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Andrew Wargo
Virginia Institute of Marine Science
College of William and Mary
http://wmpeople.wm.edu/arwargo
Impacts of Disease Management Practices on the Virulence Evolution and
Transmission of a Salmonid Virus
Infectious Hematopoietic Necrosis Virus Negative-sense single-
stranded RNA virus
Primary disease trout farming
System
Endemic salmonid fishes Pacific Coast
Family - Rhabdoviridae
Acute disease
IHNV
Waterborne Transmission
Occurs in wild and cultured fish
Disease Management Practices
Culling
Vaccination
Virulence and Transmission?
What is Culling?• Euthanize entire host population when
mortality reaches certain threshold
Culling and Virulence?Low Virulence High Virulence
Culling Threshold = 30% total population
20 % Die 80 % Die
High VirulenceLow VirulenceCulling and Virulence?
Exposure: Batch immersion Isolate fish in individual tanks
Treatments – (20 fish ea.) Sample water daily (30 days)Flush virus after sampling
Quantify viral RNAGenotype specific qPCR
IHNV Experiments
Allow for water flow each tank
Low Virulence Genotype: LV
High Virulence Genotype: HV
IHNV Data
0123456789101112131415161718192021222324252627282930
0%
20%
40%
60%
80%
100% Cumulative MortalityLVHV
CullingThreshold
0 5 10 15 20 25 300
5
10
15
20Number Fish Shedding
LVHV
Nu
mb
er
of
fish
Day post-exposure
Day post-exposure
0 5 10 15 20 25 300
2
4
6
8
LV aloneHV alone
Day post-exposure
Total Virus Shed
Log(
Viru
s/m
l H2O
)
HV WINS!
0123456789101112131415161718192021222324252627282930
0%
20%
40%
60%
80%
100% Cumulative MortalityLVHV
CullingThreshold
0 5 10 15 20 25 300
5
10
15
20Number Fish Shedding
LVHV
Nu
mb
er
of
fish
Day post-exposure
Day post-exposure
IHNV Data
0 5 10 15 20 25 300
2
4
6
8
LV aloneHV alone
Day post-exposure
Total Virus Shed
Log(
Viru
s/m
l H2O
)
WHO WINS?
S IvIH IL
Transmission Rate
Supply Rate
Culling and Virulence Mathematical Model
τvσ
0 5 10 15 20 25 300
2
4
6 Mean Daily Virus Shed
LVHV
Day post-exposure
Log(
viru
s/m
l H2O
)
V =
S Iv RIH IL
Transmission Rate
Recovery Rate
Supply Rate
τv ρvσ
0 5 10 15 20 25 300
5
10
15
20
LVHV
Num
ber
of f
ish
Number of Fish Shedding
Day post-exposure
V =
Culling and Virulence Mathematical Model
S Iv RIH IL
Transmission Rate
Recovery Rate
Death Rate
Supply Rate
τv ρvδV
σδ δ
0123456789101112131415161718192021222324252627282930
0%
40%
80%LVHV
Per
cent
Mor
talit
y
Day post-exposure
Cumulative Mortality
V =
Culling and Virulence Mathematical Model
Aquaculture Model
S I R
S I R
S I R
S I R
Migration Rate
Raceway 2
4 … Z3
1
μμ
μ
S I R
S I RS I R
Migration Rate
Raceway 2
4 … Z3
1
μμ
μS= 0I= 0R= 0
Aquaculture ModelCulling
Culling Model Results
No Culling
Log(
Num
ber
of F
ish) Susceptible
Recovered
Infected HV
Infected LV
0
1
2
3
4
5
0 50 100 1500
1
2
3
4
5
50 100 1500
Culling (Threshold= 30%)
Day Day
Culling selects for low virulence
Evolution of decreased virulence predicted
Culling
Vaccination
Virulence and Transmission?
