Viral Pathogenesis

1. Viral Pathogenesis Dr. Luka Cicin-Sain Dep. Of Vaccinology, HZI Tel. 0531 6181 4616 Luka.cicin-sain@helmholtz-hzi.de Teaching Material: Principles of Virology - Molecular Biology, Pathogenesis, and Control of Animal virus SJ Flint, LW Enquist, VR Racaniello & AM Skalka American Society of Microbiology. 2004

2. Virus Pathogenesis Lecture overview • Definition and clinical relevance • Determinants of viral pathogenesis • Methods and means to study virus pathogenesis • Role of clinical studies • Experimental methods • In vitro • In vivo

3. Virus = bad news in a protein / membrane coat Poliovirus: 28 nm 5 proteins 1 ss RNA 241 molecules C332.662 H492.388 N98,245 O131.196 P7.500 S2.340 = a chemical ? Whatis a virus? Alberts et al.; 4rd ed. (2002) Molecular Biology of the Cell

4. VIRAL PATHOGENESIS Viral pathogenesis: = processbywhich a viruscausesdisease Virulence: = capacityof a virustocausedisease Viral disease: = sumoftheeffectsof (1) thevirusreplicationanddirectdamagetocells (cytopathogenesis) plus (2) ofthe immune response on thehost (immunopathogenesis)

5. Why study viral pathogenesis? • The study of viral pathogenesis is intellectually engaging and fun • Acquire knowledge on the molecular mechanisms by which viruses cause disease • to treat and prevent viral disease • AIDS, • Rabies • Hepatitis • Influenza, etc…

6. Why were we so nervous about swine flu? 1918: Spanish Flu > 20 -50 Mio. deaths India: ca. 20 Mio USA: ca. 0,5 Mio

7. Influenza-related deaths in individuals <65 y during pandemics younger persons have a 20 fold higher risk of influenza-related mortality during a pandemic, the risk for elderly is high at any time

8. Where do the killer viruses come from?

9. Where do the killer viruses come from? Reassortment of genomic segments Double infection with avian and human influenza virus needed human virus animal virus New dangerous pathogen

10. Determinants of viral disease: Viral factors AND host factors Nature of disease:- Strain of virus (virulence) - Target tissue: where virus enters the body ability of virus to gain access to target tissue viral tropism permissivity of cells Severity of disease: - virus: ability of infection to kill cells (cytotoxic effects); quantity of virus inoculated; duration of virus infection; other infections which might affect immune response (HHV8 / HIV)

11. Incidence of Kaposi sarcoma and the HIV pandemic • - The Kaposi sarcoma was a very rare tumor • - High incidence in HIV-infected, homosexual • men • - most common tumor in Sub-Saharan Africa

12. Determinants of viral disease: Viral factors AND host factors Nature of disease:- Strain of virus (virulence) - Target tissue: where virus enters the body ability of virus to gain access to target tissue viral tropism permissivity of cells Severity of disease: - virus: ability of infection to kill cells (cytopathic effects); quantity of virus inoculated; duration of virus infection; other infections which might affect immune response (HHV8 / HIV) - immune system: immunity to virus; intact immune response; immunopathology(Hepatitis B)

13. Jaundice due to infection with hepatitis viruses mainly due to the immune reaction chronic carriers often develop a poor immune response and do not get an icterus

14. Determinants of viral disease: Viral factors AND host factors Nature of disease:- Strain of virus (virulence) - Target tissue: where virus enters the body ability of virus to gain access to target tissue viral tropism permissivity of cells Severity of disease: - virus: ability of infection to kill cells (cytopathic effects); quantity of virus inoculated; duration of virus infection; other infections which might affect immune response (HHV8 / HIV) - immune system: immunity to virus; intact immune response; immunopathology(Hepatitis B) - more host factors: general health of the host; host nutritional status (Measles!!!)

15. Mortality due to Measles Morbidity (per year): 200 – 600/100.000 Mortality: in industrialized countries: 0,2 – 0,4/100.000 in developing countries: 5 – 25/100.000 120 (-300) x more !!! Encephalitis: 0.1 – 0.25% CNS Involvement: > 50 % of the patients have an altered EEG

16. Determinants of viral disease: Viral factors AND host factors Nature of disease:- Strain of virus (virulence) - Target tissue: where virus enters the body ability of virus to gain access to target tissue viral tropism permissivity of cells Severity of disease: - virus: ability of infection to kill cells(cytopathic effects) quantity of virus inoculated; duration of virus infection; other infections which might affect immune response (HHV8 / HIV) - immune system: immunity to virus; intact immune response; immunopathology(Hepatitis B) - more host factors: general health of the host; host nutritional status (Measles!!!) host genotype (HLA !, susceptibility genes?) ageof host (influenza)

17. 1918 United States Age-dependend mortality during influenza pendemics Lederberg 1997

18. Mechanisms of viral pathogenesis Direct killing of virus infected cells by virus (e.g. HIV) Overreacting immune system (e.g. Hepatitis) Virus induced oncogenesis (e.g. Cervical Cancer in Papilloma infection, Kaposi Sarcoma) Course of the HIV infection

19. Study of viral pathogenesis (Howtoproceed?) • Clinical studies • In vitro studies (cytopathogenesis) • In vivo studies in animal models (cyto- and immunopathogenesis) • non-human primate models • mouse models • other models

20. Clinical studies Benefits • Outstanding clinical relevance Barré-Sinoussi F. et al. Science. 220, 868-71 (1983)

21. Clinical studies Benefits Course of the HIV infection • Outstanding clinical relevance • Direct information about disease

22. Clinical studies Limitations Cellular and molecular mechanisms of disease cannot be efficiently studied Course of the HIV infection

