Download Are pathogens evolving towards becoming super

Impacts of Disease Management
Practices on the Virulence Evolution and
Transmission of a Salmonid Virus
Andrew Wargo
Virginia Institute of Marine Science
College of William and Mary
[email protected]
http://wmpeople.wm.edu/arwargo
System
Infectious Hematopoietic Necrosis Virus
Negative-sense singlestranded RNA virus
Endemic salmonid fishes
Pacific Coast
Acute disease
Family - Rhabdoviridae
Occurs in wild and
cultured fish
Waterborne Transmission
Primary disease trout
farming
IHNV
Disease Management Practices
Vaccination
Culling
Virulence and Transmission?
What is Culling?
• Euthanize entire host population when
mortality reaches certain threshold
Culling and Virulence?
Low Virulence
High Virulence
Culling and Virulence?
Low Virulence
High Virulence
Culling Threshold = 30% total population
20 % Die
80 % Die
IHNV Experiments
Exposure: Batch immersion
Isolate fish in individual tanks
Allow for
water flow
each tank
Treatments – (20 fish ea.)
High Virulence Genotype: HV
Sample water daily (30 days)
Flush virus after sampling
Quantify viral RNA
Genotype specific qPCR
Low Virulence Genotype: LV
IHNV Data
LV
HV
HV WINS!
Number of fish
0
20
5
10
15
20
25
Day post-exposure
Number Fish Shedding
15
LV
HV
10
8
Log(Virus/ml H2O)
Culling
Threshold
Cumulative Mortality
100%
80%
60%
40%
20%
0%
30
Total Virus Shed
6
LV alone
HV alone
4
2
5
0
0
0
5
10
15
20
Day post-exposure
25
30
0
5
10 15 20 25 30
Day post-exposure
IHNV Data
LV
HV
Number of fish
0
20
5
WHO WINS?
10
15
20
25
Day post-exposure
Number Fish Shedding
15
LV
HV
10
8
Log(Virus/ml H2O)
Culling
Threshold
Cumulative Mortality
100%
80%
60%
40%
20%
0%
30
Total Virus Shed
6
LV alone
HV alone
4
2
5
0
0
0
5
10
15
20
25
Day post-exposure
30
0
5
10 15 20 25 30
Day post-exposure
Culling and Virulence
Mathematical Model
Supply Rate
σ
V = IH
Iv
S τ
v
Log(virus/ml H2O)
Transmission
Rate
6
Mean Daily Virus Shed
LV
HV
4
2
0
0
IL
5 10 15 20 25 30
Day post-exposure
Culling and Virulence
Mathematical Model
Supply Rate
σ
V = IH
IL
R
Iv ρ
v
S τ
v
Transmission
Rate
Number of fish
Recovery
Rate
20
Number of Fish
Shedding LV
15
HV
10
5
0
0
5 10 15 20 25 30
Day post-exposure
Culling and Virulence
Mathematical Model
Supply Rate
σ
V = IH
Percent Mortality
100%
80%
60%
40%
20%
0%
Cumulative Mortality
LV
HV
0
5 10 15 20 25 30
Day post-exposure
R
Iv ρ
v
S τ
v
Transmission
δ
Rate
Death Rate
IL
δV
Recovery
Rate
δ
Aquaculture Model
2
Raceway 1
S
3
S
I
R
S
I
R
μ Migration Rate
4…Z
μ
I
μ
R
S
I
R
Aquaculture Model
2
Raceway 1
S
3
S
I
R
S= 0
I= 0
R= 0
μ
μ Migration Rate
4…Z
μ
I
Culling
R
S
I
R
Culling Model Results
Log(Number of Fish)
No Culling
Culling
(Threshold= 30%)
Susceptible
5
5
Recovered
4
4
Infected HV
3
3
Infected LV
2
2
1
1
0
0
0
50
100
Day
150
0
50
100
150
Day
Culling selects for low virulence
Evolution of decreased virulence predicted
Disease Management Practices
Vaccination
Culling
Virulence and Transmission?
