Viral pathogenesis

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

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. 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

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

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?

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-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).

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. 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.

39. 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

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