Disease Management Practices
Vaccination and Transmission• Vaccine protection is heterogeneous• Typical vaccine trial very homogeneous
– One host population– One pathogen exposure dose– Quantify protection against disease only– Infection and transmission rarely considered
• Homogeneous trials mask protection heterogeneity
• The shape of protection heterogeneity may have major epidemiological impacts
Vaccine Protection: Heterogeneity Distribution
50% Efficacy
Susceptibility0 10.5
Pro
port
ion
of H
osts “Leaky”
“All or Nothing”
Vaccine Protection Heterogeneity:Prevalence
All or Nothing
Leaky
Exposure 2Exposure 1
All or Nothing
Leaky
Exposure 3Exposure 1
Vaccine Protection Heterogeneity:Prevalence
Gomes et. al., Plos Pathogens, 2014
Vaccine Protection Heterogeneity:Experiments
Vaccinated Sham: PBS
Isolate Fish
Pathogen dosages: 0,101,102,103,104,105,106
Measure-Mortality-Percent infected-Viral shedding
Vaccine Protection Heterogeneity:Results
0102030405060708090
100VaccinatedUnvaccinated
Pe
rce
nt
mo
rta
lity
101 102 103 105104 1060Virus Exposure Dose (pfu/ml)
Proportion SheddingCumulative Mortality
Quantity Virus Shed
Pro
port
ion
Infe
cted
Den
sity
Challenge Dose
Susceptibility
Vaccine Protection Heterogeneity:Transmission Model Framework
S = # Susceptibleλ = transmission rate W = virus concentration
I = # InfectiousR = # RecoveredD = # Deadf = proportion recoverα = shedding rate δ = virus removal rate
Culling
Vaccination
Virulence and Transmission?
Disease Management Practices
No transmission
Vaccinate TransmissionVirulence can evolve upwards
(Gandon et. al., Nature, 2001)
Non-sterilizing
Vaccination and Virulence Evolution
(Gimeno, Vaccine, 2008)
When vaccine allows transmission
Problematic
Incomplete vaccine coverage
1 2 3 4 50
2
4
6
8
Fish
Lo
g(V
iru
s/g
fis
h)
1 2 3 4 50
2
4
6
8
FishL
og
(Vir
us/
g f
ish
)
Vaccination and Transmission:IHNV Vaccine Trial
Unvaccinated Fish Vaccinated Fish
5% Mortality67% Mortality
(Kurath and LaPatra, unpublished)
No transmission
Vaccinate Transmission
When vaccine allows transmission
Virulence evolves upwards (Gandon et. al., Nature, 2001)
Non-sterilizing
(Gimeno, Vaccine, 2008)
Problematic
Incomplete vaccine coverage
Vaccination and Virulence Evolution
Incomplete Vaccine Coverage
Vaccine Virulence Evolution:Laboratory Experiments
P1 P2 P1 P2 P3P2
Vaccinate
Single
Mix
Vary: Level and timing of treatment
Quantify: Genotypic and phenotypic evolution
S Iv RIH IL … N types
Transmission Rate
Recovery Rate
Death Rate
Supply Rate
Vaccine Induced Virulence Evolution: Model Exploration
τv ρvδVδ δσ
Selective Breeding
Virulence
Co-Infection
Harnessing Evolution to Manage Disease?
IHNV BCWD
THANKS!
Rachel Breyta
Doug McKenney
Gael Kurath
Alison Kell
Tarin Thompson
Gabriella Gomes
Marc Lipsitch
Funding: NIH EEID, USDA NIFA, NSF, USGS, UW, VIMSGreg Wiens
Bill Batts
Jim Winton
MichellePenaranda
Ben Kerr
Maureen Purcell Jake ScottShannon LaDeau
Paige Barlow
Darbi Jones Barb Rutan
Jessie Viss
Questions?
Culling Model Explorations• Impact of virus migration rates (biosecurity)
• Impact of culling threshold
• Timing of strain invasion
S I R μ S I R
0123456789101112131415161718192021222324252627282930
0%
40%
80%Cumulative Mortality
CullingThreshold
Virulence Evolution in the FieldIHNV Database (WFRC)
Investigations• Virulence
• Fitness• Ecological Drivers
Genomic and Epidemiological data
Individual Fish Variation(Mixed Infections)
High levels fish-to-fish variation
Mimic natural infections in the field
12345678910
11
12
13
14
15
16
17
18
19
20
21
22
4
6
8
10
12 HVLV
Immersion
Log(
viru
s/g
fish)
Fish
Viral load in host
1 2 3 4 5 6 7 9 10
11
12
14
15
16
17
19
20
21
22
4
6
8
10
12
Log(
viru
s/g
fish)
Fish
InjectionViral load in host
12345678910
11
12
13
14
15
16
17
18
19
20
21
22
4
6
8
10
12
Log(
viru
s/m
l H2O