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
Proportion of Hosts
“Leaky”
“All or Nothing”
0
0.5
Susceptibility
1
Vaccine Protection Heterogeneity:
Prevalence
Exposure 1
All or
Nothing
Leaky
Exposure 2
Vaccine Protection Heterogeneity:
Prevalence
Exposure 1
All or
Nothing
Leaky
Exposure 3
Gomes et. al.,
Plos
Pathogens,
2014
Vaccine Protection Heterogeneity:
Experiments
Vaccinated
Sham: PBS Pathogen dosages:
0,101,102,103,104,105,106
Isolate Fish
Measure
-Mortality
-Percent infected
-Viral shedding
Vaccine Protection Heterogeneity:
Results
Cumulative Mortality
Percent mortality
100
80
Proportion Shedding
Vaccinated
Unvaccinated
60
40
20
0
0
101 102 103 104 105 106
Virus Exposure Dose (pfu/ml)
Quantity Virus Shed
Proportion Infected
Density
Challenge Dose
Susceptibility
Vaccine Protection Heterogeneity:
Transmission Model Framework
S = # Susceptible
λ = transmission rate
W = virus concentration
I = # Infectious
R = # Recovered
D = # Dead
f = proportion recover
α = shedding rate
δ = virus removal rate
Disease Management Practices
Vaccination
Culling
Virulence and Transmission?
Vaccination and Virulence Evolution
No transmission
Vaccinate
Non-sterilizing
Virulence can evolve upwards
(Gandon et. al., Nature, 2001)
(Gimeno, Vaccine, 2008)
Problematic
When vaccine allows transmission
Incomplete vaccine coverage
Transmission
Vaccination and Transmission:
IHNV Vaccine Trial
Vaccinated Fish
8
Log(Virus/g fish)
Log(Virus/g fish)
Unvaccinated Fish
6
4
2
0
8
6
4
2
0
1
2
3
4
5
Fish
67% Mortality
(Kurath and LaPatra, unpublished)
1
2
3
4
Fish
5% Mortality
5
Vaccination and Virulence Evolution
No transmission
Vaccinate
Non-sterilizing
Virulence evolves upwards
(Gandon et. al., Nature, 2001)
(Gimeno, Vaccine, 2008)
Problematic
When vaccine allows transmission
Incomplete vaccine coverage
Transmission
Incomplete Vaccine Coverage
Vaccine Virulence Evolution:
Laboratory Experiments
P1
P2
P1
P2
P2
P3
Single
Mix
Vaccinate
Vary: Level and timing of treatment
Quantify: Genotypic and phenotypic evolution
Vaccine Induced Virulence Evolution:
Model Exploration
Supply Rate
σ
IH
IL … N types
δ
R
Iv ρ
v
S τ
v
Transmission
Rate
δV
Recovery
Rate
Death Rate
δ
Harnessing Evolution to
Manage Disease?
Selective Breeding
Virulence
IHNV
BCWD
Co-Infection
THANKS!
Darbi
Jones
Rachel
Jim Breyta
Winton
Barb Rutan
Doug
McKenney
Jessie Viss
Ben Kerr
Gabriella Gomes
Gael Alison
Tarin
Kurath Kell Thompson
Michelle
Penaranda Maureen Purcell Jake Scott
Shannon
LaDeau
Bill Batts
Marc Lipsitch
Paige
Barlow
Greg Wiens
Funding: NIH EEID, USDA NIFA, NSF, USGS, UW, VIMS
Questions?
