Viral pathogenesis

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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
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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.

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Clinical latency
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Infectious progeny Cell death Signs/ symptoms Duration of infection
Acute S lt3 wks
Inapparent - S
Chronic /- L
Persistent ltlt - - L
Latent - - - L
Slowly progressive Eventually L
Tumorigenic /- - L
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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

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Virulence is a relative property

• Quantitation of virulence to compare strains
• LD50 - lethal dose for 50 kill
• ID50 - infectious dose for 50 of symptom

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alive
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Virus conc
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• How is HIV/polio/influenza transmitted?
• Why are these the only ways?
• What would it take to make HIV airborne?

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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

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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

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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

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• 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

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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

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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
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• How could you measure whether RID internalizes
  Fas?

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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

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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?

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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?

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Antibody enhancement of infection

• Dengue fever/dengue hemorrhagic fever
• Primary infection - acute, self-limiting
• Secondary infection - non-protective 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
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What part of genome is needed for virulence?

• Coxsackie virus can cause heart disease
• CVB3/0 - avirulent
• CVB3/20 - cardiovirulent
• Change in nucleotide 234

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Growth of Coxsackie in HeLa, murine fetal heart
fibroblasts, adult murine cardiomyocytes
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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

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• Pathogenicity of transfectant viruses in mice

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Virulence of chimeric and single aa substitution
PB2
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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

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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

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• Recognition of UV-irradiated extracts of
  HSV-1(KOS)-infected cells by cornea-specific CD4
  T cell clones. Cornea-reactive T cell clones
  (C1-6 and C1-15) or the OVA-specific clone O3 (2
  x 104 cells per well) were stimulated with
  UV-irradiated 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 3Hthymidine (3HTdR) and
  is expressed as mean counts per minute (cpm)
   SEM of triplicate cultures.
• Dose-dependent stimulation of cornea-specificCD4
  T cell clones by HSV UL6-(299-314) peptide. CD4
  T cell clones (C1-6 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).

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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)

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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

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• 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).

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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

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Sequenced isolates to find differences
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Contains integrated HIV with Tat defect
Secreted AP
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 LTR-driven 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.
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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
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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?

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How to make a killer virus

• What characteristics should a biological weapon
  have?
• How can it be constructed?

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• 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

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Viruses and obesity
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• 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

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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

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Other diseases with possible viral involvement

• Coronary restenosis
• Behaviorial disorders