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Using Bioinformatics
to Crack the Flu Code
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All About Inflenza
Influenza virus particles
dry out (half-life a few
hours at room
temperature).
In humans, the virus infects
cells lining the respiratory tract
(nose, throat and lungs).
Cold and dry weather
allows the virus to
survive longer outside
the body than in warm
weather.
Once infected it takes 1-3
days to get sick.
Courtesy of Centers for Disease Control and Prevention
It spreads easily by
coughing and sneezing.
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3 Flu Types:
Influenza A
Spreads fast
Influenza B
Influenza C
Mild infections
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Effect on
Society
Deaths
Medication and health care
loss of working hours
nationally. Around 75
million working
days/year are lost due to
influenza.
Table showing the mortality/10 million deaths caused
by Influenza and Pneumonia (a complication
associated with infection with influenza).
Source: http://www.cdc.gov/ncidod/EID/vol10no1/02-0705.htm
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‘Getting the Flu’
Just about everyone has been sick with ‘the Flu’
at some stage in their lives.
So, why do people get sick again and some die
when they ‘catch the Flu’ again?
Shouldn’t they have immunity to the Flu virus?
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Antigens identify Flu strains
Influenza viruses are
named according to the
antigens (proteins)
sticking out of their virus
coat.
(H)
There are two types of
antigen = N and H.
In different virus
strains, the shapes of
N and H are different.
(N)
There are 9 known N
and 16 known H types.
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The role of flu virus
antigens
The N antigen is required to
cut the virus away from the
host cell so it can spread to
infect more cells.
The N shown above has its
cutting site blocked by a drug
designed stop the flu from
spreading.
The H antigen is like a key that
allows the virus to enter into
cells with a matching lock.
This allows the virus to
replicate inside the cell.
Bird Flu H allows the virus to
infect bird intestinal cells.
Human Flu H allows the virus
to infect human lung cells.
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Virus
N cuts the links between the viruses
H attaches to cell surface
and the cell surface so virus particles
proteins so virus can enter cell
are free to go and infect more cells.
Proteins on cell surface
Virus genes are released into the
cell.
The lung cell is ‘tricked’ into
using these genes to make new
Human Lung Cell
virus particles.
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How we respond to
antigens…
The H and N antigens are like the ‘face’ of a flu
virus.
If the virus strain has infected you before, the virus
‘face’ is recognised and your immune system goes
to war fast! The virus is killed off and sometimes you
don’t even get sick.
If the virus ‘face’ changes slightly (<1% = genetic
drift), it can still be recognised quite quickly and your
immune system will fight fast. You may be sick for a
few days.
If the virus ‘face’ changes radically (genetic shift =
up to 50%), it is not recognised. it takes longer for
your immune system to prepare for war. The virus
takes hold and can make you very sick.
Major changes to the shape of the virus ‘face’ can
cause a Pandemic
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The 1918 Spanish Flu
a bigger killer than WWI
The Spanish Flu pandemic killed more
than 40 million people!
The virus antigens were extremely
different to those encountered previously.
People carried no immunity to this virus
strain so they were highly susceptible to
illness and even death.
It started in America and spread with
soldiers going to war.
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What causes a Pandemic?
Genetic Shift! Let’s use the H5N1 Bird flu as an example….
H5N1 bird flu occasionally infects humans but at this stage humans do not pass
this infection on to other humans.
The spread of infection in birds means more humans will come into contact
with and be infected by H5N1 bird flu.
The concern? Eventually a pig will be infected with a human flu and a bird flu at
the same time. They will serve as a ‘mixing pot’ for the two flu types to swap
genes.
The Result? A new flu subtype can emerge which easily spreads from person to
person. An influenza pandemic would then occur with severe symptoms, like
the lethal leakage of fluid into the lungs caused by the 1918 Spanish flu.
This process of repackaging of viral genes is called reassortment. It is illustrated
in the next slides.
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Reassortment
Influenza A infecting a human.
Can spread from human to
human due to H and N proteins
on surface.
Pig can become infected easily with bird
flu and/or human flu. Serves as a mixing
pot!
Influenza A infecting a
chicken. Can occasionally
infect humans but cannot
spread from human to
human due to H and N
proteins on surface.
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Virus coats break down and
RNA genes move to the cell
nucleus to be copied and
transcribed.
Repackaging of
genes creates a
virus that can
now transfer
from human to
human!
PANDEMIC?
Viral genes are copied and
prepared for packaging into
new virus particles.
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Naming the Flu
A/chicken/Korea/01(H9N2)
Influenza A virus was isolated from a chicken in Korea in
2001.