Culling Model Explorations
• Impact of virus migration rates (biosecurity)
S
I
R
μ
I
S
R
• Impact of culling threshold
Culling
Threshold
Cumulative Mortality
100%
80%
60%
40%
20%
0%
0
• Timing of strain invasion
5
10 15 20 25 30
Virulence Evolution in the Field
IHNV Database (WFRC)
Genomic and Epidemiological data
Investigations
•Virulence
•Fitness
•Ecological Drivers
Immersion
Log(virus/ml H2O)
12
10
8
6
4
Viral load in host
1
4
7
HV
LV
10 13 16 19 22
Fish
Shed
12
Viral load in water
10
8
Log(virus/g fish)
Log(virus/g fish)
Individual Fish Variation
(Mixed Infections)
Injection
12
Viral load in host
10
8
6
4
1
4
7
11
15
19
22
Fish
High levels fish-to-fish
variation
6
4
1
4
7
10 13 16 19 22
Fish
(Wargo and Kurath, Virus Res., 2012)
Mimic natural infections in
the field
Drivers of Variation
Coefficient of Variation (%)
600
500
400
300
200
100
Immersion
Isogenic
Immersion
Injection
Log(virus/ml H2O)
Transmission
Mean Daily Virus Shed
6
5
4
3
2
1
0
LV alone
HV alone
0
5
10
15
20
25
30
Day post-exposure
P1
P2
P1
Correlation Virus Shed and Virus Within Fish
(Log[Virus copies/ml H2O])
8
HV
LV
HV Fit Line
6
LV Fit Line
4
2
4
5
6
7
8
(Log[Virus copies/g fish])
9
10
Within Host Viral load
-Positive correlation in-host viral load and shedding (R2 = 0.76)
-HV more efficient at Shedding
11
ID 50 Results
Peak Viral Load
Infected Fish Only
LV
HV
7
6
5
All negative
Log(virus/g fish)
8
4
500
1000
2500
5000
7500
10000
100000
Virus Dosage (pfu/ml)
Exposure dosage does not impact peak viral load
ID50 & LD50
Experimental Design
1 Hour Exposure
Vary
Dosage
Day 3
Viral Load
Quantification
(ID50)
Track Mortality 30 days (LD50)
% Fish Infected
ID50 Results
% Fish Infected
100
80
60
LV
HV
40
20
0
Virus Dosage (pfu/ml)
ID50 HV: ~900 pfu/ml
ID50 LV: ~7500 pfu/ml
100
% Mortality
80
LD50 Results
HV
LV
60
40
20
0
103
104
Virus Dosage (pfu/ml)
HV LD50: ~103 pfu/ml
LV LD50: ~ 105 pfu/ml
105
Percent of Infected Fish That Die
% Mortality
100%
80%
HV
LV
60%
40%
20%
0%
4
10^4
10
10^5
10 5
Virus Dosage (pfu/ml)
HV kills more fish that it infects
Infectivity
Transmission
Duration
LD50/ID50 and Tradeoff
Hypothesis?
Virulence
?
Virulence
Virulence comes with infectivity benefit
More virulent genotypes kill larger proportion hosts,
but mortality occurs after shedding subsides,
so cost is minimal
Virulence and Other Fitness Traits
Dosage to kill & infect 50% of fish – LD 50 &
ID 50 (Undergraduate Doug McKenney)
Superinfection
(Grad student Alison Kell)
Superinfection
Ability of virus genotype to establish infection in host
with a prior established infection by another genotype
Superinfection Experiments
1 exposure
2 exposure
3 days
Interval
Harvest
1 exposure
2 exposure
HV
LV
LV
HV
HV
mock
LV
mock
mock
LV
mock
HV
mock
mock
Mixed
Single
Superinfection
groups
Primary exposure
control
Secondary exposure
control
Uninfected Control
Intervals
12 hours
24 hours
48 hours
96 hours
168 hours
Percent Fish Superinfected
Percent Superinfected
100
HV -> LV
LV -> HV
80
60
40
20
0
0 hrs
coinfection
12 hrs
24 hrs
48 hrs
96 hrs
Time Between Exposures
Kell, AM, et. al., Journal of Virology, 2013
7 days
Viral Load in Fish
10
*
8
Kell, AM, et. al., Journal of Virology, 2013
LV
Mock
Mock
2
HV
2
HV
4
Mock
4
HV
6
HV
LV
6
LV
*
LV
8
LV
HV
10
Mock
Log(virus/g fish)
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 Traits
% Fish Infected
Dosage to infect 50% of fish –ID 50
(Undergraduate Doug McKenney)
100
80
60
LV
HV
40
20
0
Virus Dosage (pfu/ml)
ID50 HV: ~900 pfu/ml
ID50 LV: ~7500 pfu/ml
Percent Superinfected
Virulence and Other Fitness Traits
100
Superinfection
(Grad student Alison Kell)
HV -> LV
80
LV -> HV
60
40
20
0
0
48
24
12
96
Time Between Exposures
(hours)
168
Could explain field maintenance of low virulence
Log(Viral Load)
3
6
2
4
1
2
0
0
1
2
3
4
Day
5
6
7
Log(MX Fold
Change)
8
Exposure Dosage Data:
Mortality
100
No Vaccine
Vaccine
80
60
40
20
0
0
10^2
10^3
10^4
10^5
10^6
Virus Exposure Dosage (pfu/ml)
Preliminary Results
Percent Mortality
100
80
Vaccinated
Unvaccinated
60
40
20
0
0.00E+00
104 1.00E+05
103 1.00E+04
102 1.00E+03
106
100 1.00E+01
101 1.00E+02
105 1.00E+06
Virus Exposure Dosage (pfu/ml)
Vaccine impacts epidemiology
non-target pathogen?
