Prof. Dr.

Martin

Messerle

Institute of Virology, MHH

Tel. x4320
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

Virus = bad news in a protein / membrane coat

Poliovirus:
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

VIRAL PATHOGENESIS
How does a viral infection cause disease in its host ?
Viral pathogenesis:
= entire 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
plus (2) of the immune response on the host

Why study viral pathogenesis?

Goal:

to treat and eliminate viral diseases

many investigators believe that viral pathogenesis

is the most exciting field in virology today
many fundamental questions remain to be
answered
acquire mechanistic knowledge how viruses lead to
disease

How to study viral pathogenesis?

progress in understanding the molecular basis of
viral pathogenesis often comes from
studies of animal models
especially mouse models
(natural virus-host models)
study the interaction of virus and host genes
the mouse genome has been sequenced
mutagenesis of viral genes as well as host genes

Transgenic & knockout mice for studying viral pathogenesis

- small animal model

- mice that express PVR
paralytic disease
no infection by ingestion

.but take care

What is true for a mouse,
may not be true for a human

Mice tell lies !

.but take care

What is true for a mouse,
may not be true for a human

Sometimes mice tell lies !

Fundamental questions of
viral pathogenesis
pathogenesis::

How does a virus enter the host?

What is the initial host response?
Where does primary replication occur?
How does the infection spread in the host?
What organs and tissues are infected?
How is the infection transmitted to other hosts?
Is the infection cleared from the host OR
is a presistent infection established?

Why are we so nervous about bird flu?

1918: Spanish Flu
> 20 -50 Mio. deaths
India: ca. 20 Mio
USA: ca. 0,5 Mio
In USA: initially a bioterroristic attack was
assumed (1918!)
1. World War:
9-10 Mio. deaths

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

Age-dependend mortality during influenza pendemics

Lederberg
Lederberg 1997
1997

1918
United States

Where do the killer viruses come from?

The pig may act as an intermediate host for the generation of humanavian
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

How virulent was the 1918 flu?

The 1918 flu virus is resurrected
Nature 437, 794-795 (2005)
50 times as many virus particles are released from human lung cells
one day after infection with the 1918 virus than with the Texas virus.
13% of body weight is lost by mice 2 days after infection with 1918
flu; weight loss is only transient in mice infected with the Texas strain.
39,000 times more virus particles are found in mouse lung tissue 4
days after infection with 1918 flu than are found with the Texas virus.
All mice died within 6 days of infection with 1918 flu; none died from
the Texas strain.

Virulence determinants: Clevage of

influenza virus hemagglutinin HA0

Some influenza viruses utilize other

proteases for cleavage of HA0

WSN/33

Sequence variation at the cleavage site between HA1

and HA2 determines tissue tropism and pathogenicity
of Avian Influenza viruses
Cleavage site
...Q S R G...

Clara
Trypsin

Protease
bronchial
epithelium

Virus
all mammalian
Influenza viruses

...R/L X R/L R... Furin,

all cells
Subtilisin
family

H5,H7 avian
Influenza viruses

...Q S R G...

Trypsin-independent

H1N1 1918 (Spanish)

...RERRRKK...

Furin

H5N1 1997 (Hong Kong)

The making of a killer virus

1997: a highly lethal influenza virus jumped from birds to humans (Hongkong)
6 of 18 patients were killed
The local poultry was completely culled (ca. 1.6 Mio. animals)

The making of a killer virus

The Hong Kong virus NS1 protein contained a glutamic acid at position 92
change of the Glu to Asp disarms the virus
NS1 is one virulence factor of the Hong Kong Virus
often virulence of influenza and of other viruses is polygenic

NS1 how does it work?

Stop of protein synthesis

( antiviral state)

Interference with IFN functions

a) Blocking of IFN induction/expression
b) Interference with the binding of the
IFN to their receptors
(decoy receptors)
c) Block of the intracellular signaling
pathway
d) Downregulation of the expression of
the ISGs

NS1 how does it work?

Why is the avian H5N1 virus NOT an

efficient killer of humans yet ?
green:
sialic acid
linked to
galactose
via a -2,6
bound

nasal mucosa

paranasal sinus

bronchos

SA2,6Gal
red:
sialic acid
linked to
galactose
via a -2,3
bound
SA2,3Gal
bronchiole

alveolus

Why is the avian H5N1 virus NOT an

efficient killer of humans yet ?
Human
H1N1 virus

Avian
H3N2 virus
similar results
with avian
H5N1 and H4N6
viruses
bronchial epithelial cells

alveolar cells

Virus entry into the host

-

respiratory tract
lung surface: 140 m2 (tennis court),
6 l air/min (10.000-20.000 l/day)
mechanical defense: mucus, ciliated
cells, alveolar M, basement memb.

alimentary tract
200 - 300 m2
hostile environment: stomach acidic;
intestine alkaline; digestive enzymes,
bile detergents
rhinovirus acid-labile; picornavirus
acid-stabile
reovirus activated by proteases
rarely enveloped viruses

Virus entry into the host

-

uro-genital tract
sexual route: HIV, hepatitis B,
papillomaviruses, herpes simplex virus
type 2

eye
tear fluid, immunoprivileged

skin
relatively impermeable barrier
(dead cells)
usually injury is needed:
a scratch or a vector bite, or
inoculation with a needle