)
Fish
ShedViral load in water
(Wargo and Kurath, Virus Res., 2012)
Drivers of Variation
Series1100
200
300
400
500
600
Coe
ffic
ient
of
Var
iatio
n (%
)
Immersion IsogenicImmersion
Injection
Transmission
P2 P1P1
0 5 10 15 20 25 300
2
4
6 Mean Daily Virus Shed LV aloneHV alone
Day post-exposureLog(
viru
s/m
l H2O
)
Correlation Virus Shed and Virus Within Fish
- Positive correlation in-host viral load and shedding (R2 = 0.76)
- HV more efficient at Shedding
4 5 6 7 8 9 10 112
4
6
8 HV
LV
HV Fit Line
LV Fit Line
(Log[Virus copies/g fish])
(Lo
g[V
iru
s c
op
ies
/ml
H2
O])
Within Host Viral load
She
d V
iral l
oad
ID 50 ResultsPeak Viral Load
500 1000 2500 5000 7500 10000 1000004
5
6
7
8
LVHV
All
nega
tive
Virus Dosage (pfu/ml)
Infected Fish Only
Log(
viru
s/g
fish)
Exposure dosage does not impact peak viral load
ID50 & LD50Experimental Design
Vary Dosage
Day 3 Viral Load
Quantification (ID50)
1 Hour Exposure
Track Mortality 30 days (LD50)
ID50 Results % Fish Infected
ID50 HV: ~900 pfu/mlID50 LV: ~7500 pfu/ml
500 1000 2500 5000 7500 10000 1000000
20
40
60
80
100
LVHV
Virus Dosage (pfu/ml)
% F
ish
Infe
cted
LD50 Results
1051041030
20
40
60
80
100
HVLV
HV LD50: ~103 pfu/mlLV LD50: ~ 105 pfu/ml
% M
orta
lity
Virus Dosage (pfu/ml)
Percent of Infected Fish That Die
10^4 10^50%
20%
40%
60%
80%
100%HV
LV
10 4 10 5
Virus Dosage (pfu/ml)
% M
orta
lity
HV kills more fish that it infects
LD50/ID50 and Tradeoff Hypothesis?
Virulence comes with infectivity benefit
Virulence
Infe
ctiv
ity
More virulent genotypes kill larger proportion hosts, but mortality occurs after shedding subsides,
so cost is minimal
Tra
nsm
issi
on
Dur
atio
n
Virulence
?
Virulence and Other Fitness Traits
Superinfection (Grad student Alison Kell)
Dosage to kill & infect 50% of fish – LD 50 & ID 50 (Undergraduate Doug McKenney)
Superinfection
Ability of virus genotype to establish infection in host with a prior established infection by another genotype
1 exposure 2 exposure
HV LV
LV HV
HV mock
LV mock
mock LV
mock HV
mock mock
Mixed
Single
Superinfectiongroups
Primary exposure control
Secondary exposure control
Uninfected Control
1 exposure 2 exposure Interval 3 days
Harvest
Intervals
12 hours 24 hours 48 hours 96 hours 168 hours
Superinfection Experiments
Percent Fish Superinfected
Kell, AM, et. al., Journal of Virology, 2013
0 hrs co-infec-
tion
12 hrs 24 hrs 48 hrs 96 hrs 7 days0
20
40
60
80
100 HV -> LVLV -> HV
Time Between Exposures
Pe
rce
nt
Su
pe
rin
fec
ted
Viral Load in Fish
LV
Moc
k
LV
HV
Mo
ck
HV
HV
M
ock
HV
L
V
Mo
ck
LV
Log(
viru
s/g
fish)
HV
LV
Kell, AM, et. al., Journal of Virology, 2013
2
4
6
8
10
2
4
6
8
10
24 Hour Delay Time
* *
Superinfection and Virulence Tradeoff?
• First virus genotype infecting host has advantage
• Virulence not associated with superinfection fitness
Could explain field maintenance of low virulence
Virulence and Other Fitness TraitsDosage to infect 50% of fish –ID 50 (Undergraduate Doug McKenney)
ID50 HV: ~900 pfu/mlID50 LV: ~7500 pfu/ml
500 1000 2500 5000 7500 10000 1000000
20
40
60
80
100
LVHV
Virus Dosage (pfu/ml)
% F
ish
Infe
cted
Virulence and Other Fitness TraitsSuperinfection
(Grad student Alison Kell)
0
20
40
60
80
100HV -> LV
LV -> HV
Time Between Exposures(hours)
Pe
rce
nt
Su
pe
rin
fec
ted
9648240 12 168
Could explain field maintenance of low virulence
0 1 2 3 4 5 6 72
4
6
8
0
1
2
3
Log(
Vira
l Loa
d)
Day
Log(MX
Fold
Change)
Exposure Dosage Data:Mortality
0 10^2 10^3 10^4 10^5 10^60
20
40
60
80
100No Vaccine
Vaccine
Per
cent
Mor
talit
y
Virus Exposure Dosage (pfu/ml)
0.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+060
20
40
60
80
100
VaccinatedUnvaccinated
Per
cent
Mor
talit
y
Virus Exposure Dosage (pfu/ml)
Preliminary Results
100 101 102 103 104 105 106
IHNV BCWD
Vaccine impacts epidemiology non-target pathogen?