The antigen types were H9 and N2
Try some yourself:
A/swine/Ehime/80(H1N1)
A/Tokyo/67(H2N2)
A/duck/Hainan/2004(H6N2)
B/Nanchang/97
NB. Occasionally you will find more in the name. For our purposes today, ignore
those letters &/or numbers
Answer question 3 in your worksheet
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The Influenza Genome
All of the genetic material found in the virus is known as its genome. The
genome is divided into 8 ribonucleoprotein (RNP) segments. The genetic
material is (-) sense RNA (this is complementary to mRNA)
Source: http://www.omedon.co.uk/influenza/influenza/
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Influenza has
8 gene
segments
Sequencing has revealed the genome
for influenza along with the proteins
it codes for.
Segment
Size
(nucleotides)
1
2341
PB2
Subunit of polymerase: Host cap binding and endonuclease
2
2341
PB1
Catalytic subunit of polymerase
3
2233
PA
Subunit of polymerase, active in vRNA synthesis
4
1778
HA
Haemagglutinin
5
1565
NP
Nucleoprotein: Part of transcriptase complex
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1413
NA
Neuraminidase: release of virus
7
1027
M1
Matrix protein: Major component of virion
M2
Integral membrane protein: Ion channel
NS1
Anti-interferon protein. Effects on cellular RNA transport
NS2
RNP nuclear export
8
890
Polypeptide
Function
Source: http://www.omedon.co.uk/influenza/influenza/
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The importance of Flu Chasers….
Scientists monitor the Flu viruses circulating in the population by looking at
changes to the virus H and N antigens.
To identify the strain of flu virus……
Send a sample off to the laboratory for Gene Sequencing. This is an
accurate way to find out the sequence of nucleotides in the viral RNA.
After sequencing the H and/or N genes they can compare them with the
gene sequences from other strains of the virus. This lets them look for
the mutations that can cause epidemics and pandemics.
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Mutant gene = Mutant Protein?
Yes or No?
But does a change or mutation in the gene
sequence always mean there will be a
change in the protein or antigen?
To work this out we must determine the
gene sequence and then the amino acid
sequence for the protein.
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What is Gene Sequencing?
Gene sequencing is identifying and determining the order of
the base pairs in a segment of RNA or DNA
A
G
T
G
C
C
T
T
A
A
A
T
A T G AG T A A T GG AGAA GA A C TT T . . .
Answer question 1 in your worksheet.
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The 1918 Spanish Flu
N Gene Sequenced and Translated
The underlined sequence codes for the signal peptide. Boxed amino acids indicate potential
glycosylation sites. Circled amino acids indicate the active site residues (3).
Reid, Ann H. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6785-6790
Copyright ©2000 by the National Academy of Sciences
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Gene Expression
This is not how transcription happens in the influenza virus! The flu virus
genome is RNA NOT DNA! But, it still makes mRNA and so we can use
the sense DNA genetic code.
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Transcription – copy DNA into RNA
Try to work out the following:
1.
2.
The second strand of DNA (complementary strand). Remember the base pairing
rule in DNA, A pairs with T and G pairs with C.
The code in RNA after copying the 1st DNA strand (the sense strand). Be careful.
In RNA, T is replaced by U. An A in the DNA sense strand will see U added to the
growing RNA strand.
Write your answer down and then click to check your answer.
DNA: AAT CTG GGG AAC TCG TTT CGC CCC CGA
TTA GAC CCC TTG AGC AAA GCG GGG GCT
mRNA: UUA GAC CCC UUG AGC AAA GCG GGG GCU
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mRNA containing the genetic code
copied from the (-) sense RNA virus genome
moves into cytoplasm of the host cell.
Ready for…….
Translation
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5’
3’
Codon = 3
bases in
mRNA
Anti-codon
= 3 bases
in tRNA
Amino Acid
TRANSLATION:
1. mRNA locks onto a ribosome.
2. The ribosome reads the mRNA message 3 bases at a time = CODON
3. Transfer RNA (tRNA) molecules carry amino acids. Each tRNA has an anticodon that will only base pair with the correct codon on mRNA.
4. Base pairing occurs between mRNA and tRNA and the new amino acid is
added to a growing chain.
Source: http://genetics.nbii.gov/Basic1.html
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A series of three nucleotides coding for an amino acid in DNA is a triplet
Nucleotides in the genetic code correspond to sense strand DNA or mRNA.
