Viral pathogenesisViral 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

Cell death Signs/ symptoms

Duration of infection

Acute + + + S <3 wks

Inapparent + + - S

Chronic + + +/- L

Persistent <<+ - - L

Latent - - - L

Slowly progressive

+ + Eventually +

L

Tumorigenic

+/- - + L

Viral VirulenceViral 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 propertyVirulence is a relative property

• Quantitation of virulence to compare strains– LD50 - lethal dose

for 50% kill– ID50 - infectious

dose for 50% of symptom

Virus conc

% alive

100

50

• 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 mayViral 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 vaccineAttenuation - 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?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 apoptosisAdenovirus 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 Adenovirus infection followed by treatment with anti-fas abanti-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 virusWest 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 ApoptosisWest 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 infectionFilovirus 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 infectionAntibody 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

What part of genome is needed for What part of genome is needed for virulence?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 Growth of Coxsackie in HeLa, murine fetal heart fibroblasts, adult murine cardiomyocytesfibroblasts, adult murine cardiomyocytes

InfluenzaInfluenza• 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 Virulence of chimeric and single aa substitution PB2PB2

Foot and Mouth DiseaseFoot 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 HSV1Molecular 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) 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 [3H]thymidine ([3H]TdR) and is expressed as mean counts per minute (cpm) ± SEM of triplicate cultures.

(B) 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).

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 Coronavirus neurovirulenceneurovirulence

• 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)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

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.

Contains integrated HIV with Tat defect

Secreted AP

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

Taxis assay: membrane with monocytes on one side and test protein on other

Count cells on filter

Viruses and multiple sclerosis?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 virusHow 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 obesityViruses 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 diabetesViruses 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 Other diseases with possible viral involvementinvolvement

• Coronary restenosis

• Behaviorial disorders