viral pathogenesis dr. luka cicin-sain dep. of vaccinology, hzi tel. 0531 6181 4616...

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 factorsDeterminants 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 pandemicIncidence 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




Severity of disease:- virus: ability of infection to kill cells (cytopathic effects);



- immune system: immunity to virus; intact immune response; immunopathology (Hepatitis B)

Jaundice due to infection with hepatitis viruses

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




Severity of disease:- virus: ability of infection to kill cells (cytopathic effects);




- more host factors: general health of the host; host nutritional status(Measles!!!)

Mortality due to MeaslesMortality 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




Severity of disease:- virus: ability of infection to kill cells (cytopathic effects)




- 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

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

http://en.wikipedia.org/wiki/Hiv


• 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




Limitations

1. Cellular and molecular mechanisms of disease cannot be efficiently studied

Clinical studies




1. Cellular and molecular mechanisms of disease cannot be efficiently studied

2. Association does not predict causality

Clinical studies

Limitations

V. C. Lombardi et al., Science 326, 585-589 (2009)

Koch's postulatesRequirements 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.

Clinical studiesExperimental models

http://en.wikipedia.org/wiki/Cell_culture

Experimental models – in vitro

Cell death

Virus Ag


• 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


Determinants of fitness

Wild type (wt) virus

Deletion ( ) D Mutant

Revertant virus

1

2

3

4

5

M36 RevDM36wt

PF

U/m

l (lo

g1

0)

Virus

Virus + zVAD-fmk (death inhibitor)

DM36

M36 rev

Active Casp-3 (cell death)


Wt/Rev DM36

HIV genomeExperimental models – in vitro

(wt)

(Dnef)


HIV genome


Negative regulators of virus replication

HIV-1 wt

HIV-1 DNef

HIV-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


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


Limitations

Comparison of HIV and SIV genomesHIV-1



Benefits 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 wt

Only 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



Benefits

Time kinetics of the immune responseTime kinetics of the immune response

window of opportunityto establish infection

► role back of the (adaptive) immune response


Testing the control of virus with immune cells

Since CD8 depletion increases the virus load, CD8 are important for the control of virus replication


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



Benefits

Transgenic & knockout mice for studying viral pathogenesisTransgenic & knockout mice for studying viral pathogenesis


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)


Transgenic virus & knockout miceTransgenic virus & knockout mice

Adapted from Luker GD et al. J Virol. 2003

N


Immune evasionImmune evasion


- 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

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 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)

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?



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)




Growth capacity of the MCMV m152 mutant in vitro and in vivo

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

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



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

No growth defect of the m152 mutant in mice lacking CD8+ T cells


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)




….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

viral pathogenesis dr. luka cicin-sain dep. of vaccinology, hzi tel. 0531 6181 4616...

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