Download influenza viruses

JJUST A VIRUS !
small viruses
– big impact
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Cover : Images from the 3D-film
« Just a Virus ! ».
Dendritic cells.
Graphic : Influenza Virus, p.3.
( Fritz Höffeler )
20-21 16-17 14-15 4-7 18-19 10-11 2-3 8-9 12-13 22-23 VIRUSES ARE EVERYWHERE
FLU VIRUSES… …INFLUENZA VIRUSES
VIRUSES ON THE ATTACK
SMALL AND FEW IN NUMBER
– BUT IMPORTANT SENTINELS
LEADING ACTORS
IN INFLUENZA INFECTIONS
FLU VIRUSES MONITORED WORLDWIDE…
…AND ANALYSED IN LABS THROUGHOUT
THE WORLD
MORE RESEARCH IS required
BIOINFORMATICS – INFLUENZA VIRUSES
GLOSSARY
CONTENTS
Capsid protein
Bacteriophage T4 – bacterial virus
Capsid seen from above
Tobacco mosaic virus (TMV) – plant virus
Single-stranded
RNA/(+)ssRNA
Double-stranded
DNA/dsDNA
Head
Capsid protein
Capsid seen from above
Tail
Tail fibre
Bacteriophage T4 – bacterial virus
Bacteriophage T4 – bacterial virus
Foot-and-mouth disease virus – animal virus
Capsid
Double-stranded
DNA/dsDNA
Double-stranded
Head
Single-stranded
DNA/dsDNA
Head RNA/(+)ssRNA
Tail
Tail fibre
Tail fibre
Foot-and-mouth disease virus – animal virus
Foot-and-mouth disease virus – animal virus
Influenza A virus – animal virus, human flu virus
Single-stranded
Bacteriophage
T4 – bacterialRNA/(+)ssRNA
virus
Envelope
Capsid
Single-stranded
Matrix proteins
RNA/(+)ssRNA
Polymerase
Double-stranded
DNA/dsDNA
Haemagglutinin
Neuraminidase
Head
Tail
Single-stranded
RNA/(-)ssRNA
Tail fibre
RNA (eight different
chromosomes)
surrounded by proteins
Influenza A virus – animal virus, human flu virus
Envelope
Influenza A virus – animal virus, human
flu virus
Human immunodeficiency virus Envelope
(HIV) proteins
Matrix
Foot-and-mouth disease virus – animal
virus
Envelope
Matrix
proteins
Capsid
RNA/(+)ssRNA
Single-stranded
RNA/(+)ssRNA
Capsid protein
Capsid protein
Capsid seen from above
Capsid seen from above
Virus ( Latin : poison,
slime, venom )
Tail
Capsid
Tobacco mosaic virus (TMV) – plant virus
Tobacco
mosaic virus (TMV) – plant virus
Single-stranded
Polymerase
Matrix proteins
Polymerase
Haemagglutinin
Glycoprotein
Single-stranded
Neuraminidase
RNA/(+)ssRNA
Haemagglutinin
RNA (two identical
Neuraminidase
chromosomes)
surrounded by proteins
RNA (eightand
different
Integrase
protease
chromosomes)
RNA (eight different
surrounded by proteins
chromosomes)
surrounded by proteins
Single-stranded
RNA/(-)ssRNA
Single-stranded
RNA/(-)ssRNA
Single-stranded
RNA/(+)ssRNA
Reverse Transcriptase
Human immunodeficiency virus (HIV)
Human immunodeficiency virus (HIV)
Envelope
Envelope
Matrix proteins
Influenza A virus – animal virus, human flu virus
Matrix
proteins
Glycoprotein
Envelope
Glycoprotein
RNA (two identical
Matrix proteins
chromosomes)
RNA (two identical
surrounded by proteins
chromosomes)
Polymerase
Integrase and
surrounded
byprotease
proteins
Haemagglutinin
Integrase and protease
Neuraminidase
Single-stranded
RNA/(+)ssRNA
Reverse Transcriptase
Single-stranded
2
RNA/(+)ssRNA
Reverse Transcriptase RNA (eight different
chromosomes)
Single-stranded
surrounded by proteins
RNA/(-)ssRNA
Viruses are tiny particles with a genome
of double-stranded or
single-stranded
DNA or RNA of very
different
sizes.
Tobacco mosaic
virus (TMV)
– plant virus
Some are
surrounded
Capsid protein
by a protein envelope
( envelopedCapsid
viruses )
seen from above
while others are naked
( non-enveloped viruses ). They can multiply
only in living cells,
onto which they dock
and enter using a “lock
and key” mechanism.
Single-stranded
RNA/(+)ssRNA
Viruses do not have
their own metabolism.
They utilise the machinery in the cells which
they have infected.
→ host-specific/organ-specific
Viruses are dangerous pathogens for plants, animals, and humans
Single-stranded
RNA/(+)ssRNA
viruses are everywhere
Tobacco mosaic virus (TMV) – plant virus
Electron microscope ( EM ) images
Tobacco mosaic virus ( TMV )
Bacteriophage T4
Influenza A virus
HIV
∅ 18 nm, length 300 nm
∅ 78 nm, length 111 nm
∅ 80-120 nm
∅ 100-120 nm
Structure of an influenza virus
Influenza virus chromosomes
Nucleoprotein
1
The genes of the eight viral chromosomes
A
G
U
C
1 Neuraminidase ( NA )
5 Ion channel ( M2 protein )
2 Haemagglutinin ( HA )
6 RNA polymerase complex
3 Matrix protein
7Nucleoprotein
4 Viral envelope
8 Viral RNA
Viruses
Viruses are very small particles,
invisible to the naked eye, that have
a great impact: not only do they infect
bacteria → bacteriophages but they
are also some of the most dangerous
pathogenic agents causing disease
in plants, animals, and humans. lllll
At the end of the 19th century, Dutch
scientists were looking for the cause
of disease in tobacco plants. Filtration
experiments clearly showed that
the disease was not caused by bacteria
→ Beijerinck. lllll In 1935, the pathogens were crystallised. The minute
particles first detected as fine dust in
the electron microscope ( p. 3 ) have
been known as tobacco mosaic viruses
ever since → Bernal → Klug. lllll
The Hershey-Chase experiments
in 1952 using bacteriophage T and
Escherichia coli clearly demonstrated
that the information contained in
the phage DNA alone was sufficient
RNA polymerase complex
(RNA polymerase
+ two binding proteins)
to allow the production of new phages
in the bacteria. This showed DNA
to be the carrier of genetic information
→ Hershey-Chase experiment.
Influenza A viruses
Influenza A viruses infect humans,
birds, pigs, and horses → avian flu
→ swine flu. lllll Between December
and March each year, seasonal
influenza causes acute respiratory
tract infection in some 200,000 people
in Switzerland → flu symptoms
→ influenza. lllll The virus is transmitted mainly by droplet ( aerosol )
spread though sneezing and coughing.
lllll In humans, the viruses affect
the bronchi and, more rarely, the alveoli of the lungs. Sitting on the cell
surfaces are membrane proteins which
influenza viruses utilise as receptors.
Viral surface proteins bind to the membrane proteins using a lock-and-key
mechanism. As soon as the virus has
M1/M2 proteins
Non-structural proteins
Ribonucleotides
Neuraminidase
4
Viral RNA
Nucleoprotein
8
Haemagglutinin
3
RNA polymerase binding protein 2
6
7
RNA polymerase binding protein 1
2
RNA polymerase + two binding proteins
5
docked, the cell wraps it in membrane
and engulfs it into the cell as a vesicle.
The replication cycle can then begin.
Structure of
an influenza virus
The genome of the influenza virus
consists of eight viral negative-sense
RNA → (–)ssRNA strands of different
lengths, which code for all the viral
proteins. Together with nucleoproteins
and an RNA polymerase complex,
they lie within a capsid of matrix proteins, over which is found the outer
viral envelope. Membrane proteins of
the envelope, such as haemagglutinin
( HA ) and neuraminidase ( NA ) are
responsible for the virus entering and
exiting the cell.
