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  • 1. 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
  • 2. 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.
  • 3. Clinical latency
  • 4.  
  • 5. L + - +/- Tumorigenic L Eventually + + + Slowly progressive L - - - Latent L - - <<+ Persistent L +/- + + Chronic S - + + Inapparent S <3 wks + + + Acute Duration of infection Signs/ symptoms Cell death Infectious progeny
  • 6. 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
  • 7. Virulence 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
  • 8.
    • How is HIV/polio/influenza transmitted?
    • Why are these the only ways?
    • What would it take to make HIV airborne?
  • 9. 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
  • 10. 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
  • 11. 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
  • 12.
    • 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
  • 13. 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
  • 14. Adenovirus infection followed by treatment with anti-fas ab E1b is a bcl2 homolog - inhibits fas mediated apoptosis 12.8 87.2 E1b-, RID  90.2 9.9 E1b-, RID  99.8 0.2 E1b+, RID  99.4 0.6 E1b+, RID  99.9 0.1 Wild type Non-apoptotic Apoptotic Virus /mutant Percent of cells
  • 15.
    • How could you measure whether RID internalizes Fas?
  • 16. 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
  • 17. 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?
  • 18. 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?
  • 19.  
  • 20.  
  • 21.  
  • 22.  
  • 23.  
  • 24. 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
  • 25. What part of genome is needed for virulence?
    • Coxsackie virus can cause heart disease
    • CVB3/0 - avirulent
    • CVB3/20 - cardiovirulent
    • Change in nucleotide 234
  • 26. Growth of Coxsackie in HeLa, murine fetal heart fibroblasts, adult murine cardiomyocytes
  • 27. 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
  • 28.
    • Pathogenicity of transfectant viruses in mice
  • 29. Virulence of chimeric and single aa substitution PB2
  • 30. 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
  • 31. 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
  • 32.
    • 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~ 10 4 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~ 10 5 cells per well). Proliferation was assessed after 2 days by 16 to 18 hours of exposure to 1 µCi of [ 3 H]thymidine ([ 3 H]TdR) 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~ 10 4 cells per well) were incubated with the indicated peptides (0.2 µM) in the presence of irradiated syngeneic BALB/c spleen cells (5 x~ 10 5 cells per well): , p292-308 (IgG2a b )closed square; , p299-314 (UL6) open square; , p200-222 (MMTV).
  • 33. 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)
  • 34. 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
  • 35.
    • 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).
  • 36. 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
  • 37. Sequenced isolates to find differences
  • 38. 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
  • 39. 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
  • 40. 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?
  • 41. How to make a killer virus
    • What characteristics should a biological weapon have?
    • How can it be constructed?
  • 42.
    • 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
  • 43. Viruses and obesity
  • 44.
    • 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
  • 45. 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
  • 46. Other diseases with possible viral involvement
    • Coronary restenosis
    • Behaviorial disorders