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 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