3
12
Viruses
are released
1
Adsorption
11
Firmly attached
to the cell membrane
Virus
Cell membrane
2
Cytoplasm
9b
Budding
of new
viruses
Endocytosis
8a
Transport
of membrane
proteins
ER
7a
Translation
of membrane
9a
proteins
Transport
of matrix
proteins
3
Opening
the endosome
Ribosome
6a
Translation
of proteins
10
4
Transport
of nucleoproteins
Migration into
the nucleus
5a
Nuclear
pore
Nucleus
mRNA
synthesis
4
5b
First
replication
step
6b
Second
replication
step
8b
From
the nucleus
to the viral
envelope
7b
Producing
viral
chromosomes
FLU VIRUSES…
1
The infectious cycle ( 1-12 ) of an influenza virus takes about four hours.
1 Adsorption In the human bronchus, influenza virus haemagglutinins
bind to specific cell membrane proteins, which act as receptors. On their
outer sides, these membrane proteins
have short sugar chains with sialic
acid at the end.
chus, dock on cell receptors, and are engulfed by the cells
2
Within the cell, the virus is packed into an
endosome
3
Viral chromosomes diffuse out of the endosome into the cytoplasm
5a
5b
The viral genetic material is replicated and
transcribed in the cell nucleus
2 Endocytosis The cells engulf
the viruses and, packed inside vesicles,
they are transported into the cytoplasm → endosomes → endocytosis.
The influenza virus enters the cell, multiplies,
and exits
Viruses approach the surface of the bron-
3 Opening the endosome The endosomes migrate to the cell
nucleus. In the process, protons ( H+ )
are pumped into the endosomes.
The pH inside the endosome falls to
about 5.0, which alters the spatial
structure ( conformation ) of the viral
haemagglutinin. The viral membrane
can then fuse with the endosomal
membrane (fusion). Openings are
created in the endosome and the viral
chromosomes flow out.
4 Migration into the nucleus The viral chromosomes pass through
nuclear pores to reach the nucleus,
where the nucleoproteins detach
themselves from the negative-sense
RNA → ( – )ssRNA strands.
5a mRNA synthesis The ( – )ssRNA
is transcribed to positive-sense RNA
→ ( + )ssRNA which constitutes
the viral mRNA. Viral RNA polymerase,
which channels the virus onto the RNA
segments in the cell, cannot start
transcription without a small piece of
RNA known as a primer. So the viral
invader grabs the “cap” from the end
of the cell mRNA → cap snatching.
5b first Replication step The viral genome is first transcribed
from ( – )ssRNA to ( + )ssRNA.
6a Translation of proteins The viral mRNA reaches the cytoplasm.
Matrix proteins and nucleoproteins,
as well as the components of the polymerase complex, are synthesised on
free ribosomes.
6b second Replication step The ( + )ssRNA acts as a template for
the production of ( – )ssRNA, which
forms the genome of the new viruses.
7a Translation of membrane
proteins Viral envelope proteins,
e.g. HA and NA, are translated
by the ribosomes into proteins that are
bound to the endoplasmic reticulum
( ER ) membranes. In this way,
the proteins are embedded directly in
the membranes during their synthesis.
7b Producing viral chromosomes Nucleoproteins and
the polymerase complex bind to
the new ( –)ssRNA produced.
8a Transport of membrane proteins Membrane vesicles, containing
viral envelope proteins such as
haemagglutinins and neuraminidases,
bud off from the ER and reach the cell
membrane, with which they fuse.
8b From the nucleus to the viral
envelope The chromosomes
produced in the cell nucleus migrate
through the nuclear pores into
the cytoplasm and, together with
the matrix proteins, find their way to
the newly forming viral envelopes.
9a Transport of matrix proteins Matrix proteins are produced on free
ribosomes in the cytoplasm. They migrate to the cell membrane. They accumulate to form a layer of matrix
proteins beneath the membrane at
the site of the viral envelope proteins.
9b Budding of new viruses The new virus forms gradually and
then buds off from the cell membrane.
10 Transport of nucleoproteins
and rna polymerases Nucleoproteins and components of the viral
polymerase complex are produced on
free ribosomes and then channelled
back into the cell nucleus.
11 Firmly attached to the cell
MEMBRANE Formation of virus
particles (virions) is complete but
the new viruses usually remain
attached to the cell receptors by their
haemagglutinin ( p. 12 ).
12 Release of viruses The viral
envelope protein, neuraminidase, cuts
the newly formed viruses off from
the receptors on the cell surface.
Viruses released from the host cell can
now infect other cells and continue
to multiply.
5
Receptor
OH
5´cap of cell mRNA
COO-
H C H
H C
HO
OH
OH
C H
O
H3C
O
O
…INFLUENZA VIRUSES
They attach themselves
to specific cell receptors and snatch the “cap” off the cell’s messenger RNA
6
Cell receptor with sialic acid
Cap snatching
C
C
H2
O
HO
NH
HO
O
HO
Cap snatching
2-3 bonds in pigs,
birds and horses;
in human alveoli
RNA polymerase
complex on viral
( – )RNA
2-6 bonds in pigs;
in human bronchi
N-acetylneuraminic acid
(sialic acid)
Galactose
2
3
2-3 bond
Viral ( + )RNA
2
2-6 bond
6
Viral ( – )RNA
Cap snatching
In order to replicate themselves,
viruses have to use the host cell
machinery for synthesis. They use
cell nucleotides and amino acids
for transcription, replication, and
translation ( pp. 4 and 5 ). lllll For this
purpose, they split the 5’ terminal
sequence off cell mRNA → 5’ cap.
Viral polymerase, which consists of
three subunits, has an endonuclease
that cleaves the 5’ cap with 10-15 nucleotides from the cell mRNA. This
fragment then serves as a primer
for the viral polymerase in the synthesis of viral mRNA. lllll The 5’ caps
are decisive in several processes.
They protect the mRNA from premature breakdown, are important for
transport out of the nucleus, and help
ribosomes at the start of translation.
By snatching the 5’ cap from the cell
mRNA, viruses paralyse the synthesis
of the cell’s own proteins.
Cell receptors
with sialic acid
Haemagglutinin, a surface protein
of the influenza virus, binds various
cell membrane proteins that have
sugar residues carrying terminal sialic
acid. Sialic acid is bound to galactose
with an a-2,6 linkage in the → epithelial
cells of human → bronchi, while it
has an a-2,3 bond in pulmonary cells. lllll There are 16 different haemagglutinins (H1, H2, H3, etc.); each virus
carries just one HA variant. The variants
sometimes have different affinities
for 2,6 and 2,3 bonds.
Influenza viruses enter the body in inspired air
Viruses approach the outer bronchial membrane, which is covered with cilia
7
Influenza viruses slide down the cilia towards the cell membrane
Natural killer cells migrate towards infected cells
Natural killer cells attack infected cells
Lymphocytes produce antibodies
Antibodies catch the viruses
Antibodies attach themselves to viruses
before they can dock on cell receptors
Dendritic cells with characteristic protrusions capture pathogens
Every day we come into contact with
viruses, so it is crucial that we have an
effective immune system. It takes
up the fight against these invaders.
It also switches off virus-infected cells
so that the viruses are no longer able
to replicate in them. lllll Vertebrates
have two forms of → immunity
to pathogens, firstly nonspecific
or → innate immunity, and secondly
specific or → adaptive immunity.
If a virus, such as the influenza virus,
succeeds in entering the human body,
it infects cells and multiplies inside
them.
Innate immunity
Natural killer ( NK ) cells ( → NK cells )
are part of our innate immunity and
can attack very quickly. They circulate
8
throughout the body and are able to
detect virus-infected cells by sensing
their surface molecules.
Personal ID
All of the body’s nucleated cells carry
a form of personal ID on their surface
→ MHC molecules ( class I ). Some viruses cause the infected cell to ( almost )
stop producing this ID. NK cells sense
this quickly and attack. They bind
the infected cells and give them the
“kiss of death”.
The “kiss of death”
→ apoptosis
On meeting an infected cell, NK cells
release a protein → perforin. As its
name implies, perforin makes holes
in the infected cell. Important ions
such as potassium (K+) now flow out
of the cell through these pores and
there is an influx of water – the infected cell bursts. NK cells cause further
damage to infected cells. They secrete
enzymes → granzymes. Granzymes
enter mainly through the pores in
the cell created by perforin and, once
inside, break down proteins.
