Viral Virulence

Relative nature of virulence

La Crosse Virus
(-)ve strand RNA virus
(Bunyaviridae)
Same family as Hantavirus, but is
arthropod-borne
neurotropic
Attenuated Clone B.5
Reduced capacity to produce
viremia and cross the blood brain
barrier
Virulence revealed by intracerebral
innoculation, but only in suckling
mice
No difference in virulence with wt
strain when innoculated
subcutaneously in adult mice
Intracerebral infection
PFU per LD50
Subcutaneous infection
PFU per LD50
La Crosse
virus strain Suckling mice Adult mice Suckling mice Adult mice
Virulent
Wild type
Attenuated
clone B.5
~1
~1
~1
>10
6
~1
>10
5
>10
7
>10
7
Choice of host and route
can dramatically influence
susceptibilty and resistance
Equal High
Virulence
Equal Low
Virulence
Attentuation
observed
Factor influencing Virulence
Viral Factors
Viral Strain
Route of Infection
Dose of Virus
Host Factors
Species
Age
Genetic Susceptibility
SARS Mortality Rate
>60 years 55.0% 45.3 to 64.7%
<60 years 6.8% 4.0 to 9.6%
Ave. 95% CI
http://www.thelancet.com/journal/vol361/iss9368/full/llan.361.9368.early_online_publication.25595.1
Measures/Quantitation of Virulence
Symptoms (e.g.)
Paralysis (poliovirus)
Jaundice (hepatitis)
Rash (measles)
Case/infection ratio
Death/Survival
# of IU/PFU per LD
50
(50%
fatality in cohort)
Pathogenic lesions
Virus Study period
Paralytic rate
per 100
primary infections Relative rates
Wild type
OPV
1931-1954
1961-1978
0.7
0.000062
~10,000
1
Virulence grade
Case fatality
Rate (%)
Mean survival
Time (days)
Percent
of isolates
I
II
IIIA
IIIB
IV
V
>99%
95%-99%
90%-95%
70%-90%
50%-70%
<50%
<13
14-16
17-22
23-28
29-50
NC
4%
18%
39%
25%
14%
1%
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Rabbit Myxoma Virus
1951
Biological control of wild rabbits:
co-evolution of viral virulence and host
resistance
1859, 12 European rabbits were introduced into
an Australia farm; by 1928 more than a billion
rabbits (>500/sq.mile) were ruining agriculture
1950, rabbit myxoma virus (>99% mortality rate)
was introduced; by 1953, >95% of rabbit
population was eliminated, by 1955, rabbit
population began to increase.
Reasons:
Virulence of rabbit myxoma virus decreased;
surviving rabbits developed increased resistance;
changes in vector activity (mosquitoes) decreased efficiency
of transmission
Measures/Quantitation of Virulence
Symptoms (e.g.)
Paralysis (poliovirus)
Jaundice (hepatitis)
Rash (measles)
Case/infection ratio
Death/Survival
# of IU/PFU per LD
50
(50%
fatality in cohort)
Pathogenic lesions
Virus Study period
Paralytic rate
per 100
primary infections Relative rates
Wild type
OPV
1931-1954
1961-1978
0.7
0.000062
~10,000
1
Virulence grade
Case fatality
Rate (%)
Mean survival
Time (days)
Percent
of isolates
I
II
IIIA
IIIB
IV
V
>99%
95%-99%
90%-95%
70%-90%
50%-70%
<50%
<13
14-16
17-22
23-28
29-50
NC
4%
18%
39%
25%
14%
1%
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Rabbit Myxoma Virus
1951
Percent
of isolates
0%
1%
5%
10%
25%
59%
1959
Experimental Manipulation of
Viral Virulence
VIRULENCE
Passage in Animals
Adaptation to survival in
host; may be tissue
specific
Passage in Cell Culture
Attentuation due to lack of
host immune response
Experimental Manipulation of
Viral Virulence
Passage in Animals
Adaptation to survival in host;
may be tissue specific
Yellow Fever
Adaptation to neurovirulence
by intracerebral passaging
SHIV (chimeric Simian-
Human Immunodeficiency
Virus)
Repeated passaging results in
severely pathogenic virus
(SHIV 89.6 to 89.6P) that
causes CD4 depletion and
death within 6 months
Passage in Cell Culture
Attentuation due to lack of host
immune response
Vaccinia (small pox vaccine strain)
MVA (Modified Vaccinia Ankara)
250 passages in Chick Embryonic
Fibroblast results in ability to infect
but not replicate in mammalian
cells
Results in loss of immune evasion
genes; more immunogenic vaccine
vector (?)
HIV
T-cell line adapted virus
More neutralization sensitive
than primary strains grown in
fresh PBMCs
Selection of Attentuated Viral Variants
Temperature Sensitive Variants
Antibody-resistant virus
Some neutralization resistant viruses can
have increased attentuation in vivo
Neutralization resistance can also lead to
increased virulence
Mutagenized viruses and selection

