Week 3 Influenza Virus

Question 1

3.6: Influenza Virus Summary

Questions to think about:
1. What determines the pathogenicity of the virus
2. What determines how easily the virus is transmitted between humans
Exam style question: how can an avian influenza A virus evolve to become one that readily infects humans

Question 2

What does ‘Orthomyxoviridae’ translate to?

How many genera are there? what are they?
How are they told apart?

Which is the major human pathogen?

Answer 2

Orthomyxoviridae
•Name is from the Greek ‘myxa’ = mucus
•There are three genera, distinguished serologically on the basis of their matrix (M) and nucleoprotein (N) antigens. They are:
•Influenzavirus, A, B;
•Influenzavirus C; and
•‘thogoto-like-viruses’ which do not infect humans
•Influenza A virus is the major human pathogen and is the topic of today’s lectures

Question 3

Influenza virus particles

Are highly ______
What shape are they?
What diameter?

Answer 3

Influenza virus particles
Are highly pleomorphic, (ability of some bacteria to alter their shape or size in response to environmental conditions) mostly spherical/ovoid, 80-120nm diameter, but many forms occur, including long filamentous particles

Viruses are impossible to see without powerful microscopes. This is a picture of influenza under high magnification

Question 4

Morphology of Virus

What does the virus inherit from the host cell?
What are the two types of glycoprotein spikes that protrude out of the cell called? what are the percentage ratios?

What is the inner side of the envolope lined by? what is each other their roles?

Answer 4

Morphology of Virus
The virus inherits the lipid envelope from the cell, this forms the outer surface of the particle, which has prominent glycoprotein spikes on them, two types:
Haemagglutinin (HA), a 135Å trimer (80%)

Neuraminidase (NA), a 60Å tetramer (20%)

The inner side of the envelope is lined by matrix proteins (MP)
M1: ion channels
M2: ion channels important in encoating the virus,
Within the MP’s you have the ribonucleic proteins

Question 5

Classification of influenza is based on?

Which are known to infect humans?
Give an example

Answer 5

Influenza A:
Classified on the basis of antigenic relationships of the external spike (protruding) from the nucleus or lipid membrane. (HA and NA spikes)
Hemagglutin (HA) and neuraminidase (NA) proteins, H1- H16 and N1 and N2 are known to infect humans and cause epidemics.
For example: H1N1

Question 6

What is the function of:

HA: Haemagglutinin

NA: Neuraminidase

Answer 6

Function of HA and NA
HAagglutinates erythrocytes - important for attachment and entry of the virus to the host cells
NA removes neuraminic acid (sialic acid) from receptors – for release of new virus from cells
à HA binds the sialic acid receptor on the surface of host cell and NA clips it off allowing release of viral particles. ** **

Question 7

Structure of HA:

Where is the receptor binding site?
Where is teh cleavage site?
When it cleaves what does it split into? what are they called?
What is the role of HA?

Answer 7

Has receptor binding site and the top, and cleavage site at the bottom
When it is cleaved it splits into two chains HA0 and HA2
The role of HA is
- binding virus to human cell
- after HA is cleaved it is involved in fusing endocytotic and viral membrane

Question 8

What is the anatomy of influenza with regards to the role of

M2

M1

Answer 8

Anatomy of influenza

Penetrating the lipid membrane of the virus are molecules of M2 which form ion channels, allowing protons to enter the interior of the virus during replication in the cells.

Inside the matrix shell (M1) are nucleoprotein and RNA transcriptase essential transcription of viral RNA to mRNA during replication

Question 9

Influenza A virus RNA and proteins

How many ribonucleoprotein complexes are there withhin influenza virus A, and how many proteins do they encode for?

Which are the subunits of RNA polymerase

Answer 9

There are 8 ribonucleoprotein complexes within influenza virus A which encode for 11 proteins

< Inventory of the 11 proteins

**PA PB1 and PB2 are all subunits of RNA polymerase **

Question 10

What is the life cycle of influenza virus?