Warning signals
Virus-infected cells secrete → interferons, which signal a viral infection
to neighbouring but as yet uninfected
cells. Acting on this information,
uninfected cells produce substances
that inhibit viral replication. This suppresses the spread of viruses from
cell to cell.
Every day we come into contact with viruses. It is crucial that we have an effective immune system
virus on the attack
Different influenza viruses have
different haemagglutinin variants ;
the fragments of each variant fit
into specific receptors on the T cells.
When a T helper cell with receptors
for a particular virus meets a dendritic
cell presenting the corresponding
viral structure ( e.g. a viral haemagglutinin fragment ), it binds to the fragment and is thereby activated.
Memory required !
Adaptive immunity
Besides innate immunity with its
non-specific immune mechanisms,
specific mechanisms exist. These are
not acquired until after an infection.
They start at the same time as
the innate defences but only come
into effect a few days later because of
the long start-up phase. While NK
cells eliminate infected cells and prevent further viral replication,→ viral
antibodies, which are specific to
a particular virus, catch viruses circulating within the body. Antibodies
are not produced until particular
→ lymphocytes ( → B cells ) have been
in contact with the virus. In order
to produce antibodies, these cells have
to be stimulated and activated. Specialised cells of the adaptive immune
system are responsible for this :
→ T helper cells, which are another
type of lymphocyte. But the T cells
themselves also have to be stimulated
beforehand.
Activated T helper cells now move to
another part of the lymph node,
where B lymphocytes in particular are
to be found. B cells possess receptors
for components of the influenza virus.
The B cells are activated once they
are bound to these structures and they
have also received signals from virusspecific T helper cells. They multiply
and mature to → plasma cells,
which can then secrete antibodies
specific to the flu virus ( → primary immune response ). lllll The antibody
fits into the viral structure ( → antigen )
like a key into a lock. Resulting antigen-antibody complexes can be engulfed and digested by → phagocytes
→ macrophages. lllll Some of
the specific plasma cells remain after
the flu infection has been overcome,
and become → memory B cells. These
cells can produce antibodies very
quickly if there is another infection
with the same strain of influenza virus
( → secondary immune response ).
Sentinels
→ Dendritic cells perform a sentinel
function. They are on sentry duty and
at the same time act as the fire alarms
of the immune system. They patrol
the body looking for interlopers.
Should they find one, in this case a flu
virus, they swallow it up. They break
down the virus into fragments and
migrate to the nearest lymph node.
Once there, dendritic cells display
degraded viral components, especially
haemagglutinins, on their surface.
9
SMall and few in number –
but
important
sentinels
Phagocytes have an important role in the immune system
10
One of the dendrocyte’s “arms” has “gripped” a virus
The cell engulfs the virus
Viral fragments are displayed on the dendritic cell
Phagocytes eliminate viruses and cell
debris. Initially the only known
phagocytes were → macrophages,
first described by Ivan Metchnikov,
a Russian biologist. lllll Immunologists later discovered another type
of cell that could take up pathogens :
→ dendritic cells (DCs). The name
originates from the many long finger-like protrusions of these cells,
which spread out like the branches
of a tree ( Greek : dendron = tree ). lllll Dendritic cells are present only in
small numbers ; they are smaller than
macrophages and distributed
throughout the body. lllll DCs occur
as immature and mature cells. When
a micro-organism invades the body,
it causes → inflammation. lllll Immature DCs residing in the tissues
scan their surroundings. They capture
any interlopers, take them up into
the cytoplasm and break them down
( → antigen processing ). They then
migrate to the nearest lymph node,
where they present the pathogenic
antigens on the cell surface. lllll On
the way to the lymph node, they
mature from antigen gathering cells to
→ antigen processing cells. lllll Dendritic cells are considered to be part
of the innate immune system but
they are, in fact, a link between that
system and adaptive immunity
( p. 8 ). lllll On the cell surface, DCs
carry receptors known as pathogenassociated molecular pattern → PAMP
receptors which record the molecular
pattern on pathogens and can then
recognise them. lllll These receptors
include toll-like receptors ( → TLRs ).
In evolutionary terms, TLRs are very
old and have been preserved with time.
The receptors were first identified
in Drosophila fruit flies, the household
pets of geneticists, and given the name
→ Toll. There are several of these TLRs
and they are found particularly on immature dendritic cells. Signals received
via the TLRs affect cell phagocytosis
Receptor
Haemagglutinin
Receptor
Haemagglutinin
Virus
Receptor
DC
The virus is bound by
its haemagglutinin
to receptors on the dendritic
cell ( DC ) and channelled
into the cell.
( → phagocytosis, → endocytosis ),
migration ( → chemotaxis ) and
the secretion of specific messenger
substances ( → cytokines and
→ chemokines ). They also influence
antigen presentation by dendritic
cells to T cells in lymph nodes. lllll As they mature, DCs lose their ability
to engulf pathogens ( phagocytosis )
but become capable of activating
T cells. They can also activate natural
killer cells ( → NK cells ). lllll DCs are
not a uniform group of cells but rather
a family with several different
members. They do not arise from just
one type of precursor cell. The best
known are the conventional myeloid
dendritic cell (mDC) and plasmacytoid
dendritic cell (pDC). Both of these arise
from blood-forming stem cells in
the bone marrow. mDCs and pDCs
circulate in the blood as DC precursors.
Attracted by → chemotactic signals,
the immature cells migrate into
the tissues, where they adhere to
→ chemokines and become resident.
→ Langerhans cells are also considered
to be a type of DC. Langerhans cells are
present in the epithelium and mucosal
membranes, which are particularly
at risk of invasion by pathogenic
organisms and therefore need effective
sentinel cells.
Haemagglutinin
Virus
DC
Virus
DC
Virus
fragment
Virus
fragment
Virus
fragment
MHC
molecule
MHC
molecule
MHC
molecule
Membrane
system
During its passage through
the membrane system,
the virus is broken down.
Specific viral fragments
are coupled to MHC molecules found on the inside of
the membrane vesicle.
Membrane
system
Virus
fragment
Virus
fragment
Virus
fragment
Membrane
system
MHC
molecule
MHC
molecule
MHC
molecule
Transport vesicles carry
the MHC molecules together
with the viral fragments
to the cell margins. Fusion
of the membranes brings
the MHC molecules to
the outer surface of the DC
so that it can now present
these antigens to other cells.
Ralph Steinman –
discoverer
of dendritic cells
In 1970, Ralph Steinman,
a Canadian immunologist,
moved to the laboratory of
the macrophage researcher
Zanvil Cohn at the Rockefeller University in New York.
While working there,
Steinman described how
cells engulf molecules.
→ endocytosis. lllll At
the beginning of the 70s,
immunologists developed
cell culture systems to facilitate their research into
the cellular basis of immunology. They soon realised
that, besides the B and
T cells, another cell type
was necessary and called
them accessory cells. In
the lab, these accessory cells
adhered to glass surfaces
and Steinman looked at
them using various
microscopic techniques.
He discovered a new type
of branching immune cell
which formed rapidly
changing protrusions.
Steinman called them dendritic cells ( DCs ) because
of their tree-like appearance
( Greek : dendron = tree ).
He was convinced that these
dendritic cells were the accessory cells. They were able
to induce T lymphocytes
to divide and T killer cells
to react against antigens.
He was also convinced that
these accessory cells were
not macrophages. lllll The
scientific community was
very slow in recognising
the significance of his discovery. Steinman came
under merciless criticism.
It seemed very far-fetched
that, at a time when molecular cell biology was coming into its own, a new
cell type could be discovered
merely by looking down
the microscope. lllll Steinmann persevered with his
research on dendritic cells,
however, and together with
his co-workers was the first
to describe the role of DCs
in immune reactions.
He demonstrated that DCs
are also present in human
blood. In animal experiments, he was able to
induce immunity against
tumours with antigenladen dendritic cells.
He recognised that DCs
could be activated by pathogenic organisms in order
to induce immunity. lllll In 1868, Paul Langerhans
was the first to describe
cells, subsequently named
Langerhans cells, which
he thought were part of
the nervous system.
