Prof. Dr. Martin Messerle
Hannover Medical School
Institute of Virology
Tel. 0511-532 4320
messerle.martin@mh-hannover.de
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
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
Virus = bad news in a protein / membrane coat
Poliovirus:
What is a virus?
28 nm
5 proteins
1 ss RNA
241 molecules
C332.662
H492.388
N98,245
O131.196
P7.500
S2.340
= a chemical ?
Alberts et al.;
4rd ed. (2002)
Molecular
Biology of the Cell
A few definitions…
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 Mio
USA: 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
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'.
Where do the killer viruses come from?
Reassortment
of genomic segments
Double infection
animal virus
with avian
and
human influenza virus
needed
human 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 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)
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)
Determinants of viral disease: viral factors AND
host factors
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)
Example: Immunopathology
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
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!!!)
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 !!!
0.1 – 0.25%
Encephalitis:
CNS Involvement:
> 50 %
of the patients
have an altered EEG
Determinants of viral disease: viral factors AND
host factors
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)
Example Host Factor: Age
Age-dependent mortality during influenza pandemics (Lederberg 1997)
1918
United States
Mechanisms of viral pathogenesis
Course of the HIV infection
• Direct killing of virus infected cells by virus
(e.g. HIV)
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)
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
Clinical studies
Advantages
1. Outstanding clinical relevance
Barré-Sinoussi F. et al. Science. 220, 868-71 (1983)
Clinical studies
Advantages
Course of the HIV infection
1. Outstanding clinical relevance
2. Direct information about disease
Clinical studies
Limitations
Course of the HIV infection
1. Cellular and molecular mechanisms of
disease cannot be efficiently studied
Clinical studies
Limitations
1. Cellular and molecular mechanisms of
disease cannot be efficiently studied
2. Association does not predict causality
V. C. Lombardi et al., Science 326, 585-589 (2009)
Experimental
models
Clinical studies
Koch's postulates
Requirements to identify an infectious cause of a disease
1. 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.
Experimental models – in vitro
Cell death
Virus Ag
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
Experimental models – in vitro
Determinants of fitness
Wild type (wt) virus
Deletion (D) Mutant
Revertant virus
Experimental models – in vitro
HIV genome
(wt)
(Dnef)
Experimental models – in vitro
Negative regulators of virus
replication
HIV-1 wt
HIV-1 DNef
HIV-1 Nef rev.
Niderman et al. PNAS 1989
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
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
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
Experimental models – in vivo
Comparison of HIV and SIV genomes
HIV-1
Experimental models – in vivo
Advantage of in vivo over in vitro assays
HIV-1 wt
HIV-1 DNef
SIV wt
Only the in vivo analysis showed that Nef promotes virus replication
HIV-1 Nef rev.
SIV DNef
Niderman et al. PNAS 1989
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
Experimental models – in vivo
Time kinetics of the immune response
window of opportunity
to establish infection
► role back
of the (adaptive)
immune response
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
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 & knockout mice for studying viral pathogenesis
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)
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
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)
Example immune evasion: Human CMV evades control
by CD8+ T cells via multiple mechanisms
viral
proteins
ER
proteasome
US3
Golgi
CMV
1/2
US6
viral
proteins
T cell
TAP
MHC I
US11, US2
nucleus
MHC I
Example immune evasion: Mouse CMV also
evades control by CD8+ T cells
viral
proteins
ER
proteasome
m152
Golgi
CMV
1/2
viral
proteins
T cell
TAP
m152I
MHC
nucleus
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
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
No growth defect of the m152 mutant in mice
lacking MHC molecules
viral
proteins
ER
proteasome
Golgi
CMV
1/2
viral
proteins
T cell
TAP
MHC I
nucleus
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
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
….but take care
What is true for a mouse,
may not be true for a human
Sometimes mice tell lies !
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