Answer 10

1. HA interacting with the terminal sialic acid sugar on the sensor: glycosylated human cell receptor
   Leads to a process –> endocytosis and the influenza A after endocytosis is in an endosome. Riobnuclear complexes are with a double membrane
2. Process of release called uncoating: requires the fusion of the lipid membrane which surrounds the virus with the membrane which was formed as a result of the endocytosis (which releases the viral contents into the host cell).
3. RNA then become available for replication
   - some make proteins (of which some are glycosylated
   - some go through the rER-Golgi
   - some of the RNA is packaged to produce budding virus

Question 11

Life cycle of influenza virus

1. HA interacting with the terminal sialic acid sugar on the sensor: glycosylated human cell receptor
   Leads to a processà endocytosis and the influenza A after endocytosis is in an endosome. Riobnuclear complexes are with a double membrane

**Describe attachment
Describe endocytosis **

Answer 11

Attachment

Entry into the cell is facilitated by binding of the HA spikes to mucoproteins (sialic acid) containing terminal N-acetyl neuraminic acid (NANA = sialic acid) groups. This interaction can be reversed by polysaccharide cleavage by NA spikes - prevents virus being ‘sequestered’ by inappropriate cell types - NANA is very common on cell surfaces/in mucus and would otherwise swamp the virus particle, effectively ‘neutralizing’ it.

Endocytosis

After binding, the particle is engulfed by endocytosis via coated pits into endocytotic vesicles and finally into endosomes.
These are acidified by the cell; at about pH 5.0, the HA monomers are cleaved in that polybasic region by trypsin-like enzymes in the endosome at the base of the ‘stem’ into the HA1 (upper NH2 portion) N terminal part and HA2 (trans-membrane COOH portion) polypeptides. (Lower part)

Question 12

Life cycle of influenza virus

2. Process of release called uncoating: requires the fusion of the lipid membrane which surrounds the virus with the membrane which was formed as a result of the endocytosis (which releases the viral contents into the host cell).

Answer 12

Fusion

This cleavage causes a conformational change in the HA spike which activates the membrane-fusion function located in HA2. The combination of the close proximity of the virus envelope (viral) and the membrane of the endosome and the active membrane-fusion domain of HA2 results in fusion of the two membranes and passage of the nucleocapsid into the cytoplasm.

Uncoating

pH activated ion-channels made up of M2 protein are also important in uncoating
Sequence specific nuclear targeting sequences in the NP protein result in translocation of the nucleocapsid into the nucleus.
Result of protonation and conformational change

Question 13

3. RNA then become available for replication
   - some make proteins (of which some are glycosylated
   - some go through the rER-Golgi
   - some of the RNA is packaged to produce budding virus

Describe replication?

Answer 13

Replication

During the initial phase of infection (ca. 2h), active host cell DNA synthesis is required. The reason is that the initial step in replication is that PB2 attaches to the m7G cap of host mRNAs. This structure is cleaved from the mRNA by PB1, remaining attached to PB2. The cap serves as a primer for RNA synthesis and 11-15 nucleotides (complementary to the conserved sequence at the 3’ end of the vRNA) are added by PB1, after which PB2 dissociates from the growing strand. PB1 + PA then complete the synthesis of the (+)sense strand.

Two classes of (+) sense RNA are made in infected cells:

Incomplete, 3’ polyadenylated transcripts which are exported to the cytoplasm and serve as mRNAs – used to make protein
cRNA = complete, non-polyadenylated (+)sense copies of the (-)sense vRNA, which serve as template for the synthesis of progeny (-)sense vRNAs.

Question 14

Describe

**Translation of the virus **

**Budding of the virus **

Answer 14

Translation:
Most of the proteins made (e.g. HA, NA) remain in the cytoplasm or become associated with the cell membrane. However, the nuclear protein migrates back into the nucleus, where it associates with newly-synthesized vRNA to form new nucleocapsids. These migrate back out into the cytoplasm and towards the cell membrane. NP level is thought to be a crucial switch in the replication cycle between expression and assembly.

Budding

~4hours after infection, patches of M1 protein form on the cell membrane, which appears to thicken, incorporating HA and NA on the outside of the membrane. The nucleocapsid segments are incorporated into the particle as it buds out through the membrane. NA is thought to have a role in release of budding particles (inhibited by anti-NA Abs).

Question 15

List examples of strains of influenza A

What is the most well-known virus?

Answer 15

The most well-known and tragic in terms of loss of human life is the ‘Spanish Flu’ – H1N1 – killed more people than were killed in the whole of the first world war

Question 16

What does acquired immunity regarding B cells do?