They belonged, however,
to the dendritic cells of
the immune system,
first discovered by Ralph
Steinman and Zanvil
A. Cohn in 1973. lllll Steinman was a basic research
scientist but nevertheless
he understood the enormous challenge involved in
transferring laboratory
findings into practice with
patients. With the aid
of dendritic cells he tried to
produce → vaccines. For
his work on dendritic cells,
Steinman (1943-2011)
was awarded the Nobel
Prize in Physiology or Medicine in 2011.
11
Haemagglutinin ( HA )
This protein regulates three important
steps in viral infections. 1. Haemagglutinin enables the influenza
virus to bind to → epithelial cells in
the bronchi or possibly the lungs. lllll 2. It ensures that the viral membrane fuses with the endosomal membrane within the cell. In the process,
holes are created in the endosome,
allowing the RNA segments of
the influenza genome to diffuse into
the cytoplasm and reach the cell
nucleus. lllll 3. When the virus leaves
the cell, it initially remains attached
to the cell surface because the haemagglutinin is still bound to the sialic
acid/sugar residues of the receptors.
Neuraminidase ( NA )
This surface protein is an enzyme.
It cleaves a-2,3 or a-2,6 glycosidic
bonds between the terminal sialic acid
and the receptor sugar residues
(p. 13). lllll Neuraminidase allows
newly formed viruses finally to leave
the cells. In cutting off the sialic acid,
it releases viruses still attached by
their haemagglutinins to the sialic
acid/sugar residues on the receptors.
Studies have shown that viruses
with low NA activity cannot leave
the cells efficiently. lllll The frequent
mutations of the neuraminidase
also pose challenges to producing vaccines → mutations.
12
HA and NA often mutate
Along with their RNA genome,
influenza viruses also introduce their
RNA-dependent RNA polymerases,
without which they cannot replicate.
Errors are made in the synthesis
of the complementary strand, as
the polymerases insert the wrong
bases (nucleotides). A corrective mechanism exists for DNA polymerases,
but this is lacking for RNA polymerases. This is the main reason
why RNA viruses mutate so rapidly.
The virus adapts to the mutation.
Defence measures such as immunisation are less effective or do not work
at all. New vaccines therefore have to
be developed and produced each year. lllll Viral RNA synthesis is the most
susceptible to error. lllll The error rate
for RNA polymerases is about one
error in 104-105 nucleotides. By
comparison, the error rate for DNA
polymerases is about 1:107-109.
LEADING ACTORS IN INFLUENZA
Haemagglutinins and neuraminidases: the two
most important envelope proteins of the influen- infeCtionS
za virus are the leading actors in a flu infection
Influenza viruses have two surface
proteins on their envelopes –
haemagglutinin and neuraminidase.
The haemagglutinins are far more
numerous. lllll These two membrane
proteins, exposed on the surface
of the virus, are potent → antigens,
which provoke a strong immune
response. They are also subject
to frequent mutations. The production
of vaccines is concerned mainly
with these two proteins.
Haemagglutinin unfolds and “pierces” the membrane with three prongs. This creates holes in the endosome and the viral chromosomes diffuse into the cytoplasm.
Neuraminidase (NA)
Haemagglutinin (HA) in action
After the influenza virus has attached itself to the cell receptor
by its haemagglutinin, it is taken up into an endosome
N-acetylneuraminic acid
(sialic acid)
Galactose
membrane
of the virus
2
membrane
of the
endosome
3
2-3 bond
Site of cleavage
by neuraminidase
2
1. An endosome
with an influenza virus
2. A change in pH allows
the HA to start unfolding
2-6 bond
3. The haemagglutinin
“pierces” the endosomal
membrane with
a three-pronged arm
6
Pore
On leaving an infected cell, newly formed viruses remain attached
to the cell receptors by the sialic acid. A viral envelope protein, neuraminidase, acts as an enzyme to cut the sialic acid off from the receptor sugar residue and in this way releases the virus.
4. Conformational rearrangement of the haemagglutinin causes the viral
membrane to fuse with
the endosomal membrane.
5. Openings (pores) appear
in the endosome
6. The viral genome
diffuses into
the cytoplasm
13
A
B
C
This is called → antigen shift. The new
viral strains could then reinfect
humans and possibly cause worse
symptoms. In addition, the virus could
spread from person to person. lllll These are just possible scenarios.
Exactly how the virus would have to be
constructed to realise these scenarios
is not completely clear and remains a
current research problem.
WHO
Influenza viruses are found worldwide,
causing illness and death. Seasonal
flu usually occurs locally, with varying
degrees of severity ( from colds to
episodes of high fever ). → Pandemics
occur periodically.
no reports of human-to-human transmission. lllll In 2009-2010, all
the talk was of swine flu. This influenza virus, H1N1, spread rapidly from
one person to the next, but fortunately
its geographical range was limited.
Spanish flu 1918-1919
Hong Kong flu 1957
Asian flu 1968
Focus on pigs
Spanish flu claimed millions of lives
throughout the world. lllll In 1997,
avian influenza ( “bird flu” ) caused by
the H5N1 strain, broke out in poultry
in Hong Kong. Since then, hundreds
of people who came into direct contact
with these birds (via droppings, feathers, secretions etc.) have been infected
and about half of them have died.
Transmission from birds to humans is
still rare, however, and so far there are
14
Pigs can be infected with various
influenza viruses, whether through
contact with infected birds ( A) or
humans ( B ). Viruses replicating in pigs
can then reinfect birds or humans. lllll If pigs are simultaneously infected with viruses of different origin,
the two viral strains can exchange and
recombine their genetic information
to create new viral variants ( C ). This is
quite easy, as the influenza genome
consists of eight pieces of RNA.
When different viruses infect the same
cell, they can combine freely.
The goal of the World Health Organization is to promote and sustain
human health throughout the world.
It supports national health authorities
with coordinated information
and advice on programmes to combat
disease, especially infectious diseases. lllll WHO was founded in 1948 as
a special organisation of the United
Nations. Its headquarters are in
Geneva. lllll With 194 member states,
WHO is divided into six geographical
regions. Copenhagen is the main centre for Europe. lllll Each year (in February for the northern hemisphere and
September for the southern hemisphere), WHO publishes recommendations on flu vaccine formulations. It is,
however, left to the national health
authorities to produce vaccines with
the updated composition. In contrast
to other vaccines, those for influenza
have to be produced anew each year,
as the viruses change so rapidly ( p. 12 )
→ mutation.
flu viruses monitored
Swiss Federal Office of Public Health (SFOPH),
Bern – National Influenza Reference Centre, worldwide…
Geneva – World Health Organization (WHO), Geneva
Red arrows : bird migration paths from east to west, and to Africa
Blue arrows : bird migration paths from Central America eastwards towards Europe and Africa
What are the WHO
recommendations
based on ?
Doctors in Switzerland, who have
voluntarily joined the Sentinella notification network, submit information
on the number of patients they have
with flu. They send nasopharyngeal
swabs to the influenza monitoring
lab in Geneva, which analyses, characterises, and evaluates the number
of samples on behalf of the SFOPH.
Each year, between 1000 and 1500
samples are analysed in the following
ways :
–Genome analysis of the influenza
virus → RT-PCR
–Virus replication in cell culture
–Characterisation using the haemagglutination test ( p. 17 ). This method
allows the → serotype of the virus
to be determined and compared with
the type used for the vaccine
–Sequencing of the haemagglutinin
gene to determine the virus subtype
–Sequencing of the neuraminidase
gene to identify possible resistance
to available medications.
The results are forwarded to the Global
Influenza Surveillance and Response
System ( GISRS ), a network of public
health laboratories, which acts as
an information centre on influenza
virus spread. On the basis of information collected by the GISRS, WHO
issues warning of epidemics or pandemics that are approaching or
have already broken out, and proposes
relevant countermeasures.
15
…AND ANALYSED IN LABS
Various methods
allow us to char- THROUGHOUT THE WORLD
acterise influenza virus strains
Blood vessel with red blood cells, lymphocytes and antibodies
Red blood cells = erythrocytes
The white coloured cell = a dendritic cell
A
B
16
Antibodies labelled with green dye make influenza viruses visible in cell cultures. A. The negative control shows that no
viruses are present. B. Green fluorescent areas indicate influenza viruses
1 Haemagglutination test
2 Haemagglutination inhibition test
Virus
Virus
Red blood cell (RBC)Red blood cell (RBC)
B.