Heterogeneity in protection correlated?
Infection Classes
0 5 10 15 20 25 300
2
4
6 Mean Daily Virus Shed
LV
HV
Day post-exposure
Log(
viru
s/m
l H2O
)
“Acute”
“Chronic”
S Iv RIH IL … N types
Transmission Rate
Recovery Rate
Death Rate
Supply Rate
Density
Aquaculture Model Effects
τv ρvδVδ δσ
Other Developing Projects
Virulence Evolution after Pathogen Emergence?
Perkinsusmarinus
Haplosporidiumnelsoni
Crossostrea virginica
IHNV O. mykiss
Topics
Evolutionary Drivers of Virulence Epidemiology
Animal Husbandry Impacts on Virulence Epidemiology
Virulence EvolutionTheory
Tra
nsm
issi
on
Dur
atio
n(O
ppor
tuni
ties)
Infected Host lifespanVirulence
Infe
cted
Hos
t lif
espa
n
Pathogens should evolve towards benign coexistence with host- Conventional Wisdom (May and Anderson, In: Coevolution, 1983)
Multitude virulent pathogens
Morbidity and mortality due to
infection
Virulence Associated with Pathogen Fitness Traits
Replication
Viru
lenc
e
ReplicationTra
nsm
issi
on R
ate
(Virulence)ReplicationC
ompe
titiv
e F
itnes
s
(Virulence)
Contradicts conventional wisdom
Competitive Fitness: Relative ability of co-infecting genotypes to produce infectious progeny in a given environment (Domingo et. al., Rev. Med. Virol., 1997)
(Alizon et. al., J. Evol. Biol., 2009)
Current ParadigmThe Virulence-Tradeoff
Few in vivo studies on the nature of virulence-fitness trait associations and tradeoffs
Virulence
Tra
nsm
issi
on R
ate
(Rep
licat
ion)
VirulenceTra
nsm
issi
on D
urat
ion
(Hos
t lif
espa
n)
Objective
• Determine if virulence is associated with pathogen fitness traits in vivo:
Competitive Fitness
Replication
Shedding
Entry
IHNV Genotype DiversityOver 240 genetic variants N. America
(R. Breyta, unpublished)
M
U
L
M
U
L
0 5 10 15 20 25 30 35 400%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Experiment Day
IHNV Virulence Diversity C
umul
ativ
e P
erce
nt M
orta
lity
(Fis
h)
020406080
100
0 10 20 30Day post infection
Perc
ent m
orta
lity
HV
LV
Characterize Virulence-Fitness Association
Cumulative MortalityP
erce
nt
12 hr3 days
1
2
3
5
6
4
Exposure type 1: Immersion Exposure type 2: Injection
Sample day 3
Viral load quantification:
Genotype specific qPCR
Experimental Design Host: Rainbow trout - Oncorhynchus mykiss
3
as before
4 5 6
1
2
3
4
5
6
12 hr
HV + LV 1:1 ratio
Treatments – (14-28 fish)
LV aloneHV alone
Experimental Design - Continued
ENTRYENTRY
ININ--HOSTHOSTREPLICATIONREPLICATION
SHEDDINGSHEDDING
Injection bypasses host entry
Infection Cycle Fitness
Replication OR Entry
Viru
lenc
e
VirulenceT
rans
mis
sion
Rat
eVirulence
Com
petit
ive
Fitn
ess
Testing Virulence-Fitness Associations
Alone Mix Alone5
6
7
8
9
10 HVLV
Immersion
Log
(viru
s co
pie
s/g
fish
)
(Wargo, et. al., J. Virology, 2011)
Results: Mean Viral Load HV always produces more virus than LV
ReplicationOr
Entry
Viru
lenc
e
Com
petit
ive
Fitn
ess
Virulence
Alone Mix Alone5
6
7
8
9
10 HVLV
Immersion
Log
(viru
s co
pie
s/g
fish
)
(Wargo, et. al., J. Virology, 2011)
Alone Mix Alone5
6
7
8
9
10 Injection
Log
(viru
s co
pie
s/g
fish
)
HV always produces more virus than LV
Viral load differences?
Replication? Entry?