Study of Attentuated Viruses
Variant viruses (wt. vs attentuated) should be
genetically pure
Variant viruses should differ by as little as
possible
Variant viruses should differ only under non-
permissive conditions; i.e. there should be
culture or innoculation conditions where
replication is comparable
Comparative pathogenesis
(Virulent vs attentuated viruses)
Portal of entry
Upper vs lower respiratory tract for
influenza; ability to replicate in lower
respiratory tract (higher temp.) results
in increased pathogenictiy
Viremia
Most viremic can be most pathogenic
(not always) (poliovirus strains)
Ability to produce peripheral viremia
may affect end-organ pathology (La
Crosse vs Tahyna virus)
Neural Spread
Target Organ
Tropism
relative pathogenicity for different
tissues
Evasion of host immune responses
Comparative pathogenesis
(Virulent vs attentuated viruses)
Portal of entry
Upper vs lower respiratory tract for
influenza; ability to replicate in lower
respiratory tract (higher temp.) results
in increased pathogenictiy
Viremia
Most viremic can be most pathogenic
(not always) (poliovirus strains)
Ability to produce peripheral viremia
may affect end-organ pathology (La
Crosse vs Tahyna virus)
Neural Spread
Target Organ
Tropism
relative pathogenicity for different
tissues
Evasion of host immune responses
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Tahyna virus actually replicates better than La Crosse
virus in brain, but inability to produce fatal encephalitis
after subcutaneous injection is due to lack of replication
in periphery
Comparative pathogenesis
(Virulent vs attentuated viruses)
Portal of entry
Upper vs lower respiratory tract
for influenza; ability to replicate in
lower respiratory tract (higher
temp.) results in increased
pathogenictiy
Viremia
Most viremic can be most
pathogenic (not always)
Neural Spread
IM injection of wt vs avirulent strain of
rabies virus (e.g. MAR variant RV 194-2)
results in equal speed of spread to
CNS, but once there, spreads more
slowly to contiguous neurons
Target Organ
Tropism
relative pathogenicity for different
tissues
Evasion of host immune responses
Spread of Infection
(Neuroblastoma Cells)
0
20
40
60
80
100
120
0 2 4 6 8 10
Log2 Viral Dilutions
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Vi rulent (wt)
Avirul ent (RV 194-2)
Spread of Infection
(Baby Hamster Kideney-21 Cells)
0
20
40
60
80
100
120
0 2 4 6 8 10
Log2 Viral Dilutions
P
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Vi rulent (wt)
Avirul ent (RV 194-2)
avirulent
virulent
Comparative pathogenesis
(Virulent vs attentuated viruses)
Portal of entry
Upper vs lower respiratory tract for
influenza; ability to replicate in lower
respiratory tract (higher temp.) results
in increased pathogenictiy
Viremia
Most viremic can be most pathogenic
(not always)
Neural Spread
IM injection of wt vs avirulent strain of rabies
virus (e.g. MAR variant RV 194-2) results in
equal speed of spread to CNS, but once there,
spreads more slowly to contiguous neurons
Target Organ
Bunyavirus neurotropism
Neurotropism = neuroinvasiveness
Poliovirus enterotropism vs neurotropism
Tropism
relative pathogenicity for different
tissues
Evasion of host immune responses
Type 1
poliovirus strain
TCD50
per ml
Enterotropism
TCD50 per po
ID50
Neurotropism
TCD50 per ic
PD50
Virulent Mahoney
(CNS suspension)
10
6
10
3.3
(monkeys)
10
1.9
Attenuated LSc
(Tissue culture
fluid)
10
7.6
~10
4
(humans)
>10
7.6
Bunyavirus
Poliovirus
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LD
50
based
on IC injection
Comparative pathogenesis
(Virulent vs attentuated viruses)
Portal of entry
Upper vs lower respiratory tract for
influenza; ability to replicate in lower
respiratory tract (higher temp.) results
in increased pathogenictiy
Viremia
Most viremic can be most pathogenic
(not always)
Neural Spread
IM injection of wt vs avirulent strain of rabies
virus (e.g. MAR variant RV 194-2) results in
equal speed of spread to CNS, but once there,
spreads more slowly to contiguous neurons
Target Organ
Bunyavirus neurotropism
Poliovirus enterotrpism vs neurotropism
Tropism
relative pathogenicity for different
tissues
Evasion of host immune responses
CD4+
CD4+
Macrophages T cell Line
Primary T cells
CD4+
M-tropic
T-tropic
CCR5+ CXCR4+
CCR5+CXCR4+
(Early)
(Late)
(~50%)
Clinical AIDS Sexual
Transmission
HIV
Comparative pathogenesis
(Virulent vs attenuated viruses)
Portal of entry
Upper vs lower respiratory tract for influenza;
ability to replicate in lower respiratory tract
(higher temp.) results in increased
pathogenictiy
Viremia
Most viremic can be most pathogenic (not
always)
Neural Spread
IM injection of wt vs avirulent strain of rabies virus
(e.g. MAR variant RV 194-2) results in equal speed of
spread to CNS, but once there, spreads more slowly
to contiguous neurons
Target Organ
Bunyavirus neurotropism
Poliovirus enterotrpism vs neurotropism
Tropism
relative pathogenicity for different tissues
Evasion of host immune responses
LCMV (Clone 13 vs Armstrong strain)
Clone 13 replicates better/faster in
macrophages; rapid destruction of macs
leads to atttenuation of antigen
presentation, suppression of immune
response and thus results in viral escape
Armstrong strain leads to immunizing
infection and viral clearance
SIV/HIV (wt vs Dnef)
Also, Sidney Blood Bank cohort example