Virus
Virus
Red blood cell
Red blood cell
B.
Anti-HA antibody Anti-HA antibody
Non-specific antibodies
Non-specific antibodies
A.
A.
Negative
Negative
Positive
Positive
Negative
Negative
Positive
Positive
This test demonstrates the presence of influenza viruses.
The influenza virus haemagglutinin binds to cell membrane
proteins with sugar chains containing sialic acid. If viruses adhere to receptors on the RBCs, the components join together
to form a pale red meshwork of cells. This process is called haemagglutination. lllll A. If there are no viruses in the → serum,
the RBCs sink to the bottom and form a red button (clump). lllll B. Viruses are present in the serum and a pale red meshwork forms.
This test looks for antibodies that recognise the influenza
virus haemagglutinin.
A. If the antibody does not recognise the viral haemagglutinin, the viruses dock on the membrane proteins of the red
blood cells and a pale red meshwork of cells forms. lllll B.
When the antibodies recognise the viral HA and bind to it, the
viruses can no longer dock on the red blood cells. The cells sink
to the bottom and form a red clump. Haemagglutination is
therefore inhibited by the antibodies present in the serum.
Serial dilutions show the relative antibody concentration.
Laboratory tests with titre wells
Laboratory tests with titre wells
2
4
8 16 32 64 1282565121024 2048
8 16 32 64 1282565121024
2048
Sample 1
A/Victoria/
361/11
Sample 2
A/Wisconsin/
15/09
Serial dilutions are made of the sample (1:2, 1:4 etc.) to determine the relative viral concentration, known as the → titre. lllll Sample 1 ( Virus 1 ) causes haemagglutination to a dilution of 1:128 – a titre of 128 – while Sample 2 ( Virus 2 ) has a
titre of 256.
A/Perth/
16/09
Virus 1
Virus 1 has a similar antigen ( haemagglutinin ) to influenza
A/Victoria/361/11.
8 16 32 64 1282565121024
2048
A/Victoria/
361/11
A/Wisconsin/
15/09
A/Perth/
16/09
Virus 2
Virus 2 has a similar antigen ( haemagglutinin ) to influenza
A/Wisconsin/15/09.
17
It is assumed throughout the world that another influenza pandemic could
occur. No-one can predict which virus will be responsible or when or where it
will break out.
more research is required
18
Three major pandemics were caused
by influenza A in the twentieth century. The envelope protein combination
was H1N1 in 1918, H2N2 in 1957,
and H3N2 in 1968. lllll An influenza
pandemic can break out whenever
humans are infected by viruses with
new HA and NA combinations arising
from → antigen shift. The human
immune system is not armed against
the new strain and the pathogen
can spread rapidly, affecting people
of all ages ( p.14 ) → pandemic.
Epidemic
Seasonal → epidemics are recurrent
events. If flu is diagnosed in at least
1.5% of patients, it is referred to
as an epidemic. lllll The effects of
the annually recurring flu epidemic on
society are often underestimated :
days off work, high healthcare costs
( doctor’s visits, hospital stay ), and
deaths. The severity of a flu epidemic
varies from year to year. It has not
yet been explained why there are differences in → virulence and it remains
one of the unanswered research
questions. Viruses do not change only
through new combinations of HA
and NA ( → antigen shift ) but also by
→ point mutation in the HA and
NA genes (→ antigen drift). Segments
of envelope proteins which act as
particularly potent antigens are called
→ epitopes. HA and NA mutations
in these segments have a strong influence on the immune response. lllll The faster the immune response,
the less damage the virus can cause.
When there are new antigens, the immune system needs considerably
more time to eliminate pathogens.
It is therefore worthwhile stimulating
the slow learning process by immunisation with the new antigens. WHO
and the SFOPH recommend an annual
flu jab for specific risk groups, e.g. elderly or immunocompromised people.
Immunisation
( vaccination ) remains
the cornerstone of
flu prevention
→ Vaccines with inactivated flu viruses
have been used for more than 60 years.
Vaccines represent a real breakthrough
in medicine. They reduce the risk of
catching flu at the same time as helping to reduce the spread of the virus
in the population.
Production of vaccines
The production of → vaccines, the immunisation (vaccination) programme,
and its implementation are left to each
individual country. Healthcare professionals inform the population about the
advantages and risks of immunisation
( www.bag.admin.ch/influenza ). lllll It takes 4-6 months to produce a vaccine. The following material may be used :
a. Selected viral strains are grown
in embryonic hens’ eggs, isolated,
inactivated and prepared as
a vaccine. These vaccines no longer
contain any infective viruses.
b. The viral components are further
processed and most of them
removed ( split → virion vaccines ).
c. Only the two most important viral
antigens, HA and NA, are used
for the vaccines ( subunit vaccines ).
Vaccines are produced either with
or without substances intended
to enhance the immune response
( → adjuvant ).
A vaccine mimics
the natural infection
The vaccine induces an immune
response which remains active for
a long time or can be reactivated
much later ( → memory cells ).
A vaccine’s efficiency is determined
by measuring how high the specific
antibody concentration in the serum
becomes after the vaccine has been
administered, compared with serum
antibody levels after a natural
infection. The specificity of the vaccine
is verified with the haemagglutination
inhibition test, determining the antibody titre ( p. 17 ). A vaccine does not
Antibodies capture viruses. They are usually targeted against viral haemagglutinin and block this antigen
Viruses captured by the antibodies are no longer able to infect cells
provide adequate protection
until about two weeks after it has
been given. One prerequisite for
it to be effective is, of course, that
the viruses circulating in the population correspond to those strains
targeted by the vaccine.
New strategies
Vaccines can now be produced in cell
cultures using biotechnological
methods. Cell cultures are less time
consuming and less expensive
than production in embryonic eggs.
They do not contain any traces
of the egg protein which may cause
allergic reactions in some people. lllll Individual viral proteins can also be
produced in isolation. In the case
of influenza, this means HA and NA
antigens in particular. The genes
are inserted into → plasmids as DNA
sequences. The plasmids are multiplied and placed in yeast cells where
the → recombinant protein is produced.
Another type of approved vaccine
contains → virosomes. lllll Vaccines
are generally injected, but nasal sprays
have now also been produced. lllll Antiviral medications affect the viral
replication cycle: on the entry of
the virus into the cell, in the endosome,
or when the virus exits the cell. lllll These medicines have been on
the market for only a few years and
are continuously being developed
further. The focus is on their side
effects and viral resistance. lllll M2 ion channel inhibitors only work
against influenza A, as M2 is not found
in → influenza B or C. The active substance binds directly to the M2 ion
channel, blocks its activity, and
in this way increases the pH within
the virus-containing endosomes.
The higher pH prevents the structural
changes in the haemagglutinin which
are essential for the virus to open
the endosome ( p.13 ). lllll Neuraminidase inhibitors interact with the neuraminidases of influenza A and B.
They inhibit all subtypes N1-N9, preventing the virus from reaching
the bronchi through the mucous covering the epithelial cells, which delays
infection. The inhibitors prevent
the viruses budding from the infected
cells ( p. 12 ), which delays the virus
reaching the expired air and subsequent person-to-person transmission.
Work is in progress on methods for
the targeted presentation of antigens,
e.g. by coupling them to dendritic
cells, which will accelerate antibody
production and result in a quicker and
stronger immune response. lllll Faster and more cost-effective processes
for vaccine production are urgently
required, to allow a more rapid
response to serious situations. lllll In addition, there is a demand for novel
and even more specific medications. lllll Better understanding of the basic
principles of viral infection, the various
modes of transmission, the effects
of an infection, and the activation of
the immune response is still important. Research has to meet the challenges of the future.
A scientific challenge
The aim of research is to produce
vaccines that provide sustained universal immunity against all strains of influenza virus.
19
other animal species, including pigs,
horses, cats, seals and whales. In contrast, influenza B is found only in
humans and has no different HA or NA
subtypes. lllll Viral strains ( subtypes )
that have been analysed are designated
in the following way : Influenza A or
B/origin ( Which animal ? If no species
is stated, then the origin is human )
/place where it was first isolated ( country or state )/number ( determined by
the lab )/year of isolation. HN subtypes
are given in brackets.
