506070

Control PRRS

% Dead (Gilt Level)

98%

1% 1%

VIAMECDECAUT

50%

9% 8%

33%

Variability in fetal preservation Control

Viable (VIA)

Meconium stained (MEC)

Decomposed (DEC)

Autolysed (AUT)

PRRS

Legend: INOC=1 CTRL=0 Preservation: 1=viable 1.5=meconium

stained (viable) 2=decomposed 3=autolysed 4=mummy <20cm (died < inoculation)

STATION 7 FETAL PRESERVATION SCORE ID_gilt Grp Inoc_Ctrl L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 R12 R11 R10 R9 R8 R7 R6 R5 R4 R3 R2 R1 Comments

16 1 1 1,5 3 1 3 2 1 1 1 1 3 2 1 1 1 17 1 1 1 1 1 1 1 1,5 1,5 1 3 1 1 1 1 3 3 18 1 1 3 2 3 3 3 2 2 3 3 3 2 3 3 3 2 2 3 1,5 19 1 1 3 2 1 1 1 1 1 1 3 3 3 1 1,5 3 2 3 20 1 1 3 2 1,5 2 1,5 1 3 3 3 3 3 21 1 1 1 1 1 1 1 3 3 1 1 3 3 3 1 22 1 0 1 1 1 1 1 1 1 1 1 23 1 1 Not pregnant 24 1 1 3 3 2 1 1 1 1 2 2 1 1 3 1 25 1 1 3 1 1 3 1 3 1 1 1 1 1 1 3 2 3 3 3 26 1 1 1,5 2 3 2 4 3 2 3 3 3 3 1 2 2 27 2 1 1 1,5 1 1 4 3 1 1 2 1,5 3 3 28 2 1 1 2 1 1 3 1 1 1 No left horn 29 2 0 1 1 1 1 1 1 1 1 1 1 30 2 1 1 1 1 1 1 1 3 1 1 1 3 3 1,5 3 3 31 2 1 3 3 3 3 4 1 1 3 3 3 1 3 3 1 1 3 3 3 32 2 1 4 4 1 1 1 1 1 3 3 3 1 1 3 1 1 1,5 33 2 1 3 1 1,5 2 1 3 1 1 1 3 1 3 1 1 1 1 1 1 34 2 1 3 1 1 2 3 1 1 1 1 3 3 3 3 1 1 35 2 1 1 1 1 1 1 1,5 1 1 1 1 1 1 1 1 1 1 1 36 2 1 1,5 3 1,5 1,5 1 3 2 1,5 3 3 3 37 2 1 Died post inoculation - PRRS 11dpi 38 3 1 1,5 3 3 1 1 3 2 1 1 1 3 3 39 3 1 1 1 1 1 4 4 1 3 1 2 2 1 40 3 1 2 2 3 3 3 4 4 1 1,5 1,5 1 1,5 3 1 1 1 1 1 41 3 1 3 3 3 3 3 3 4 1 1 1 1 1 42 3 1 3 3 3 3 1 1 1 1 43 3 1 2 1,5 1 1 4 4 3 3 3 3 1 3 1 1 1 3 1 44 3 1 3 1,5 1,5 3 1,5 3 3 3 3 3 1 1 1,5 45 3 0 1 1 1 1 1 1 1 1 1 1 1 1 46 3 1 1 1 1 1 1 1 1 No left horn 47 3 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 48 3 1 1 1 1 1 4 1 1 1 1 49 3 1 1 1 1 1 1 1 3 1 3 2 1 1 1 1,5 2 50 4 1 1 3 3 3 3 2 1,5 3 3 2 51 4 1 1 1 1 2 1 1 1 3 1 1 1 1 1 1 1 52 4 1 1 1 1 1 1 1 1 1 1 53 4 1 1 1 1 1 1 1 1 1 2 3 1 3 1 1 1 1 54 4 1 3 1 1,5 3 1 2 3 3 1 3 3 55 4 1 1 1 1 1 1 1 1 1 56 4 1 3 1 1 1 3 3 1 1 1 1 1 1 1 1 1 1 3 57 4 1 3 2 1 1 2 1 4 1 4 3 1 2 1 3 3 3 2 58 5 1 3 3 2 1 1,5 3 1,5 3 3 1 1 59 5 1 3 1,5 3 1,5 3 3 3 3 3 1,5 3 3 3 3 1 60 5 1 1 1 1 1 1 3 3 1,5 4 4 1 1 1 1 1 1 1 61 died 0dpi . died - cardiomyopathy 0dpi 62 5 1 3 3 1,5 1 2 3 3 3 3 3 3 1 1 3 4 4 1 1 1 63 5 1 1,5 2 3 3 1 1 1 1 3 3 2 1,5 64 5 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 65 5 1 3 1,5 3 1,5 1 1 1 1 1 1 3 1 1,5 3 66 5 1 1,5 3 1 1 3 1,5 3 1,5 1 1 1 1 1 3 1 3 67 5 0 1 1 1 1 1 1 1 1 1 1 1 1 1 68 5 1 1 1 3 3 1 1 1 1,5 1,5 3 69 5 0 1 1 3 1 1 1 1 1 1 4 1 1 70 5 1 1 1 3 1 1 1 1 1 1,5 2 3 1,5 1 71 6 1 1,5 1 2 3 3 3 1 2 3 1,5 1 1,5 1,5 3 2 3 3

72 6 1

Aborted: 16 fetuses (#1-8 placed on left, #916 on right)

73 6 1 1 1 1 1 1 3 3 1 1 2 3 3

Variability in spatial clustering with uterus

Large variation in fetal (litter) susceptibility

Proportion of litters that are PRRS positive in thymus or serum

Conclusions (so far) 1. Fetal mortality and viral load cluster in uterus 2. Dramatic decrease in all WBC subsets by 2 dpi 3. BW dam not related to PRRSV severity or fetal outcome 4. IUGR fetuses appear to be less susceptible than large fetuses 5. Most important phenotypic predictors of fetal death and viral load: Viral load at maternal fetal interface Number adjacent dead and PCR positive fetuses Presence of virus in fetus (fetal death)

6. GWAS identified a number of genomic markers associated with reproductive outcome (fetal death, fetal viral load) Not associated with WUR10000125 on SSC4

7. Transcriptomic analyses identified a number of differentially expressed genes associated with: Early immune responses in gilts with low fetal mortality rate Host responses in endometrium & fetus potentially associated with fetal

and placental compromise 8. Metabolomic analyses promising preliminary results – analyses ongoing

PRRSV resilience – ongoing work 1. Genotyping and GWAS on autoysed fetuses 2. Additional transcripomic analyses: (resistant /susceptible fetuses) 3. Metabolomic analyses of gilts and fetuses associated to identify profiles

associated with resistance and disease progression 4. New experiment with earlier termination point: How and when virus crosses placenta (uterine glands/areolae) Endometrial and fetal immune responses associated with resistance Validation of genomic biomarkers Epigenetic mechanisms

5. Evaluation and potential adoption of genetic biomarkers into breeding programs

Acknowledgements and Funders

• Fast Genetics

• VIDO animal care + research scientists (Drs. Wilson, Gerdts, Walker)

• Prairie Diagnostic Services

• WCVM molecular lab

• Animal care unit, WCVM

• Bob Rowland

• Jack Dekkers

• Suzy Carman

• Many laboratory staff & students

Funding