Different routes of viral entry

Initiation of a viral infection

- sufficient virus must be available
single droplet of aerosol produced by sneezing:
1 l blood from hepatitis patient:

108 rhinovirus particles

108 hepatitis B virus particles

- cells at site of infection must be accessible,

susceptible, and permissive for the virus
- local host antiviral defense system must be
absent or at least initially ineffective

Viral Spread: Dissemination

- following replication at site of entry, viral particles
(1) can remain localized,
localized
rhinovirus infection in respiratory epithelium

or (2) spread to other tissues

measles virus spread
from respiratory epithelium to other tissues

Viral Spread:
- local spread in epithelium:
newly released virus infects neighboring cells
contained by physical constraints
controlled by the intrinsic & immune defenses

- disseminated infection:
infection
infection spreads beyond the primary site of infection
physical and immune barriers breached
basement membrane compromised by destruction & inflammation
access to subepithelial tissue = clearance or dissemination
(tissue fluid, lymphatic system, phagocytes)

- systemic infection:
infection
if many organs become infected

Viral hematogenous spread:

spread
target organs for viruses that enter at epithelial surfaces and spread via the blood

Viral hematogenous spread:

spread

Viral neural spread:

spread
- viral spread from the primary site of infection
by entering local nerve endings
- very characteristic for certain viruses:
rabies virus, alphaherpesviruses
- for others, invasion of nervous system less frequent diversion
from their normal site of replication and destination
poliovirus, reovirus
- some may replicate in the brain, but spread via the
hematogenous route
mumps virus, HIV, measles virus
- molecular mechanisms that dictate spread
by neural or hematogenous pathways
are not well understood for most viruses

Viral infection of the nervous system

neutrotropic virus:
can infect neuronal cells
infection may occur by neural or hematogenous spread
initiating from a peripheral site

neuroinvasive virus:
enter the central nervous system (spinal cord and brain)
after infection of a peripheral site

neurovirulent virus:
causes DISEASE of nervous tissue
manifested by neurological symptoms and often death

Viral infection of the nervous system

Herpes simplex virus:
low neuroinvasiveness of the CNS, but high neurovirulence
replicates normally in mucuous membranes and enters the
peripheral nervous system
rarely enters the CNS; when, severe, if not fatal consequences

Mumps virus:
high neuroinvasiveness, but low neurovirulence
most infections lead to invasion of central nervous system,
system
but neurological diseases are mild

Rabies virus:
high neuroinvasiveness and high neurovirulence
readily infects peripheral nervous system and spreads to the CNS
with 100% lethality unless antiviral therapy is administered shortly
after infection (vaccination; antibodies)

Viral spread to the central nervous system

Entry into the central nervous system

by olfactory neurons

Determinants of viral disease

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)

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 Southern Africa

Determinants of viral disease

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:

- immune system:

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

Jaundice due to infection with

heaptitis 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

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:

- immune system:
- more host factors:

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)
immunity to virus; intact immune response;
immunopathology (Hepatitis B)
general health of the host; host nutritional status
(Measles!!!)

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:

Vaccination !

0.1 0.25%

CNS Involvement:

> 50 %
of the patients
have an altered EEG

Determinants of viral disease

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:

- immune system:
- more host factors:

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)
immunity to virus; intact immune response;
immunopathology (Hepatitis B)
general health of the host; host nutritional status
(Measles!!!)
host genotype (HLA !, susceptibility genes?)
age of host (influenza)

Successfully infecting viruses

must evade the host defense
- primary infection:

no immediate immune response, no memory

- immunocompromised patients (transplant patients, BMT; HIV)

- viral mechanisms for countering the host defenses
- host factors: some patients are unable to mount an efficient immune
response
- the immune system is unable to clear an infection, gets exhausted
(LCMV; hepatitis C, HIV)
- to avoid immune pathology, a balance between virus and host is
established (chronic, persistant infection; regulatory T cells)

Time kinetics of the immune response

window of opportunity
to establish infection
role back
of the (adaptive)
immune response

Targets of viral immunomodulatory functions

- Humoral response (antibodies, complement)
- Interferons
- Cytokines and Chemokines
- Apoptosis
- Cellular response
Natural Killer Cells (innate)
Cytolytic T lymphocytes (CTL)

Human CMV evades control by CD8+ T cells

via multiple mechanisms
viral
proteins

proteasome

ER
US3

Golgi

CMV
1/2

US6

viral
proteins

T cell
TAP

MHC I

US11, US2

nucleus

MHC I

Mouse CMV also evades control by CD8+ T cells

viral
proteins

proteasome

ER
m152

Golgi

CMV
1/2
viral
proteins

T cell
TAP

nucleus

m152I
MHC

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: Koszinowskis 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: Koszinowskis 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

proteasome

ER
Golgi

CMV
1/2
viral
proteins

T cell
TAP

nucleus

MHC I

No growth defect of the m152 mutant in mice

lacking CD8+ T cells
viral
proteins

proteasome

ER
Golgi

CMV
1/2
viral
proteins

T cell
TAP

nucleus

MHC I

How to study the biological significance of

viral virulence factors?
Basic rules: Koszinowskis 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)