Answer 16

Acquired immunity: B cells
B cells become plasma cells and release antibodies, which recognise and bind to different parts of the virus. Act as markers for immune systems to destroy them

Question 17

Genetic variation

What is the mutation rate for RNA viruses?
Viral RNA polymerase is low-fidelity - what does this mean?
Is human DNA more stable than virus RNA?
What is gene reassortment?

Answer 17

Genetic drift - RNA viruses tend to have high mutation rates – 10⁴ times higher than that of human DNA. Viral RNA polymerase is low-fidelity, so transcription errors accumulate. Also there is no proof reading activity. HA (250 residues) undergoes 2 or 3 amino-acid substitutions per year. Human DNA is much more stable than in viruses ** **

Gene ‘swapping’ or reassortment of genes is known as antigenic shift, if a cell were infected with 2 viruses – then there are 28 (256) possible combinations of the 8 RNA segments

Question 18

Vaccines

How does vaccines work?

Answer 18

Vaccines
Work by tricking your immune system into responding to something that looks like something bad but is actually something pretty harmless
Vaccines
Expose immune system to a defective virus or viral component
Immune system responds and is then ready for next infection –> IMMUNITY.

Question 19

What was the method used?

Answer 19

Vaccine Production
Live virus is injected into fertilized hen’s egg
Virus replicates as embryo develops
Virus-containing fluid is harvested from egg

A vaccine for influenza was first developed in 1945. Traditionally, influenza vaccines have involved growing the virus inside fertilized hen’s eggs. This method is still practiced today.

In this method, live influenza viruses are injected into the eggs. As the embryo develops, the virus replicates to high numbers within the egg. Virus-containing fluid is then harvested from the eggs for use in vaccines.

Question 20

Killed Vaccines

How is the virus killed?
Can the immune system sense the virus?

Live Vaccines ?

Answer 20

Killed Vaccines

Also called inactivated vaccines
Virus is killed by injecting a chemical into the egg
Immune system senses virus, but virus is unable to replicate and spread

Live Vaccines
Also called attenuated vaccines
Virus is still alive, but can’t cause disease

Question 21

FluMist™ nasal spray

Virus designed to grow best at colder temperatures
Flu virus weakened at body temperature

Elaborate on this:

Answer 21

One biotechnology company is developing a vaccine that doesn’t require a needle. The FluMist nasal spray contains a live influenza virus that only grows well at cold temperatures. Inside the body, the virus is unable to grow properly and your immune system fights it off easily.

All the vaccines we have described so far have the same disadvantage: they all need to be grown in eggs. This takes time, effort, and a LOT of eggs.

Question 22

Subunit Vaccines

What do you need to initiate an immune response?

Answer 22

You don’t need the whole virus to get an immune response. As you may recall, your immune system responds to the surface H and N proteins. So, you can create a vaccine containing only H or N. Biotechnology companies use genetic engineering techniques to produce large quantities of H and N proteins in bacteria or yeast cells.

Question 23

Drug functioning as Inhibitors

What are the two types of inhibitors?
What do they do?

Answer 23

Antiviral drugs can be one of two types: neuraminidase inhibitors or hemagglutinin inhibitors. With neuraminidase inhibitors, influenza can still enter host cells but they are unable to leave. Because of this, the viruses can’t spread and infect other cells. Zanamivir and Oseltamivir are the two neuraminidase inhibitors on the market. In contrast, hemagglutinin inhibitors prevent the virus from entering host cells. This results in a decreased infection. The two hemagglutinin inhibitors available are Amantadine and Rimantadine.

Question 24

Why so few drugs?

What is the virus life-cycle dependent on?

Answer 24

•There are far fewer anti-viral drugs than antibacterial drugs because so much of the virus life cycle is dependent on the machinery of its host. There are many agents that could kill off the virus, but they would kill off host cell as well. So the goal is to find drugs that target molecular machinery unique to the virus. The more we learn about these molecular details, the better the chance for developing a successful new drug.
•

Question 25

In 2005 what influenza virus was reconstructed?
What origin was infered?

Why was there alot of media attention?

