Transcript Viral pathogenesis - California State University, Fullerton

Viral pathogenesis
“No virus is known to do good. It has been well
said that a virus is a piece of bad news wrapped up
in protein.”
Medawar and Medawar
Learning objectives
• Describe mechanisms that viruses use to damage host
cells.
• Explain how the host contributes to damage resulting from
virus infection.
• Design an experiment to determine what virus genes are
involved in pathogenesis.
Clinical latency
Infectious
progeny
Acute
+
Inapparent +
Chronic
+
Persistent <<+
Latent
Slowly
+
progressive
Tumorigen +/ic
Cell death
+
+
+
+
-
Signs/
symptoms
+
+/Eventually
+
+
Duration of
infection
S <3 wks
S
L
L
L
L
L
Viral Virulence
• The ability of a virus to cause disease in an infected host
• A virulent strain causes significant disease
• An avirulent or attenuated strain causes no or reduced
disease
• Virulence depends on
– Dose
– Virus strain (genetics)
– Inoculation route - portal of entry
– Host factors - eg. Age SV in adult neurons goes
persistent but is lytic in young
Virulence is a relative property
• Quantitation of
virulence to compare
strains
– LD50 - lethal dose
for 50% kill
– ID50 - infectious
dose for 50% of
symptom
100
% alive
50
Virus conc
• How is HIV/polio/influenza transmitted?
• Why are these the only ways?
• What would it take to make HIV airborne?
Viral genes that affect virulence may
• Affect the ability of the
virus to replicate
• Enable the virus to spread
within host or between
hosts
• Defeat host defense
mechanisms
• Produce products that are
directly toxic
Attenuation - polio vaccine
•
3 serotypes of Sabin virus
(attenuated) changed in 5’
NTR
– Affects ability to
replicate in neurons
– Affects translation of
mRNA in neuronal
culture cells but not
other cells
– Replicate poorly in gut
so less is produced to
spread
What damage do viruses do?
• Direct damage to cells due
to cell death/apoptosis
– Paralysis
– Immune deficiency
• Disruption of normal cell
functions (eg protein
synthesis, secretion,
membrane trafficking)
• Immune response to virus
infected cells
• Immune cell release of
cytokines
• Virus hijacking/expressing
host genes
• Evoking an autoimmune response that affects
uninfected cells
– Mimicry
– Exposing protected sites
– Infecting immune cells - B cell antibody
production against variety of proteins
– Hyperexpression of MHC
Adenovirus and apoptosis
• Binding to Fas receptor
triggers apoptosis (even
ab)
• RID is Ad protein that
internalizes epidermal
growth factor receptor
• Hypothesis: RID
internalizes Fas receptor
and protects from
apoptosis
Adenovirus infection followed by treatment with
anti-fas ab
Percent of cells
Virus /mutant
Apoptotic
Non-apoptotic
Wild type
0.1
99.9
E1b+, RID
0.6
99.4
E1b+, RID
0.2
99.8
E1b-, RID
9.9
90.2
E1b-, RID
87.2
12.8
E1b is a bcl2 homolog - inhibits fas mediated apoptosis
• How could you measure whether RID internalizes
Fas?
West Nile virus
• Flavivirus (like hepC)
• Vector borne
• Appeared in US in
1999 and spread
across country
• Symptoms include
neurologic and may
lead to paralysis and
death
West Nile Virus and Apoptosis
• Hypothesis: Capsid protein
expression in cells results in
apoptosis through
mitochondrial pathway
• Inflammation follows as a
response to apoptosis
• How do you show apoptosis as
a result of capsid expression?
• How could you show it is the
mitochondrial pathway?
Filovirus infection
• Ebola and Marburg
• Hemorrhagic fever, shock
and death
• Hypothesis: Shock is
often associated with
release of cytokines by
macrophage/monocyte
• What do you need to
show?
Antibody enhancement of infection
• Dengue fever/dengue
hemorrhagic fever
• Primary infection - acute, selflimiting
• Secondary infection - nonprotective antibodies bind and
facilitate entry to monocytes
through Fc receptor
• Causes cytokine release that
leads to hemorrhage, shock and
death
• Ebola/HIV similar affect
Ebola pseudotyped VSV
What part of genome is needed for
virulence?
• Coxsackie virus can
cause heart disease
• CVB3/0 - avirulent
• CVB3/20 cardiovirulent
• Change in nucleotide
234
Growth of Coxsackie in HeLa, murine fetal heart
fibroblasts, adult murine cardiomyocytes
Influenza
•
•
•
•
•
Avian H5N1 appeared in 1997
Until then most H1, H2, H3
Fatal with distribution in several tissues
HA determines binding to host and virulence
Basic amino acids at cleavage site increase protease
susceptibility
Pathogenicity of
transfectant viruses in
mice
Virulence of chimeric and single aa substitution
PB2
Foot and Mouth Disease
• Picornavirus
• OTai strain infects swine but not cattle; OCamp is virulent
for swine and cattle
• Chimeric viruses used to infect BHK (same responses on
porcine) and BK
Molecular mimicry by HSV1
• Herpes keratitis may cause
blindness
• T cell destruction of
corneal tissue
• Hypothesis: Damage is
due to autoimmune
response caused by
molecular mimicry
• Disease elicited by CD4 T
cells for corneal antigen in
mouse model
(A)
(B)
Recognition of UV-irradiated extracts of
HSV-1(KOS)-infected cells by corneaspecific CD4+ T cell clones. Corneareactive T cell clones (C1-6 and C1-15) or
the OVA-specific clone O3 (2 x~ 104 cells
per well) were stimulated with UVirradiated extracts of HSV-1-infected or
uninfected Vero cells in the presence of irradiated syngeneic BALB/c spleen cells
(5 x~ 105 cells per well). Proliferation was
assessed after 2 days by 16 to 18 hours of
exposure to 1 µCi of [3H]thymidine
([3H]TdR) and is expressed as mean counts
per minute (cpm) ± SEM of triplicate
cultures.
