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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

7 Nucleoprotein

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 64 128 256 512 1024 2048

8 16 32 64 128 256 512 1024 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 64 128 256 512 1024 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/


Just a virus! Small viruses - big impact  

This booklet accompanies the 3D film “Just a Virus !” ( 8 minutes ) and goes into more detail on the subject of flu viruses. The material is...

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