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Viral Pathogenesis. Dr. Luka Cicin-Sain Dep. Of Vaccinology, HZI Tel. 0531 6181 4616 [email protected]. Teaching Material: Principles of Virology - Molecular Biology, Pathogenesis, and Control of Animal virus SJ Flint, LW Enquist, VR Racaniello & AM Skalka - PowerPoint PPT Presentation
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Viral Pathogenesis
Dr. Luka Cicin-SainDep. Of Vaccinology, HZITel. 0531 6181 [email protected]
Teaching Material:Principles of Virology -
Molecular Biology, Pathogenesis, and Control of Animal virusSJ Flint, LW Enquist, VR Racaniello & AM Skalka
American Society of Microbiology. 2004
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
What is a virus?
Alberts et al.; 4rd ed. (2002)
Molecular Biology of the Cell
Virus = bad news in a protein / membrane coat
Poliovirus:28 nm5 proteins1 ss RNA241 moleculesC332.662
H492.388
N98,245
O131.196
P7.500
S2.340
= a chemical ?
VIRAL PATHOGENESIS
Viral pathogenesis: = 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)
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…
Why were we so nervous about swine flu?
1918: Spanish Flu > 20 -50 Mio. deaths
India: ca. 20 MioUSA: ca. 0,5 Mio
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
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'.
Where do the killer viruses come from?
Where do the killer viruses come from?
Reassortmentof genomic segments
Double infectionwith avianand human influenza virusneeded
human virusanimal virus
New dangerous pathogen
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 tissueviral tropismpermissivity 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)
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
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 tissueviral tropismpermissivity 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)
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
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 tissueviral tropismpermissivity 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!!!)
Mortality due to MeaslesMorbidity (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
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 tissueviral tropismpermissivity 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)
Age-dependend mortality during influenza pendemicsLederberg 1997
1918United States
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
• Clinical studies • In vitro studies (cytopathogenesis)• In vivo studies in animal models (cyto- and
immunopathogenesis)– non-human primate models– mouse models – other models
Study of viral pathogenesis(How to proceed?)
1. Outstanding clinical relevance
Clinical studiesBenefits
Barré-Sinoussi F. et al. Science. 220, 868-71 (1983)
Clinical studiesBenefits
1. Outstanding clinical relevance
2. Direct information about disease
Course of the HIV infection
Limitations
1. Cellular and molecular mechanisms of disease cannot be efficiently studied
Clinical studies
Course of the HIV infection
1. Cellular and molecular mechanisms of disease cannot be efficiently studied
2. Association does not predict causality
Clinical studiesLimitations
V. C. Lombardi et al., Science 326, 585-589 (2009)
Koch's postulatesRequirements to identify an infectious cause of a disease1. The microorganism must be found in abundance in all
organisms suffering from the disease, but should not be found in healthy hosts.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.
Clinical studiesExperimental models
Experimental models – in vitro
Cell death
Virus Ag
Experimental models – in vitro
• 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
Benefits
Menard et al. J. Virol 2003
Experimental models – in vitroDeterminants of fitness
Wild type (wt) virus
Deletion (D) Mutant
Revertant virus
1
2
3
4
5
M36 RevDM36wt
PFU
/ml (
log 1
0)
Virus
Virus + zVAD-fmk (death inhibitor)
DM36
M36 rev
Active Casp-3 (cell death)
Experimental models – in vitro
Wt/Rev DM36
HIV genomeExperimental models – in vitro
(wt)
(Dnef)
Experimental models – in vitro
HIV genome
Experimental models – in vitro
Negative regulators of virus replication
HIV-1 wtHIV-1 DNefHIV-1 Nef rev.
Niderman et al. PNAS 1989
• 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
Experimental models – in vitro
Limitations
• 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
Experimental models – in vivo
Benefits
• 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
Experimental models – in vivo
Limitations
Comparison of HIV and SIV genomesHIV-1
Experimental models – in vivo
Experimental models – in vivoBenefits of in vivo assays over in vitro
Binninger et al. J. Virol 1991
HIV-1 wt
SIV DNef
HIV-1 Nef rev.
HIV-1 wt
HIV-1 DNef
HIV-1 Nef rev.
Niderman et al. PNAS 1989
SIV DNef
SIV wtOnly the in vivo analysis showed that Nef promotes virus replication
• 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
Experimental models – in vivo
Benefits
Time kinetics of the immune response
window of opportunityto establish infection
► role back of the (adaptive) immune response
Experimental models – in vivo
Testing the control of virus with immune cells
Since CD8 depletion increases the virus load, CD8 are important for the control of virus replication
Experimental models – in vivo
CD8 depleted Control monkeys
• 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
Experimental models – in vivo
Benefits
Transgenic & knockout mice for studying viral pathogenesis
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)
Experimental models – in vivo
Transgenic virus & knockout mice
Adapted from Luker GD et al. J Virol. 2003
N
Experimental models – in vivo
Immune evasionExperimental models – in vivo
- Apoptosis - Interferons- Cytokines and Chemokines- Cellular response - Natural Killer Cells (innate)- Cytolytic T lymphocytes (CTL)
- Humoral response (antibodies, complement)
Ability of the virus to evade detection and or antiviral activity by the immune system.
proteasomeER
Golgi
MHC I
T cell
viralproteins
TAP
1/2
MHC I
US11, US2
US6
US3
CMV
viralproteins
nucleus
Human CMV evades control by CD8+ T cellsvia multiple mechanisms
proteasomeER
Golgi
T cell
viralproteins
TAP
1/2
MHC I
m152
CMV
viralproteins
nucleus
Mouse CMV also evades control by CD8+ T cells
m152
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
Disabling the gene reduces the fitness of the mutant virus in vivoThe ability to replicate in tissue culture is not affected
Basic rules: Koszinowski´s postulates (KP II)
How to study the biological significance of viral virulence factors?
Disabling the gene reduces the fitness of the mutant virus in vivoThe 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)
Basic rules: Koszinowski´s postulates (KP II)
How to study the biological significance of viral virulence factors?
Growth capacity of the MCMV m152 mutant in vitro and in vivo
Disabling the virulence gene reduces the fitness of the mutant virus in vivoThe ability to replicate in tissue culture is not affected
Reduced virulence (attenuation) of the MCMV mutant in vivo
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
proteasomeER
Golgi
T cell
viralproteins
TAP
1/2
MHC I
CMV
viralproteins
nucleus
No growth defect of the m152 mutant in mice lacking MHC molecules
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
proteasomeER
Golgi
T cell
viralproteins
TAP
1/2
MHC I
CMV
viralproteins
nucleus
No growth defect of the m152 mutant in mice lacking CD8+ T cells
Disabling the gene reduces the fitness of the mutant virus in vivoThe 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)
Basic rules: Koszinowski´s postulates (KP II)
How to study the biological significance of viral virulence factors?
….but take care
What is true for a mouse, may not be true for a human
Sometimes mice tell lies !
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
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