Dose-dependent stimulation of corneaspecificCD4+ T cell clones by HSV UL6(299-314) peptide. CD4+ T cell clones (C16 and C1-15) (2 x~ 104 cells per well) were
incubated with the indicated peptides (0.2
µM) in the presence of irradiated syngeneic
BALB/c spleen cells (5 x~ 105 cells per
well): , p292-308 (IgG2ab)closed square; ,
p299-314 (UL6) open square; , p200-222
(MMTV).
Mutant Ul6
• A - T cell proliferation
• B - virus replication
• C - immunization and
adoptive transfer of T cells
to nude mice; infection
with WT (open circle:
control; closed circle:
mutant virus; square: wt
virus)
Coronavirus
neurovirulence
• Mouse hepatitis virus
• Neurotropic strains - acute
meningoencephalitis then
chronic demyelination;
noneurotropic - acute
meningitis
• Acute phase - virus
replicates in neurons and
glial cells; then low levels
of viral RNA persist in
glial cells and chronic
inflammation
• Hypothesis: cytokine
response of brain immune
cells determines disease
outcome
•
Analysis of mRNA levels of cytokines
24 h following infection of astrocytes
with a neurotropic (MHV-A59) and a
nonneurotropic (MHV-2) virus
compared with an uninfected control
culture. The blots of mouse cytokine
array assays are shown. The cytokine
key is as follows: A, colony-stimulating
factor granulocyte; B, gamma
interferon; C, IL-1; D, IL-1ß; E, IL-2; F,
IL-3; G, IL-4; H, IL-5; I, IL-6; J, IL-7;
K, IL-9; L, IL-10; M, IL-11; N, IL-12
p35; O, IL-12 p40; P, IL-13; Q, IL-15;
R, IL-16; S, IL-17; T, IL-18; U,
lymphotoxin B; V, TNF-; W, TNF-ß; X,
GAPDH; Y, ß-actin; Z, bacterial plasmid
(pUC18).
HIV associated dementia (HAD)
• Occurs in ~ 15 - 30% of cases
of subtype B but only 1-2% of
subtype C
• Migration of monocytes to
brain correlated to HAD
• Extracellular Tat protein
exhibits strong monocyte
chemotactic properties
• Hypothesis: Differences in
Tat between subtypes B and C
may account for different rates
of HAD
Sequenced isolates to find differences
Secreted
AP
Contains
integrated
HIV with Tat
defect
Functional evaluation of Tat transactivation (A) expression vectors encoding the isogenic C-Tat
proteins. Differences within the dicysteine motif of these vectors are highlighted.. (B) Transactivation
of LTR-driven GFP expression by different Tat vectors in 293 cells. (C) Transactivation of LTRdriven SEAP expression by different Tat vectors in 293 cells. SEAP in the culture medium was
quantified on day 1 (open bars) and day 3 (filled bars). (D) Rescue of the Tat-defective virus by
isogenic C-Tat proteins. HLM-1 cells were transfected with different C-Tat variant expression vectors.
Culture supernatants were collected on days 1, 3, 5, and 7 following transfection, and p24 levels in the
culture supernatants were determined. Results of experiments using samples from day 3 are
presented; similar results were observed for samples from other days. Abs, absorbance; -VE, parental
vector.
Taxis assay:
membrane with
monocytes on one
side and test
protein on other
Count cells on
filter
Monocyte migration induced by isogenic Tat proteins. f-MLP peptide was used as a
positive control at 10-7 and 10-8 M concentrations. Tat proteins were used at
concentrations of 100 and 20 ng/ml (12 and 2.4 nM, respectively) as indicated. No
grad, wells with 100 ng of CC-Tat protein/ml in both the compartments. Differences in
the numbers of monocytes that migrated with Tat-CC and Tat-CS were statistically
significant
Viruses and multiple sclerosis?
• Protein database search
for virus gene products
with similarity to myelin
basic protein
• Used a variety of aa
substitutions accounting
for those that are not
essential for function
• Why protein and not
nucleic acid sequence?
How to make a killer virus
• What characteristics should a biological weapon
have?
• How can it be constructed?
• Ectromelia virus causes
mousepox
• Recovery due to CTL
death of infected cells via
perforin pathway
mousepox virus produces
inhibitors of caspases
• Vaccinia virus does not
inhibit caspases so they
are killed by two
mechanisms
• Il4 skews immune
response to ab production
and shuts down perforin
pathway
Viruses and obesity
• Canine distemper virus - hypothalamic damage?
• Rous associated virus, borna virus
• Chicken adenovirus - excessive fat accumulation but lower
cholesterol and triglycerides
• Ad36 - human ad that causes obesity in chickens and mice
and lower chol/triglyc
Viruses and diabetes
• Mouse model
• B - decrease in diabetes
with expression of Ad
early genes
• square expressing all E3
genes), DL704/NOD
(triangle expressing E3
apoptosis-inhibitory
genes), DL309/NOD (x
expressing E3 MHC class
I suppressive gene), and
nontransgenic controls diamond
Other diseases with possible viral
involvement
• Coronary restenosis
• Behaviorial disorders