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.
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
Quantitation of virulence to compare strains LD50 - lethal dose for 50% kill ID50 - infectious dose for 50% of symptom Virulence is a relative property 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 E1b is a bcl2 homolog - inhibits fas mediated apoptosis
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, 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 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
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
Recognition of UV-irradiated extracts of HSV-1(KOS)-infected cells by cornea-specific CD4+ T cell clones. Cornea-reactiveT cell clones (C1-6 and C1-15) or the OVA-specific clone O3 (2x~ 104 cells per well) were stimulated with UV-irradiated extracts ofHSV-1-infected or uninfected Vero cells in the presenceof -irradiated syngeneic BALB/c spleen cells(5 x~ 105 cells per well). Proliferation was assessed after 2 days by 16to 18 hours of exposure to 1 µCi of [3H]thymidine ([3H]TdR) and is expressed as mean counts per minute (cpm) ± SEMof triplicate cultures. • Dose-dependent stimulationof 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 (0.2µM) in the presence of irradiated syngeneic BALB/c spleencells (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
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.
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
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