Answer 25

2005 Spanish influenza reconstructed in 1918 being potentially of avian origin

Avian-like origin of flu is cause for concern

Phylogenetic analysis of PB2 gene
J.K. Taubenberger et al Nature 437, 889-893 (2005) 440, E9-E10 (2006)
Found 1918 sequence might have originated from an avian form

A lot of media fuzz regarding this – ethical consideration, but they were at a very high level of containment i.e safety lab

Question 26

**Species barrier birds to man **

What is the species barrier?

What is the difference in receptor specificity?
For example on H3 and H1, HA, how many amino acid substituions willl switch receptor specificity?

What is swine considered as?

Answer 26

A switch in receptor specificity from sialic acid connected to galactose a2-3 linkages (avian) to a2-6 linkages (human) is a major species barrier. T

For example on H3 & H1 HA frameworks, only need two amino acid substitutions can switch receptor specificity.
Origin of the species barrier: why a human virus will not infect a bird and vice versa. Due to the glycosylation patterns being different

Swine – mixing pot as they can be infected by both as they have receptors for both

Sialic acid linking in humans (2,6) and in avian species is (2,3)

Question 27

Structure and Receptor Specificity of the Hemagglutinin from an H5N1 Influenza Virus
Stevens et al. (2006) Science 312, 404-410

What is the H-N name form Vietnamese influenza virus?
What type of microarray analysis was it?

Answer 27

HA from Vietnamese H5N1 influenza virus is more closely related to the 1918 and other human H1 HAs than to duck H5.
H1 binding specificity can be converted by 2 point mutations.
Glycan microarray analysis of this Viet04 HA reveals it binds the avian a-2-3 sialic acid receptor
Mutations that convert H1 sereotype to human receptor specificity reduced the binding to avian receptor.
However, mutations that convert H2 and H3 to human receptor specificity did permit human receptor binding, which suggests a path for this H5N1 virus to gain a foothold in the human population

Question 28

**Summary 1 **

What does influenza inherit from its host?

What is the role of HA?
What is the role of NA?
Does influenza have a proof reading correction?

Polyrmase is high or low fidelity? what is the mutation rate therefore?

Answer 28

Influenza has a lipid coat, inherited from its host which hides all but the surface HA (haemagglutinin) and NA (neurominidase) proteins.

HA (haemagglutinin) is important in viral attachment to the sialic acid receptor of the host cell.

NA (neurominidase) is important in viral release from the host receptor.

Influenza is a single stranded RNA virus i.e. no proof reading correction

The polymerase is low fidelity which gives rise to high rate of mutation (antigenic drift).

Reassortment of the 8 genetic segments is an important further source of diversity and ability

Question 29

Summary 2

Receptor for HA in sialic acid linked to galactose

…in avian receptor by _______
…in humans by_______

Do humans have any other linkage, and where is it?
Why is swine refered to as mixing pot?
What are the two functions of HA?

Answer 29

The avian receptor for HA is sialic acid linked to galactose by an α1,2 linkage, the human receptor has α2,6 linkage.

Humans have some α2,3 linked sialic acid receptors, but these are buried deep in the lungs and relatively inaccessible.

Swine can act as a “mixing pot” for reassortment as they have both avian and human receptors.

HA has two functions (1) receptor (sialic acid) attachment and (2) after cleavage by a host protease the released fusion peptide of HA brings the viral membrane and endosome membrane together releasing the 8 genetic components into the cell cytoplasm.

H1N1 swine flu (2007) is a reassortment of three swine viruses.

Question 30

Summary 3

What is the evidence that suppourts origin of H1N1 from human/swine and what is the contrast from avian?

What mutations are linked to H1N1 that increase transmissibity?

Answer 30

Origin of H1N1 Spanish flu (1918) is controversial; evidence suggests that the HA and NA segments are closer to human and swine, but the polymerase segments are closer to avian proteins. It seems possible there is a combination of drift & reassortment. Clearly a link to avian influenza.

The presence of a polybasic region in HA means that HA is cleaved (and the fusion function activated) by a wider range of host proteases, this leads to infection of a wider range of tissues and organs.

Transmissibility of the 1918 H1N1 virus is linked to HA and PB2 mutations.

NS1 involved in antagonizing host type I interferon responses.

Eliciting a cytokine storm (which the 1918 Spanish flu did so effectively) damages host tissues; proteins implicated: NS1, PB2, HA and NA