Viral Pathogenesis Dr. Luka Cicin-Sain Teaching Material: Dep. Of Vaccinology, HZI Tel. 0531 6181 4616 Luka.cicin-sain@helmholtz-hzi.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

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? 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 C332.662 H492.388 N98,245 O131.196 P7.500 S2.340 = a chemical ? What is a virus?

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)

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 6

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 with avian and human influenza virus needed human virus animal 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 (cytotoxic effects); quantity of virus inoculated; duration of virus infection; other infections which might affect immune response (HHV8 / HIV) 10

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 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

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 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

Mortality due to Measles 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

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

Age-dependend mortality during influenza pendemics 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) Overreacting immune system (e.g. Hepatitis) Virus induced oncogenesis (e.g. Cervical Cancer in Papilloma infection, Kaposi Sarcoma) 18

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

Benefits Clinical studies Outstanding clinical relevance Barré-Sinoussi F. et al. Science. 220, 868-71 (1983) Outstanding clinical relevance 20

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

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

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

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.

Experimental models – in vitro Cell death Virus Ag

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

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

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)

Experimental models – in vitro HIV genome

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

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

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 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

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

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

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.

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

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

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

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

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

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)

Transgenic virus & knockout mice Experimental models – in vivo Transgenic virus & knockout mice N Adapted from Luker GD et al. J Virol. 2003

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)

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

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

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

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

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)

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

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

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

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 CD8+ T cells viral proteins ER proteasome Golgi CMV 1/2 T cell viral proteins TAP MHC I nucleus

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)

Sometimes mice tell lies ! ….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 58

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