Bioinformatics
for monitoring influenza
viruses
Bioinformatics allow us to analyse
and compare DNA, RNA or amino acid
sequences. The findings can be used
to follow the evolution of genes.
Bioinformatics is therefore an important
tool for monitoring rapidly evolving
organisms such as influenza viruses
on an international basis. lllll Evaluation of this information is only possible
through the close cooperation of medical professionals, information technologists, biologists and chemists. lllll Influenza viruses belong to the Orthomyxoviridae family and mutate rapidly.
One of the main reasons for this is
the lack of a corrective mechanism
during replication. In its absence, approximately one base ( nucleotide )
in 10,000 is inserted incorrectly, giving
a very high error rate ( p. 12 ). lllll Mutations in the antigenic envelope
proteins, haemagglutinin ( HA ) and
neuraminidase ( NA ), are the most
important for our immune systems
and for the development of vaccines
and antiviral medications. Sixteen
HA subtypes ( H1, H2, H3 … H16 ) and
nine NA subtypes are known for
influenza A ; these subtypes are serologically different. Antibodies to
one subtype react poorly or not at all
with another subtype. Vaccination
against H1N1 viruses provides hardly
any protection against infection
with H3N2 virus. All these subtypes
circulate in waterfowl. Only a few of
them have been identified in humans,
in particular H1N1, H2N2 and H3N2,
which were responsible for the three
major → pandemics ( p. 14 ). Influenza
A has also been demonstrated in
20
For example :
A/Switzerland/7729/98 (H3N2)
A/swine/Iowa/157/30 (H1N1)
A/Puerto Rico/8/34 (H1N1)
B/Yamagata/16/88 (H3N2)
Current viruses
in close-up
Haemagglutinin mutations have
great effects on the severity of a flu
epidemic, and are therefore the subject
of intensive research. lllll Doctors
send nasopharyngeal swabs to dedicated laboratories. If viruses are present,
their genome is analysed. In the case
of influenza viruses, whose genome
consists of single-stranded RNA divided
into eight chromosomes, the information is first transcribed to DNA and
then sequenced. The haemagglutinin
gene consists of some 1700 nucleotides and, as a protein, haemagglutinin
is about 570 amino acids long.
The aim of this task is to
1.Look for mutations in the viral
sequences presented
2.Demonstrate mutations that could
have effects on the haemagglutinin
protein sequence.
3.Determine the HA subtype by
comparing it with reference strains.
One interesting thing here is that
the latest information becomes available on influenza A viruses recently
detected in Switzerland, as analysed
in the Influenza Reference Centre
in Geneva. lllll You will find relevant
documents and worksheets on
the interpharma website at : www.
biotechlerncenter.interpharma.ch
( Just a Virus ! – Bioinformatics ).
Have fun.
Timeline
of flu infections
Day 1
Contact with people infected with
the flu virus
Days 1-3
Incubation period. Viruses attack
bronchial cells. Viral replication
Days 2-8
Infectious phase without flu
symptoms. Although infected
persons don’t have any symptoms
for 3-5 days ( up to 7 days in
children ), they can still pass
the infection on to other people
during this time.
Days 4-10
Signs and symptoms of flu: cough,
runny nose, fever, tiredness etc.
Complications such as pneumonia
may also occur.
After 2-3 weeks
Antibody production
Bioinformatics
–Influenza Viruses
Bioinformatics are extremely useful for
monitoring influenza viruses
Within the cell: view of nuclear pore and filaments
An endosome with virus near a nuclear pore
Within the nucleus : loosely packed DNA
21
DNA surrounded by histoproteins forms a “string of pearls”
→ Beijerinck, Martinus Willem Dutch mi-
→ Endosome A membrane-enclosed vesicle
nity develops after contact with a foreign sub-
crobiologist ( 1851-1931 ), who developed enrich-
( small bubble ) with an acid pH, found within
stance ( antigen ) and is adapted to the particular
ment cultures for micro-organisms, researched
cells; it contains enzymes which break down pro-
infection. In contrast to innate immunity, it is
tobacco mosaic disease, and recognised that the
teins.
very specific. T and B lymphocytes are the key
pathogen (later identified as the tobacco mosaic
→ Epidemic An infectious disease affecting
cells involved, as well as the memory cells arising
virus) could pass through bacterial filters.
many people at the same time in one locality.
from them.
→ Bernal, John Desmond British physicist
→ Epithelial cells Polar cells with an apex and
→ Adjuvant A substance added to a vaccine,
( 1901-1971 ), who investigated viral structures
a base. The apical side is directed towards the
to enhance the reaction to the antigen. Adjuvants
with the aid of X-rays.
outside of the body ( in the skin ) or inwards into
also make it possible to use smaller quantities of
→ Bronchi The trachea ( windpipe ) divides into
the lumen of the gastrointestinal tract. The api-
antigen in a vaccine, something that may be
two branches, the right and left main bronchi.
cal and basal cell membranes of epithelial cells
important in reducing costs.
These divide tree-like into more branches leading
differ in structure and function.
→ Antibody A protein molecule produced by
into the alveoli of the lungs.
→ Epitope The part of the antigen which is
plasma cells in response to antigens. An anti-
→ 5’ cap Chemical changes in RNA molecules
bound by T or B cell receptors. Synonym : anti-
body binds to its specific antigen. Antibodies are
( after transcription ) in eucaryocytes. The terminal
gen determinant
also called immunoglobulins.
sequence ( “cap” ) increases the stability of mRNA
→ Flu symptoms General malaise, high tem-
→ Antigen A substance which the immune
and is important for the translation of mRNA to
peratures, chills, fatigue, headache, joint pains,
system recognises as foreign. Antibodies and
proteins on the ribosomes.
loss of appetite, nausea, vomiting etc.
lymphocytes bind specifically to their target an-
→ Cap snatching Viruses, including influen-
→ Granzymes Enzymes that are present in
tigen. If the antigen triggers an immune response,
za A, have developed a mechanism to steal mRNA
the granules ( tiny particles ) of cytotoxic T lym-
it is known as an immunogen.
caps: if there is a 5’ cap, they split it off the end
phocytes and natural killer ( NK ) cells ; they serve
→ Antigen drift Changes in viral antigens ( the
of the cell mRNA together with about 10-15 nu-
to eliminate other cells through apoptosis.
main antigens of influenza viruses are the enve-
cleotides.
→ Hershey-Chase experiment With this
lope proteins HA and NA ). Antigen drift arises from
→ Chemokines Small proteins which stimu-
historic experiment, Alfred Hershey and Martha
point mutations in the viral genome. It is due to
late the migration and activation of phagocytes
Chase proved that genetic information is stored
the erroneous replication of the viral genome.
and lymphocytes. They have a key role in inflam-
in DNA and not in protein. They used T4 bac-
Antigen drift in haemagglutinin is responsible
matory reactions.
teriophages with radioactively-labelled sulphur
for the flu epidemics that occur each year.
→ Chemotactic signals ( chemokines ) and phosphorus.
