Viral Pathogenesis

1. Viral Pathogenesis Prof. Dr. Martin Messerle Hannover Medical School Institute of Virology Tel. 0511-532 4320 messerle.martin@mh-hannover.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. Pathogenesis 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. A few definitions… Viral pathogenesis: = process by which a virus causesdisease Virulence: = capacity of a virus to causedisease Viral disease: = sum of the effects of (1) the virus replication and direct damage to cells (cytopathogenesis) plus (2) of the immune response on the host (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. 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) Determinants of viral disease: viral factors AND host factors

11. Example: Consequences of double infection Incidence of Kaposi sarcoma and the HIV pandemic • - The Kaposi sarcoma was a very rare tumor • - high incidence in HIV-infected, homosexual • men (Europe, USA) • nowadays, most common tumor • in Sub-Saharan Africa • (due to HIV-1 pandemic)

12. 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) Determinants of viral disease: viral factors AND host factors

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 Example: Immunopathology

14. 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!!!) Determinants of viral disease: viral factors AND host factors

15. Example: host factor, Nutritional status 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. 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?) ●age of host (influenza) Determinants of viral disease: viral factors AND host factors

17. 1918 United States Example Host Factor: Age Age-dependent mortality during influenza pandemics (Lederberg 1997)

18. Mechanisms of viral pathogenesis Direct killing of virus infected cells by virus (e.g. HIV) Course of the HIV infection

19. 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)

20. Study of viral pathogenesis (how to proceed?) • clinical studies • in vitro studies (cytopathogenesis) • in vivo studies in animal models (cyto- and immunopathogenesis) • non-human primate models • mouse models • other models

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

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

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

24. Clinical studies Cellular and molecular mechanisms of disease cannot be efficiently studied Association does not predict causality Limitations Kaiser J. Chronic fatigue syndrome. Studies point to possible contamination in XMRV findings. Science. 2011 Jan 7;331(6013):17. V. C. Lombardi et al., Science 326, 585-589 (2009)

25. 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 pureculture. • 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.

26. Experimental models – in vitro Cell death Virus Ag

27. Experimental models – in vitro Advantages • 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

28. Experimental models – in vitro Determinants of fitness Wild type (wt) virus Deletion (D) Mutant Revertant virus

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

30. Experimental models – in vitro Negative regulators of virus replication HIV-1 wt HIV-1 DNef HIV-1 Nef rev. Niderman et al. PNAS 1989

31. 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

32. Experimental models – in vivo Advantages • „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

33. 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

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

35. Experimental models – in vivo Advantage of in vivo over in vitro assays SIV wt Only the in vivo analysis showed that Nef promotes virus replication HIV-1 wt HIV-1 DNef HIV-1 Nef rev. SIV DNef Niderman et al. PNAS 1989

36. Experimental models – in vivo Advantages • „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

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

38. Experimental models – in vivo Testing the control of virus with immune cells Control monkeys CD8 depleted Since CD8 depletion increases the virus load, CD8 T-cells are important for the control of virus replication

39. Experimental models – in vivo 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) Advantages of the mouse models

40. Experimental models – in vivo Transgenic & knockout mice for studying viral pathogenesis

41. 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)

42. How to study the biological significance of viral virulence factors? Disabling the virulence gene reduces the fitness of the mutant virus in vivo The ability to replicate in tissue culture is not affected

43. 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)

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

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

46. Growth capacity of the MCMV m152 mutant in vitro and in vivo Krmpotic A et al. J Exp Med 1999;190:1285-1296 Disabling the virulence gene reduces the fitness of the mutant virus in vivo The ability to replicate in tissue culture is not affected

47. No growth defect of the m152 mutant in mice lacking MHC molecules or CD8+ T cells mutant wildtype The fitness of the mutant virus is restored in hosts that are genetically deficient for the target molecule or the effector cells

48. viral proteins viral T cell proteins No growth defect of the m152 mutant in mice lacking MHC molecules ER proteasome Golgi CMV 1/2 TAP MHC I nucleus

49. No growth defect of the m152 mutant in mice lacking MHC molecules or CD8+ T cells mutant wildtype The fitness of the mutant virus is restored in hosts that are genetically deficient for the target molecule or the effector cells

50. Study of viral pathogenesis(What to study)? Define cause-effect relationships between infections and pathologies Define mechanisms by which viruses harm target cells Define viral genes that are relevant for the pathogenic process Define pathologies caused by an overreacting immune system