IHNV
BCWD
Heterogeneity in protection correlated?
Log(virus/ml H2O)
Infection Classes
6
Mean Daily Virus Shed
4
“Acute”
LV
HV
“Chronic”
2
0
0
5
10 15 20
Day post-exposure
25
30
Aquaculture Model Effects
Supply Rate
σ
IH
IL … N types
Transmission
δ
Rate
Death Rate
R
Iv ρ
v
S τ
v
δV
Density
Recovery
Rate
δ
Other Developing Projects
Virulence Evolution after Pathogen Emergence?
IHNV
O. mykiss
Crossostrea virginica
Haplosporidium
nelsoni
Perkinsus
marinus
Topics
Evolutionary Drivers of Virulence Epidemiology
Animal Husbandry Impacts on Virulence
Epidemiology
Morbidity and
mortality due to
infection
Virulence
Transmission
Duration
(Opportunities)
Infected Host lifespan
Virulence Evolution
Theory
Infected Host lifespan
 Pathogens should evolve towards benign coexistence with host
-Conventional Wisdom (May and Anderson, In: Coevolution, 1983)
Multitude virulent pathogens
Replication
(Virulence)
Competitive Fitness
Replication
Transmission Rate
Virulence
Virulence Associated with
Pathogen Fitness Traits
Replication
(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)
Virulence
Transmission Duration
(Host lifespan)
Transmission Rate
(Replication)
Current Paradigm
The Virulence-Tradeoff
Virulence
Few in vivo studies on the nature of virulence-fitness trait
associations and tradeoffs
Objective
• Determine if virulence is associated with
pathogen fitness traits in vivo:
Entry
Replication
Competitive Fitness
Shedding
IHNV Genotype Diversity
Over 240 genetic variants N. America
U
M
M
L
U
L
(R. Breyta, unpublished)
Cumulative Percent Mortality (Fish)
IHNV Virulence Diversity
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
5
10
15
20
Experiment Day
25
30
35
40
Percent
Percent
mortality
Characterize Virulence-Fitness
Association
Cumulative Mortality
HV
LV
100
80
60
40
20
0
0
10
20
Day post infection
30
Experimental Design
Host:
Rainbow trout - Oncorhynchus mykiss
Exposure type 1: Immersion
12 hr
Exposure type 2: Injection
22
12 hr
33
11
3 days
Sample day 3
Viral load
quantification: 55
Genotype
specific qPCR 66
44
3
4
5
as before
6
Experimental Design - Continued
Treatments – (14-28 fish)
LV alone
HV alone
HV + LV 1:1 ratio
Infection Cycle Fitness
IN-HOST
REPLICATION
ENTRY
Injection bypasses host entry
SHEDDING
Competitive Fitness
Replication OR Entry
Transmission Rate
Virulence
Testing Virulence-Fitness Associations
Virulence
Virulence
Immersion
10
9
HV
LV
8
HV always produces
more virus than LV
7
6
5
Mix
Replication
Or
Entry
Alone
Competitive Fitness
Alone
Virulence
Log(virus copies/g fish)
Results: Mean Viral Load
Virulence
(Wargo, et. al., J. Virology, 2011)
Log(virus copies/g fish)
10
Log(virus copies/g fish)
Results: Mean Viral Load
10
Immersion
HV
LV
9
8
HV always produces
more virus than LV
7
6
5
Alone
Mix
Alone
Viral load differences?