T
T
C
A
TTT Phe [F]
TTC Phe [F]
TTA Leu [L]
TTG Leu [L]
TCT Ser [S]
TCC Ser [S]
TCA Ser [S]
TCG Ser [S]
TAT Tyr [Y]
TAC Tyr [Y]
TAA Ter [end]
TAG Ter [end]
CCT Pro [P]
CCC Pro [P]
CCA Pro [P]
CCG Pro [P]
CAT His [H]
CAC His [H]
CAA Gln [Q]
CAG Gln [Q]
ACT Thr [T]
ACC Thr [T]
ACA Thr [T]
ACG Thr [T]
AAT Asn [N]
AAC Asn [N]
AAA Lys [K]
AAG Lys [K]
F
i
r
CTT Leu [L]
s
CTC Leu [L]
t C
CTA Leu [L]
CTG Leu [L]
P
Back to page 28
o
ATT Ile [I]
s
i A ATC Ile [I]
t
ATA Ile [I]
i
ATG Met [M]
o
n
GTT Val [V]
GTC Val [V]
G
GTA Val [V]
GTG Val [V]
GCT Ala [A] GAT Asp [D]
GCC Ala [A] GAC Asp [D]
GCA Ala [A] GAA Glu [E]
GCG Ala [A] GAG Glu [E]
G
TGT Cys [C] T
TGC Cys [C] C
TGA Ter [end] A
TGG Trp [W] G T
h
CGT Arg [R]
T i
CGC Arg [R]
C r
d
CGA Arg [R]
A
CGG Arg [R]
G P
o
AGT Ser [S]
T s
AGC Ser [S]
C i
AGA Arg [R]
A t
AGG Arg [R]
G i
o
GGT Gly [G]
T n
GGC Gly [G]
C
GGA Gly [G]
A
GGG Gly [G]
G
Source: http://psyche.uthct.edu/shaun/SBlack/geneticd.html
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The Genetic Code
Since RNA is constructed from four types of nucleotides, there are 64 possible codons
(4x4x4).
Three of these codons specify the termination of the polypeptide chain = STOP codons.
That leaves 61 codons to specify only 20 different amino acids.
Most amino acids have more than one codon.
Exceptions to this rule are the START transcription amino acid Methionine (Met)
and the amino acid Tryptophan (Trp)
The genetic code is said to be degenerate.
NB/ The sequences coding for the protein N in this activity are shown as sense
DNA rather than mRNA.
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Cracking the Flu Code
STUDENT ACTIVITY
Now you will use Comparative Genomics
to
Look for changes or mutations in the gene for influenza N antigen.
Use the student instructions
And
Answer the questions in your student worksheet
as you complete these activities.
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N Sequences
Click on the ‘i’ button above to open the text file containing the N
sequences you will use in this activity.
Save this file to your desktop for later use in Biology Workbench.
Click the URL below to open The Biology WorkBench to run sequence
alignments:
1.
2.
3.
http://workbench.sdsc.edu/
Follow the student instructions to:


Import the N sequences from the text file on your desktop into
Biology WorkBench, and
Run a multiple sequence alignment.
Genetic Code
The button on the left will take you back to the
slide containing the genetic code used for
translating your protein sequences.
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INVESTIGATING MUTATIONS
TEACHER LED DISCUSSION
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Mutations
Mutations are events which change the sequence of DNA. They can be very small changes
(1 base pair) or very large changes (hundreds of base pairs). Mutations can involve base
substitutions, deletions or insertions. They can also involve sequence inversions.
Mutations can:
• affect how genes are expressed
• how RNAs fold
• how mRNAs are spliced (introns
removed), and
• how chromosomes segregate.
We will discuss the impact of
mutations on proteins:
Substitution, deletion,
insertion and inversion events
Diagram showing different mutation events in DNA.
Source: http://www.uoguelph.ca/mbgwww/courses/94200/Toxicology3.html
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Point Mutations
Single nucleotide mutations or Point Mutations and their Consequences on the genetic code:
-Synonymous mutation: code for same amino acid (i.e. the genetic code is degenerate)
GCU – Alanine
GCC - Alanine
-Missense mutation: codes for different amino acid
- Conservative: chemically similar amino acid (Lys Arg)
AAG – lysine
AGG – Arginine
- Nonconservative: chemically different amino acid (Phe Ser)
UUC – Phenylalanine
UCC - Serine
-Nonsense mutation: stop codon
UGG – Tryptophan
UAG - STOP
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Frameshift Mutations
Frameshift mutations occur when
nucleotides are inserted into or
deleted from a coding sequence.
The worst mutations occur when
insertions and deletions are not in
multiples of three.
A frameshift mutation in the sequence
of the flu’s NA gene would alter the
active site of this enzyme drastically.
Sialic acid residues would no longer
bind with the active site. The virus
would stick to the host cell (see
diagram below) so it could no longer
spread.
The reading frame changes and the
amino acids being added
downstream of the mutation alter
drastically. This changes the shape
of a protein and generally stops it
from doing its job.
Source:
http://www.roche.fr/rochefr/planete/internet/in
ternet.jhtml?ssRubrique=1700014
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Investigating Mutations
STUDENT ACTIVITY

Go back into Biology Workbench to:
Run a multiple sequence alignment using H3N2, H1N1 and H5N1 influenza
A viral sub-strains.
 Use the sequence alignment to construct a phylogenetic tree.
Follow the student instructions
And
Answer questions in the student worksheet
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