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Alveolar macs
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Genetic Determinants of Virulence
Mutant vs wt Clones
Attenuation or virulence can be due to changes in viral proteins or
UTR of viral genomes
Generally, increased number of mutations is correlated with
increased attenuation and reduced chance of reversion
Consideration for recombinant life-virus vaccine devlopment
SIV Dnef






Reversion to virulence does not necessarily require back
mutation; compensatory mutations in same protein (or even
different proteins) is possible
Attenuating mutations are generally host range alterations
(replication is affected only in some tissues, or cells)


Virulence Genes of Cellular Origin
Virokines
Mimic the action of cytokines; increases
host cell proliferation and virus production
Viroceptors
Cytokine decoys
Ab or Complement scavenger
Virulence Genes of Cellular Origin
Pox Viruses
VCP (Vaccinia Complement
Control Protein)
Abrogates complement mediated
atttack on viral infected cells
Homolog of C4-BP that inactivates
C4b, a critical player in the
complement cascade
TNF Viroceptors
TNF is proinflammatory cytokine that
activates immune networks
Soluble TNF-receptor homology
encoded by poxviruses can TNF
secreted by host cell and dampen
subsequent immune response
(IL-4): Super-pox
TH2 cytokine which suppresses Th1
(cell-mediated) immunity
Mousepox engineered to express IL-4
becomes extremely virulent (Super-
pox)

Herpesviruses
gE/gI glycoprotein can act
as Fc receptors; prevent
effector functions of antiviral
antibodies produced by the
host

AUSTRALIA:
Engineered Mouse Virus Spurs Bioweapon Fears
Elizabeth Finkel

MELBOURNE, AUSTRALIA--The surprising virulence of a virus genetically altered to reduce rodent
infestations in Australia has raised alarm over whether such research could be hijacked to produce
biological weapons. In an unusual twist, those sounding the alarm are not environmental activists but
the scientists themselves. Despite their warning, it's not clear whether the unexpected result, which
turned a vector into a potent killer, could be duplicated in viruses that affect humans. But scientists say
it should serve as a warning to the community to be more aware of the potentially harmful
consequences of their work.
Science 2001 Jan 26;291(5504):585
Mousepox-sensitive
(BALB/c)
Mousepox-resistant
(C57BL/6))
Early activation of virus-specifc CTLs
Production of high levels of type 1 cytokines
IL-2, IL-12, IFN-g and TNF-a
Mousepox-IL-4+
Mousepox-sensitive
(BALB/c)
Mousepox-resistant
(C57BL/6))
Immunized with attenuated
wt mousepox strain
% Mortality in Immunized mice
Wt IL-4+
C57BL/6 0 60
Balb/c 0 60
IL-4 is a Type 2 cytokine
enhances humoral immunity
Can Vaccinia (the attenuated version of smallpox
that is used in smallpox vaccine) engineered to
express IL-4 become a super smallpox--overcome
people who has already been vaccinated??

Bioterrorism agent??