Injection
IN-HOST
REPLICATION
9
8
7
ENTRY
SHEDDING
6
5
Alone
Mix
Alone
(Wargo, et. al., J. Virology, 2011)
Replication?
Entry?
Percent of total virus
Importance of Host Entry
100
Percent of Genotype HV in Mixed Infection
Population
90
80
70
60
Immersion
Injection
• Proportion of HV reduced when bypass entry (P<0.05)
• Advantage of HV partially due to more efficient entry
• HV has replication advantage
Log(virus copies/g fish)
10
Log(virus copies/g fish)
Mean Viral Load
10
Immersion
HV
LV
9
8
6
5
Alone
Mix
Alone
Virulence
7
Injection
9
Replication
AND
Entry
8
7
6
5
Alone
Mix
Alone
(Wargo, et. al., J. Virology, 2011)
10
Immersion
HV
LV
9
8
7
6
5
Alone
Mix
Alone
Log(virus copies/ml H2O)
Log(virus copies/g fish)
10
Log(virus copies/g fish)
Mean Viral Load
Shed
7
6
5
4
3
Alone
Mix
Transmission
Rate
Injection
9
8
7
6
Virulence
5
Alone
Mix
Alone
(Wargo, et. al., J. Virology, 2011)
Alone
Summary: Experimental Observations
Competitive
Fitness
Shedding
Transmission rate
Replication
Virulence
Entry
Competitive Fitness
• The more virulent genotype (HV) had an advantage in each
fitness trait examined:
Replication
Virulence
(Replication)
Virulence
(Replication)
• 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
Transmission
Duration
Demonstrated
Benefit
Shed
7
6
5
4
3
Alone
Mix
Hypothesis
Cost
Virulence
(Host lifespan reduction)
Virulence
Transmission
Log(virus copies/ml H2O)
Transmission rate
Trade-off Hypothesis?
High Virulence Type
Low Virulence Type
Alone
Time
Transmission
Duration
Transmission
Tradeoff Prediction Vs. Data
High Virulence Type
Low Virulence Type
Log(Virus/ml H2O)
Virulence
(Host lifespan reduction)
8
Time
Total Virus Shed
LV alone
6
HV alone
4
2
0
0
5
10
15
20
Day post-exposure
25
30
100%
Cumulative Mortality
80%
Number of fish
Percent Mortality
Shedding Kinetics – Second Genotype Pair
Mer95
60%
LR80
40%
20%
0%
0
5
10 15 20 25 30
Log(Virus/ml H2O)
Day post-exposure
8
20
Number of Fish
Shedding
15
Mer95
LR80
10
5
0
0
5
10 15 20 25 30
Day post-exposure
Total Virus Shed
Same conclusions
6
4
2
0
0
5
10
15
20
25
Day post-exposure
30
More virulent type
(LR80) sheds at higher
quantities for longer
Conclusions
• The more virulent genotype had shedding advantage over
infection period examined
Transmission
Duration
Lifetime Transmission
Potential
• Virulence correlated with lifetime transmission potential
Virulence
(Replication)
Conventional
Wisdom
Virulence
(Host lifespan reduction)
Conclusions
• The more virulent genotype had shedding advantage over
infection period examined
Transmission
Duration
Lifetime Transmission
Potential
• Virulence correlated with lifetime transmission potential
Virulence
(Replication)
Virulence
No cost to virulence
Lack of Virulence-Tradeoff?
Fitness
Fitness
• Isolates don’t represent spectrum of virulence
Virulence
• In a trait not measured
Virulence
Graduate
Undergrad
Alison
Kell
Doug
McKinney
– Environmental stability
– Super-infection fitness
– Minimum Infectious Dose
• No cost to virulence
– Investigate field patterns
Culling Creates Virulence Tradeoff
Likelihood Culled
Transmission Rate
Virulence
Virulence
Transmission Duration
Cost
Benefit
Virulence