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Using Using BioinformaticBioinformatics to Crack the s to Crack the

Flu Code Flu Code

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All About InflenzaAll About InflenzaInfluenza virus Influenza virus

particles dry out particles dry out (half-life a few hours (half-life a few hours at room at room temperature). temperature).

Cold and dry weather Cold and dry weather allows the virus to allows the virus to survive longer survive longer outside the body outside the body than in warm than in warm weather.weather.

Once infected it takes Once infected it takes 1-3 days to get sick.1-3 days to get sick.

Courtesy of Centers for Disease Control and Prevention

It spreads easily by It spreads easily by coughing and coughing and sneezing.sneezing.

In humans, the virus In humans, the virus infects cells lining the infects cells lining the respiratory tract (nose, respiratory tract (nose, throat and lungs).throat and lungs).

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3 Flu 3 Flu Types: Types: Influenza A

Spreads fast

Influenza B Influenza C

Mild infections

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Effect Effect on on

SocietySocietyDeathsDeaths

Medication and Medication and health care health care

loss of working hours loss of working hours nationally. Around nationally. Around 75 million working 75 million working days/year are lost days/year are lost due to influenza.due to influenza. Table showing the mortality/10 million deaths caused Table showing the mortality/10 million deaths caused

by Influenza and Pneumonia (a complication by Influenza and Pneumonia (a complication associated with infection with influenza).associated with infection with influenza).

Source: http://www.cdc.gov/ncidod/EID/vol10no1/02-Source: http://www.cdc.gov/ncidod/EID/vol10no1/02-0705.htm0705.htm

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‘‘Getting the FluGetting the Flu’’

Just about everyone has been sick with Just about everyone has been sick with ‘‘the Fluthe Flu’’ at some stage in their lives. at some stage in their lives.

So, why do people get sick again and So, why do people get sick again and some die when they some die when they ‘‘catch the Flucatch the Flu’’ again?again?

ShouldnShouldn’’t they have immunity to the Flu t they have immunity to the Flu virus?virus?

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Antigens identify Flu Antigens identify Flu strainsstrains

There are two types of antigen = N and H.

In different virus strains, the shapes of N and H are different.

There are 9 known N and 16 known H types.

Influenza viruses are named according to the

antigens (proteins) sticking out of their virus

coat.(H)

(N)

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The role of flu The role of flu virus antigensvirus antigens

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.

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.

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Human Lung Cell

Virus Proteins on cell

surface

H attaches to cell surface proteins so virus can enter cell

Virus genes are released into the cell.

The lung cell is ‘tricked’ into using these genes to make new virus particles.

N cuts the links between the viruses and the cell surface so virus particles are free to go and infect more cells.

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How we respond to How we respond to antigens…antigens…

The H and N antigens are like the The H and N antigens are like the ‘‘faceface’’ of a flu virus. of a flu virus.

If the virus strain has infected you before, the virus If the virus strain has infected you before, the virus ‘‘faceface’’ is recognised and your immune system goes to is recognised and your immune system goes to war fast! The virus is killed off and sometimes you war fast! The virus is killed off and sometimes you dondon’’t even get sick.t even get sick.

If the virus If the virus ‘‘faceface’’ changes slightly (<1% = genetic changes slightly (<1% = genetic drift), it can still be recognised quite quickly and your drift), it can still be recognised quite quickly and your immune system will fight fast. You may be sick for a immune system will fight fast. You may be sick for a few days.few days.

If the virus If the virus ‘‘faceface’’ changes radically (genetic shift = up changes radically (genetic shift = up to 50%), it is not recognised. it takes longer for your to 50%), it is not recognised. it takes longer for your immune system to prepare for war. The virus takes immune system to prepare for war. The virus takes hold and can make you very sick.hold and can make you very sick.

Major changes to the shape of the virus Major changes to the shape of the virus ‘‘faceface’’ can can cause a Pandemiccause a Pandemic

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The 1918 Spanish FluThe 1918 Spanish Flua bigger killer than WWIa 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?What causes a Pandemic?Genetic Shift! LetGenetic Shift! Let’’s use the H5N1 Bird flu as an s use the H5N1 Bird flu as an

example….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 The spread of infection in birds means more humans will come into contact with and be infected by H5N1 bird flu. 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 The Result? A new flu subtype can emerge which easily spreads from person to person. An influenza pandemic would then from person to person. An influenza pandemic would then occur with severe symptoms, like the lethal leakage of fluid occur with severe symptoms, like the lethal leakage of fluid into the lungs caused by the 1918 Spanish flu.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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ReassortmentReassortment

Influenza A infecting a human. Can spread from human to human due to H and N proteins on surface.

Influenza A infecting a chicken. Can occasionally infect humans but cannot 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!

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Viral genes are copied and prepared for packaging into new virus particles.

Repackaging of genes creates a virus that can now transfer from human to human!

PANDEMIC?

Virus coats break down and RNA genes move to the cell nucleus to be copied and transcribed.

