1. Viral Pathogenesis Dr. Luka Cicin-Sain Teaching Material:
Dep. Of Vaccinology, HZI
Tel
Viral Pathogenesis
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. What is a virus? Virus = bad news in a protein / membrane coat
Alberts et al.;
4rd ed. (2002)
Molecular
Biology of the Cell
Poliovirus:
28 nm
5 proteins
1 ss RNA
241 molecules C H
N98,245 O
P7.500
S2.340
= a chemical ?
What is a virus?

4. VIRAL PATHOGENESIS Viral pathogenesis: Virulence: Viral disease:
= process by which a virus causes disease
Virulence:
= capacity of a virus to cause disease
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
> Mio. deaths
India: ca. 20 Mio
USA: ca. 0,5 Mio 6

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?
The pig may act as an intermediate host for the generation of human−avian reassortant influenza viruses with pandemic potential. Observations of humans infected with avian influenza A (H5N1) virus in Hong Kong in 1997 suggest that man himself may act as a 'mixing vessel'.

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) 10

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) 12

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!!!) 14

15. Mortality due to Measles
Morbidity
(per year): – 600/
Mortality:
in industrialized countries:
0,2 – 0,4/
in developing countries:
5 – 25/
120 (-300) x more !!!
Encephalitis: – 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?)
age of host (influenza) 16

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

18. Mechanisms of viral pathogenesis
Course of the HIV infection
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) 18

19. 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

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

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

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

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

24. Koch's postulates Clinical studies Experimental models
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 Determinants of fitness
Wild type (wt) virus
Deletion (D) Mutant
Revertant virus

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

29. Experimental models – in vitro
HIV genome

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

31. Negative regulators of virus replication
Experimental models – in vitro
Negative regulators of virus replication
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. Comparison of HIV and SIV genomes
Experimental models – in vivo
Comparison of HIV and SIV genomes
HIV-1

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

37. 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

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. Testing the control of virus with immune cells
Experimental models – in vivo
Testing the control of virus with immune cells
CD8 depleted
Control monkeys
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
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

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

42. Advantages of the mouse models
Experimental models – in vivo
Advantages of the mouse models
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. Transgenic virus & knockout mice
Experimental models – in vivo
Transgenic virus & 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. Human CMV evades control by CD8+ T cells via multiple mechanisms
viral
proteins ER
proteasome
Golgi
US3
CMV
1/2
US6
T cell
viral
proteins
TAP
MHC I
MHC I
US11, US2
nucleus

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

51. Reduced virulence (attenuation) of the MCMV mutant
in vivo

52. 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

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

54. 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

55. No growth defect of the m152 mutant in mice
lacking CD8+ T cells
viral
proteins ER
proteasome
Golgi
CMV
1/2
T cell
viral
proteins
TAP
MHC I
nucleus

56. 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)

57. Sometimes mice tell lies !
….but take care
What is true for a mouse,
may not be true for a human
Sometimes mice tell lies !

58. 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 58

59. THANK YOU! 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