23. Clinical studies Cellular and molecular mechanisms of disease cannot be efficiently studied Association does not predict causality Limitations V. C. Lombardi et al., Science 326, 585-589 (2009)

24. Clinical studies Experimental models Koch's postulates Requirements to identify an infectious cause of a disease • The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy hosts. • The microorganism must be isolated from a diseased organism and grown in pure culture. • The cultured microorganism should cause disease when introduced into a healthy organism. • The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

25. Experimental models – in vitro Cell death Virus Ag

26. Experimental models – in vitro Benefits • Infection of cells at high frequency (high MOI) • In situ study of virus in infected cells • Study of virus proteins and their interaction partners • Study of substances that block virus replication • Study of virus fitness determinants Huang et al. J. Virol 2008 Menard et al. J. Virol 2003

27. Experimental models – in vitro Determinantsoffitness Wild type (wt) virus Deletion (D) Mutant Revertant virus

28. Experimental models – in vitro M36 rev DM36 Virus Virus + zVAD-fmk (death inhibitor) Active Casp-3 (cell death) Wt/Rev DM36 5 4 3 PFU/ml (log10) 2 1 wt DM36 M36 Rev

29. Experimental models – in vitro HIV genome

30. Experimental models – in vitro HIV genome (wt) (Dnef)

31. Experimental models – in vitro Negative regulatorsofvirusreplication HIV-1 wt HIV-1 DNef HIV-1 Nef rev. Niderman et al. PNAS 1989

32. Experimental models – in vitro Limitations • It is not possible to study immune pathogenesis • It is not possible to study the pathology affecting multiple cell types • In vitro results may not reflect in vivo phenomena

33. Experimental models – in vivo Benefits • In vivo veritas • It is possible to study the mechanisms by which the immune system controls viruses • It is possible to study the pathology affecting multiple cell types in an organ and in situ • It is possible to study immune pathogenesis

34. Experimental models – in vivo Limitations • The results may not reflect human disease (e.g. mice infected with HCV will not develop hepatitis) • Some viruses are restricted to humans (e.g. Human herpesviruses) • These viruses are studied by using homologue viruses that coevolved with the animal host • The infection of animals with animal model viruses may not entirely reflect the clinical conditions

35. Experimental models – in vivo Comparison of HIV and SIV genomes HIV-1

36. Experimental models – in vivo Benefitsof in vivo assaysover in vitro HIV-1 wt SIV DNef HIV-1 Nef rev. Only the in vivo analysis showed that Nef promotes virus replication SIV wt HIV-1 wt HIV-1 DNef HIV-1 Nef rev. SIV DNef Binninger et al. J. Virol 1991 Niderman et al. PNAS 1989

37. Experimental models – in vivo Benefits • In vivo veritas • Itispossibletostudythemechanismsbywhichthe immune systemcontrolsviruses • Itispossibletostudythepathologyaffecting multiple celltypes in an organand in situ • Itispossibletostudy immune pathogenesis

38. Experimental models – in vivo Time kinetics of the immune response window of opportunity to establish infection ► role back of the (adaptive) immune response

39. Experimental models – in vivo Testingthecontrolofviruswith immune cells Control monkeys CD8 depleted Since CD8 depletion increases the virus load, CD8 are important for the control of virus replication

40. Experimental models – in vivo Benefits • In vivo veritas • Itispossibletostudythemechanismsbywhichthe immune systemcontrolsviruses • Itispossibletostudythepathologyaffecting multiple celltypes in an organand in situ • Itispossibletostudy immune pathogenesis

41. Experimental models – in vivo Transgenic & knockout miceforstudying viral pathogenesis

42. Experimental models – in vivo Advantages ofthemousemodels • Smallest and cheapest mammals • Advanced genetic tools are readily available (transgenic and knockout mice) • Cell biology tools are readily available (mouse specific monoclonal antibodies, proteins and sequences)

43. Experimental models – in vivo Transgenicvirus & knockout mice N Adapted from Luker GD et al. J Virol. 2003

44. Experimental models – in vivo Immune evasion Ability of the virus to evade detection and or antiviral activity by the immune system. • Apoptosis • Interferons • Cytokines and Chemokines • Cellular response • Natural Killer Cells (innate) • Cytolytic T lymphocytes (CTL) • Humoral response (antibodies, complement)

45. viral proteins US3 US6 viral T cell proteins US11, US2 Human CMV evades control by CD8+ T cells via multiple mechanisms ER proteasome Golgi CMV 1/2 TAP MHC I MHC I nucleus

46. viral proteins m152 m152 viral T cell proteins Mouse CMV also evades control by CD8+ T cells ER proteasome Golgi CMV 1/2 TAP MHC I nucleus

47. MCMV wildtype infected cells are NOT recognized and lysed by specific T cells (Cr-release assay) Deletion of the virulence factor m152 restores CD8+ T cell lysis

48. How to study the biological significance of viral virulence factors? Basic rules: Koszinowski´s postulates (KP II) Disabling the gene reduces the fitness of the mutant virusin vivo The ability to replicate in tissue culture is not affected

49. How to study the biological significance of viral virulence factors? Basic rules: Koszinowski´s postulates (KP II) Disabling the gene reduces the fitness of the mutant virus in vivo The ability to replicate in tissue culture is not affected Reinserting the gene into the mutant virus (generating a "rescuant") restores fitness The fitness of the mutant virus is restored in hosts that are genetically deficient for the target molecule or have been treated to abrogate the target molecule or effector cell (e.g. by antibody depeletion). Fitness is defined by transmission (surrogate: viral titers in organs)

50. Growth capacity of the MCMV m152 mutant in vitro and in vivo Disabling the virulence gene reduces the fitness of the mutant virus in vivo The ability to replicate in tissue culture is not affected