→ Antigen presenting cells Dendritic cells,
Signals which trigger chemotaxis in certain cells
→ Host-specific Species specificity of a
macrophages, and B lymphocytes can present
and, for example, fix them at the site of infection.
pathogenic organism. A pathogen such as a virus
antigens. They can, for example, break down pro-
→ Chemotaxis Tissue injury releases sub-
infects only one biological species ( the host ). In
teins into fragments and, together with other
stances that fix phagocytes at the affected site.
higher animals and humans, viruses often infect
molecules necessary for stimulation, present them
The movement of cells specifically towards these
particular organs, e.g. hepatitis viruses attack the
to T cells.
substances is called chemotaxis.
liver, herpes simplex viruses target the lips; these
→ Antigen processing The breakdown of an-
→ Cytokines Soluble substances which are re-
viruses are termed organ-specific.
tigens to fragments which bind to MCH mole-
leased from the cell and have multiple effects on
→ Immunisation ( vaccination ) A distinc-
cules and together are presented to the T cells.
other cells. This group includes interferons.
tion is made between active and passive immu-
→ Antigen shift Exchange of gene segments
→ Dendritic cells ( DCs ) DCs are cells special-
nisation. Active immunisation : dead or inactivated
( RNA molecules ) between viruses when cells are
ised in antigen presentation. Their name stems
pathogens are injected with the intention of
simultaneously infected with more than one
from their tree-like branching appearance ( Greek :
stimulating immunity against specific patho-
strain of virus. The next generation of viruses then
dendron = tree ).
genic organisms. Passive immunisation : antise-
contains new combinations of RNA segments
→ Endocytosis The cell takes up substances
rum that already contains antibodies to the
and has new properties. This mechanism is par-
or particles by engulfing them : the cell mem-
pathogen is administered.
ticular well-known from influenza viruses.
brane surrounds the particle, invaginates, and
→ Immunity Ability to resist certain organisms
→ Apoptosis Programmed cell death : the cell
pinches off to form a vesicle within the cell. All
that cause disease.
is broken down in such a way that the cell con-
cells have the ability of endocytosis. Endocyto-
→ Inflammation The typical tissue response
tents do not spill out onto surrounding cells.
sis by phagocytes is called phagocytosis.
to injury or infection. It is intended to overcome
Compare with : necrosis
→ Endoplasmic reticulum ( ER ) The ER
the irritation and prevent it from extending, as
→ Avian flu ( bird flu ) Influenza in birds ; the
membrane is in connection with the nuclear en-
well as to repair any damage. The characteristics
H5N1 influenza A variant can be transmitted to
velope. It is divided into smooth endoplasmic re-
of inflammation are redness, warmth, swelling
humans and cause life-threatening disease.
ticulum ( sER ) and rough endoplasmic reticulum
and pain.
→ B cells Also called B lymphocytes. Together
( rER ). Ribosomes (protein factories) orientated to-
→ Influenza Influenza/flu is caused by influ-
with T cells, B cells are one of the main groups of
wards the cytoplasm are found on the rER mem-
enza viruses.
lymphocytes. B cell antigen receptors are anti-
branes ; protein synthesis occurs in the ribosomes.
→ Influenza A, B, and C viruses Influenza
body molecules sitting on the cell membrane.
The ER is particularly well developed in cells spe-
A and B are the main pathogens that cause flu in
Once stimulated by an antigen, they become
cialised in exporting proteins, e.g. antibody-pro-
humans. Influenza C is seldom the causative
plasma cells and produce antibodies.
ducing plasma cells ( mature B lymphocytes ).
agent in humans. Influenza A and B have hae-
→ B memory cells see → memory cells
→ Bacteriophage The term literally means
The sER does not have any ribosomes, hence the
magglutinin ( HA ) and neuraminidase ( NA) as
word smooth. It contains numerous enzymes,
their envelope proteins. Instead of HA and NA,
“bacteria eater”. A virus that infects bacteria and
produces fatty acids and steroid hormones ( e.g.
influenza C carries haemagglutinin-esterase fu-
kills them.
sex hormones ), and is responsible for detoxify-
sion factor ( HEF ). All these proteins are important
ing alcohol and medicines.
for viral uptake into cells.
22
glossary
→ Adaptive immunity This type of immu-
→ Innate immunity A series of non-specific
→ Phagocytes Cells able to engulf and digest
defence mechanisms that are old in evolutionary
pathogens. These cells include macrophages.
→ Titre The dilution step giving the relative
concentration of an antibody or antigen ( e.g.
terms. Unlike adaptive immunity, these defences
→ Phagocytosis The process of engulfing
virus ). The titre is determined by serial dilution :
do not require any previous contact with the an-
particles or bacteria by phagocytic cells.
the sample is progressively diluted in fixed steps.
tigen in order to be effective. The innate immune
→ Plasma cells Mature B cells that produce
→ TLR ( toll-like receptor ) Receptors of the
system includes phagocytes, natural killer cells,
antibodies.
innate immune system, present on macrophages
messenger substances ( cytokines ) and the com-
→ Plasmids Small ring-shaped double-strand-
and dendritic cells, and which trigger an immune
plement system.
ed DNA molecules, found mainly in bacteria.
response. TLRs are proteins similar to the
→ Interferons Interferons are messenger sub-
They can replicate independently of the bacteri-
Drosophila toll protein.
stances that are produced in response to viral or
al chromosomes and are passed on by a cell to
→ Toll receptor Toll describes a mutation in
bacterial infections. There are three subtypes of
its daughter cells. Genes for antibiotic resistance
the fruit fly Drosophila. The embryos have a very
interferon: -, -, and -.
are found on plasmids.
unusual appearance, as they develop mainly ab-
→ Klug, Aaron (1926- ) British biochemist and
→ Point mutation Permanent changes in a
dominal structures. When Christiane Nüsslein
molecular biologist who explained the structure
gene, affecting only one base of a nucleic acid.
Volhard, who later won a Nobel Prize, first saw
of the tobacco mosaic virus using X-rays.
→ Primary response Specific immune reac-
this phenomenon under the microscope, she ex-
→ Langerhans cells Dendritic cells that are
tion after the first contact with an antigen. It is
claimed “Toll!” [German for “great”, “amazing”].
not yet active are called Langerhans cells : they
not as strong as a response following the second
In Drosophila, toll protein is responsible for the
are present in the upper layers of the skin and the
or subsequent antigen contact.
development of the mesoderm. It was also later
mucous membranes.
→ Recombinant protein Proteins produced
found to have a role in immunity.
→ Lymphocytes Subgroup of white blood
through genetic engineering.
→ Vaccine Substance used for immunisation
cells ( leucocytes ). They are further divided into T
→ RT-PCR Acronym for reverse transcriptase
( vaccination ). The name derives from the Latin
and B lymphocytes.
polymerase chain reaction, a method for demon-
vaccina = coming from cows. The first vaccines in
→ Macrophages Macrophages are phago-
strating RNA. The RNA is first transcribed to DNA,
human history came from the pustules of peo-
cytes. They carry pathogen-associated molecular
and this is amplified using PCR.
ple with harmless cowpox. The fluid obtained
pattern receptors which allow them to recognise
→ Secondary response Immune response
was used to prevent infection with smallpox
and engulf bacteria. They also eliminate cell de-
after repeat contact with an antigen. It comes
viruses.
bris and dead cells.
into action more quickly and more strongly.
→ Virion A virus particle outside of a cell. The
→ Memory cells There are both memory T
→ Serotype Variations in viral or bacterial sub-
infective form of a virus.
cells and memory B cells. They emerge during an
groups that can be distinguished by serological
→ Virosome Literally means “viral body”.
immune response, are extremely long-lived cells,
tests. These tests use the properties of antibodies
These are synthetically produced vesicles con-
and allow the immune system to respond much
that bind specifically to certain surface structures
sisting of viral membrane proteins etc. The viro-
more rapidly on re-exposure to the same antigen.
on the pathogen.
some structure is similar to that of the original
They are therefore responsible for the sustained
→ Serum The liquid component of clotted blood,
virus. Virosomes are not replicated but are pure
immunity acquired from vaccination or infec-
without any cells or fibrin; it does, however, con-
active fusion vesicles. They can be used as vac-
tious diseases of childhood.
tain antibodies.
cines. Influenza virosome envelopes contain hae-
→ MHC molecules Proteins with sugar chains
→ (+)ssRNA Positive-sense RNA : this single
magglutinin (HA) and neuraminidase ( NA ).
( glycoproteins ), which are coded in the major his-
stranded RNA (ssRNA) has the same polarity as
→ Virulence The property of a pathogenic
tocompatibility complex ( MHC ) and are important
cell mRNA, so it can be translated directly into pro-
agent to cause infection and illness : the infectious
for antigen presentation to T cells. They are also
teins by the cell’s natural transcription machinery.
potential of a virus.
referred to as histocompatibility antigens ( H an-
→ (–)ssRNA Negative-sense RNA : the single
tigens ). They are divided into MHC class I mole-
stranded RNA ( ssRNA ) of these viruses has the
cules, which are found in all nucleated cells in
opposite polarity to the cell mRNA so, unlike
the body, and MHC class II molecules, which are
positive-sense RNA, it cannot be translated
present only on antigen-presenting cells.
directly into proteins. This RNA has therefore to
→ Mutation A permanent change in the ge-
be transcribed into complementary RNA before
netic material.
translation. The enzyme needed for this is not
→ Natural killer ( NK ) cells Cells of innate
found in the cell, so the virus brings it in as part
immunity, which form the first-line defences
of the virion. Flu viruses belong to the group of
when viruses invade the body.