ENTRYENTRY
ININ--HOSTHOSTREPLICATIONREPLICATION
SHEDDINGSHEDDING
Results: Mean Viral Load
• Proportion of HV reduced when bypass entry (P<0.05)
• Advantage of HV partially due to more efficient entry
Importance of Host Entry
• HV has replication advantage
Immersion Injection60
70
80
90
100 Percent of Genotype HV in Mixed Infection Popu-lation
Pe
rce
nt
of
tota
l vir
us
Alone Mix Alone5
6
7
8
9
10 HVLV
Immersion
Log
(viru
s co
pie
s/g
fish
)
(Wargo, et. al., J. Virology, 2011)
Alone Mix Alone5
6
7
8
9
10 Injection
Log
(viru
s co
pie
s/g
fish
)
ReplicationANDEntry
Viru
lenc
e
Mean Viral Load
Alone Mix Alone5
6
7
8
9
10 HVLV
Immersion
Log
(viru
s co
pie
s/g
fish
)
Alone Mix Alone3
4
5
6
7 Shed
Log
(viru
s co
pie
s/m
l H2O
)
(Wargo, et. al., J. Virology, 2011)
Alone Mix Alone5
6
7
8
9
10 Injection
Log
(viru
s co
pie
s/g
fish
)
Tra
nsm
issi
on R
ate
Virulence
Mean Viral Load
• The more virulent genotype (HV) had an advantage in each fitness trait examined:
• Suggests virulent genotype has overall fitness advantage
• However, fitness is ultimately driven by lifetime transmission– Examined traits at peak viral load– Lifetime shedding kinetics important
ReplicationV
iru
len
ceVirulence
Co
mp
etit
ive
Fitn
ess
Virulence
Tra
nsm
issi
on
rat
e
(Replication) (Replication)
Competitive Fitness
Replication
Shedding
Entry
Summary: Experimental Observations
Trade-off Hypothesis?
Virulence
Tra
nsm
issi
on r
ate
Tra
nsm
issi
on
Dur
atio
n
Virulence(Host lifespan reduction)
Alone Mix Alone3
4
5
6
7 Shed
Log
(viru
s co
pie
s/m
l H2O
)
Time
Tra
nsm
issi
on High Virulence Type
Low Virulence Type
DemonstratedBenefit
HypothesisCost
Time
Tra
nsm
issi
on High Virulence Type
Low Virulence Type
Tra
nsm
issi
on
Dur
atio
n
Virulence(Host lifespan reduction)
0 5 10 15 20 25 300
2
4
6
8
LV aloneHV alone
Day post-exposure
Lo
g(V
iru
s/m
l H2O
) Total Virus Shed
Tradeoff Prediction Vs. Data
Shedding Kinetics – Second Genotype Pair
Same conclusions
More virulent type (LR80) sheds at higher
quantities for longer
0 5 10 15 20 25 300%
20%
40%
60%
80%
100%
Mer95
LR80
Cumulative Mortality
Day post-exposure
Per
cent
Mor
talit
y
0 5 10 15 20 25 300
5
10
15
20
Mer95LR80
Nu
mb
er
of
fish
Day post-exposure
Number of Fish Shedding
0 5 10 15 20 25 300
2
4
6
8
Day post-exposure
Total Virus Shed
Log(
Viru
s/m
l H2O
)
Conclusions• The more virulent genotype had shedding advantage over
infection period examined
• Virulence correlated with lifetime transmission potential
Virulence
Life
time
Tra
nsm
issi
on P
oten
tial
(Replication)
Tra
nsm
issi
on
Dur
atio
nVirulence
(Host lifespan reduction)
Conventional Wisdom
• The more virulent genotype had shedding advantage over infection period examined
• Virulence correlated with lifetime transmission potential
Virulence
Life
time
Tra
nsm
issi
on P
oten
tial
(Replication)
Tra
nsm
issi
on
Dur
atio
nVirulence
No cost to virulence
Conclusions
Lack of Virulence-Tradeoff?
• Isolates don’t represent spectrum of virulence
• In a trait not measured– Environmental stability– Super-infection fitness– Minimum Infectious Dose
• No cost to virulence– Investigate field patterns
Fitn
ess
Virulence
Fitn
ess
Virulence
UndergradGraduate
Doug McKinney
Alison Kell
Culling Creates Virulence Tradeoff
Virulence
Like
lihoo
d C
ulle
d
VirulenceTra
nsm
issi
on D
urat
ion
Tra
nsm
issi
on R
ate
Virulence
CostBenefit