(–) strand RNA viruses.
→ PAMP Pathogen-associated molecular pat-
→ Swine flu Influenza in pigs, which caused
tern. Molecules that can be found on certain groups
a pandemic in 2009/2010 when the H1N1 strain
of pathogenic agents, recognised by PAMP recep-
infected humans.
tors on cells belonging to the innate immune
→ T cells These cells are also known as T lym-
system.
phocytes. B cells and T cells are the two main
→ Pandemic Spread of an infectious disease
groups of lymphocytes. Functional subgroups of
across countries and continents.
T cells include T helper cells, cytotoxic T cells and
→ Perforin A protein used by cytotoxic T cells
regulatory T cells.
and natural killer cells in order to spring a leak in
→ T helper cells Subgroup of T lymphocytes.
target cells. It causes the death of the target cell
They cooperate with cytotoxic T cells or with B
by making pores in the cell membrane.
cells. Via their T cell receptors, helper cells recognise the antigen bound to MHC class II molecules. See → T cells
23
Learning objectives
By the end of each chapter, you will
be aware of the topics it covers and
be able to answer related questions.
Viruses are everywhere
Viruses attack bacteria, plants,
animals, and human beings.
They are unable to replicate without
a specific host.
Structure of a flu virus.
For advanced students
Viral silhouettes can be seen
under the microscope – what are
the noticeable differences ?
Influenza virus chromosomes –
what is special about the influenza
virus genome ?
Flu viruses…
Influenza viruses
Flu viruses penetrate specific cells
in the body. They need to utilise
a wide range of the cell’s functions in
order to replicate. It is important for
flu viruses to reach the cell nucleus
for this purpose. Flu viruses exit
the cells without destroying them.
The replication ( infectious ) cycle
takes about four hours.
For advanced students
How exactly do influenza viruses
multiply ?
Influenza viruses snatch
the caps off cell mRNA – why ?
Influenza viruses bind specifically
to the cells which they infect – how
do they do this ?
Viruses on the attack
In humans, we make a distinction between innate and adaptive immunity.
Natural killer cells recognise virusinfected cells and eliminate them.
Infected cells secrete molecules
(interferons) as warning signals
to neighbouring cells. The adaptive
immune system produces antibodies
which are targeted against specific
viruses and able to detect them.
Macrophages catch, engulf, and digest
viruses with antibodies attached.
Small and few in number –
but important sentinels
For advanced students
Phagocytes such as macrophages and
dendritic cells have key functions.
They combat invaders and deal with
cell debris. Dendritic cells are found
throughout the body; they are smaller
than macrophages and there are not
so many of them. They belong to
the innate immune system but also
form a bridge to the adaptive system.
Dendritic cells catch invaders such as
viruses and present fragments of these
organisms on their surfaces. Why ?
Leading actors
in influenza infections
Influenza viruses have two important
proteins on their surfaces – haemagglutinin ( HA ) and neuraminidase ( NA ).
These two surface proteins are responsible for efficient viral replication.
They are extremely important in
the production of vaccines, as
they provoke strong responses in
the human immune system. These
two proteins change quickly, i.e.
mutate rapidly.
For advanced students
Haemagglutinin is active, but how ?
Flu viruses monitored
worldwide…
The goal of the World Health Organization ( WHO ) is to promote and maintain
human health. WHO receives
the latest data on the spread of influenza viruses from dedicated laboratories organised on a national scale.
Based on these data, WHO issues
instructions about new vaccines each
year. Each country is at liberty
to follow these instructions. As flu
viruses mutate rapidly, new vaccines
are needed every year, with different
compositions for the northern and
southern hemispheres.
…and analysed in labs
throughout the world
For advanced students
How are influenza viruses
demonstrated in labs throughout
the world ? How does this test
function?
24
More research is required
Seasonal and locally spreading
infectious diseases are called epidemics
when more than 1.5% of patients
show the corresponding symptoms.
Pandemics occur when the infection
spreads across countries and
continents. Immunisation ( vaccination ) mimics the natural infection
and is therefore an important way of
protecting high-risk groups, such
as elderly or immunocompromised
people, against infection. The aim of
research is to develop a vaccine that
can be used effectively for many years.
For advanced students
What is current research doing
to achieve this aim?
Bioinformatics
– influenza viruses
For advanced students
Initial experience of interactive
programmes which compare genes
and amino acid sequences of different
viral strains and, in this way, demonstrate changes (mutations). Such
analysis allows viral gene segments
to be synthesised in the laboratory
and potentially used for vaccine
production. Visit our website : www.
biotechlerncenter.interpharma.ch
( Just a Virus ! – Bioinformatics ).
Imprint
Useful links
Concept, project manager
Scenes from the 3D film
Interpharma, Basel, Switzerland
Dr Esther Schärer-Züblin
“Just a Virus !”
www.interpharma.ch
BioRes Sàrl, Blonay, Switzerland
Nyade, Angoulême, France
www.biotechlerncenter.interpharma.ch
Text
Fluorescent microscopy photos
SFOPH : Swiss Federal Office of Public Health,
Esther Schärer, Dr ès sci.
on page 3 and antibody tests
Berne, Switzerland
Bärbel Häcker
on pages 16 and 17 by kind per-
www.bag.admin.ch/index.html?lang=en
Dr. rer. nat., Leonberg, Germany
mission of
Dr Yves Thomas
Nationales Influenza Referenzzentrum,
Text editors
National Influenza Reference
Geneva, Switzerland
Fritz Höffeler, Biologist
Centre, Geneva, Switzerland
[National Influenza Reference Centre]
Esther Schärer
http://virologie.hug-ge.ch/
Janine Hermann
Bioinformatics, pages 20-21
René Gfeller, PhD
In cooperation with
World Health Organization ( WHO )
Dr Thomas Werner
www.who.int/influenza/
Translation
Kantonsschule Wettingen,
Clipper Uebersetzungen AG,
Switzerland
WHO regions
Zurich
Dr Yves Thomas
www.who.int/about/structure/en/
National Influenza Reference
Scientific graphics
Centre, Geneva, Switzerland
pages 2, 3, 4, 6, 11, 13, 14
Vaccines
www.who.int/influenza/vaccines/
Fritz Höffeler
Revision of the manuscript
Art for Science, Hamburg,
Dr Samuel Ginsburg
Germany
Nora Sandmeier
SF Portal : Impfung
Marc Zünd
[TV programmes: Immunisation]
Layout
www.who.int/immunization_safety/
www.videoportal.sf.tv/
We would like to thank Inter-
www.einslive.de/medien/
pharma, Basel, Switzerland, for
html/1live/2009/11/14/
Electron microscope images on
their support, especially
wissen-macht-ah-impfung.xml
page 3 by kind permission of
Janine Hermann
Karin Palazzolo , www.krnp.ch
Head of Educationals
Centers for Disease Control
and Prevention ( CDC )
( TMV, bacteriophage T4, HIV )
© 2013
www.cdc.gov/flu/
Deutsche Ausgabe : Just a Virus !
Robert Koch Institute, Berlin, Germany
Kleine Viren – grosse Wirkung
www.rki.de/
Dr Takeshi Noda
Version française : Juste un virus !
Myths and facts about flu
Dr Yoshihiro Kawaoka
A petits virus grands effets
Myths about flu : Get the facts
Dr Hans R. Gelderblom
Paul Ehrlich Institute, Berlin,
Germany
( Influenza A )
Institute of Medical Science
www.columbia.edu/cu/studentservices/
University of Tokyo, Japan
preparedness/docs/myths-facts/
www.who.int/vaccine_safety/initiative/
detection/immunization_misconceptions/en/
INFOMED
www.infomed.ch/
pk_template.php?pkid=692
The Rockefeller University
www.rockefeller.edu/about/awards/nobel/
rsteinman/
link to : Lab web page
Virology course
www.virology.ws/virology-101/