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Naming the FluNaming the Flu

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 NB. Occasionally you will find more in the name. For our purposes today, ignore those letters &/or numberspurposes today, ignore those letters &/or numbers

Answer question 3 in your worksheetAnswer question 3 in your worksheet

A/chicken/Korea/01(H9N2)

Influenza A virus was isolated from a chicken in Korea in 2001.

The antigen types were H9 and N2

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The Influenza GenomeThe Influenza Genome

All of the genetic material found in the virus is known as its All of the genetic material found in the virus is known as its genome. The genome is divided into 8 ribonucleoprotein genome. The genome is divided into 8 ribonucleoprotein (RNP) segments. The genetic material is (-) sense RNA (RNP) segments. The genetic material is (-) sense RNA (this is complementary to mRNA)(this is complementary to mRNA)

Source: http://www.omedon.co.uk/influenza/influenza/

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Influenza Influenza has 8 has 8 gene gene

segmentssegments

Sequencing has revealed the Sequencing has revealed the genome for influenza along genome for influenza along with the proteins it codes for.with the proteins it codes for.

Segment Size (nucleotides)

Polypeptide Function

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

6 1413 NA Neuraminidase: release of virus

7 1027 M1 Matrix protein: Major component of virion

M2 Integral membrane protein: Ion channel

8 890 NS1 Anti-interferon protein. Effects on cellular RNA transport

NS2 RNP nuclear export

Source: http://www.omedon.co.uk/influenza/influenza/

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The importance of Flu The importance of Flu Chasers….Chasers….

Scientists monitor the Flu viruses circulating in the population Scientists monitor the Flu viruses circulating in the population by looking at changes to the virus H and N antigens.by looking at changes to the virus H and N antigens.

To identify the strain of flu virus……To identify the strain of flu virus……

Send a sample off to the laboratory for Send a sample off to the laboratory for Gene SequencingGene Sequencing. This . This is an accurate way to find out the sequence of nucleotides is an accurate way to find out the sequence of nucleotides in the viral RNA.in the viral RNA.

After sequencing the H and/or N genes they can compare After sequencing the H and/or N genes they can compare them with the gene sequences from other strains of the them with the gene sequences from other strains of the virus. This lets them look for the mutations that can cause virus. This lets them look for the mutations that can cause epidemics and pandemics.epidemics and pandemics.

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Mutant gene = Mutant Mutant gene = Mutant Protein?Protein?

Yes or No?Yes or No?But does a change or mutation in But does a change or mutation in

the gene sequence always mean the gene sequence always mean there will be a change in the there will be a change in the protein or antigen?protein or antigen?

To work this out we must To work this out we must determine the gene sequence and determine the gene sequence and then the amino acid sequence for then the amino acid sequence for the protein.the protein.

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What is Gene What is Gene Sequencing?Sequencing?

Gene sequencing is identifying and Gene sequencing is identifying and determining the order of the base pairs in a determining the order of the base pairs in a

segment of RNA or DNAsegment of RNA or DNA

A TGAGT

T AA C T C

AT GA TG AA ATGG GAAGAACTTT. ..

Answer question 1 in your worksheet.

20Copyright ©2000 by the National Academy of Sciences

Reid, Ann H. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6785-6790

The 1918 Spanish FluThe 1918 Spanish FluN Gene Sequenced and TranslatedN 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).

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Gene Gene ExpressionExpression

This is This is notnot how transcription happens in the influenza how transcription happens in the influenza virus! The flu virus genome is RNA NOT DNA! But, it virus! The flu virus genome is RNA NOT DNA! But, it still makes mRNA and so we can use the sense DNA still makes mRNA and so we can use the sense DNA

genetic code.genetic code.

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Transcription – copy DNA Transcription – copy DNA into RNAinto RNATry to work out the following:Try to work out the following:

1.1. The second strand of DNA (The second strand of DNA (complementary strandcomplementary strand). Remember the base ). Remember the base pairing rule in DNA, A pairs with T and G pairs with C.pairing rule in DNA, A pairs with T and G pairs with C.

2.2. The code in RNA after copying the 1The code in RNA after copying the 1stst DNA strand (the sense strand). Be DNA strand (the sense strand). Be careful. In RNA, T is replaced by U. An A in the DNA sense strand will see careful. In RNA, T is replaced by U. An A in the DNA sense strand will see U added to the growing RNA strand.U added to the growing RNA strand.

Write your answer down and then click to check your answer.Write your answer down and then click to check your answer.

DNA:DNA: AAT CTG GGG AAC TCG TTT CGC AAT CTG GGG AAC TCG TTT CGC CCC CGACCC CGA

TTA GAC CCC TTG AGC AAA GCG TTA GAC CCC TTG AGC AAA GCG GGG GCTGGG GCT

UUA GAC CCC UUG AGC AAA GCG UUA GAC CCC UUG AGC AAA GCG GGG GCUGGG GCUmRNA:

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mRNA containing the mRNA containing the genetic codegenetic code

copied from the (-) sense RNA virus genome copied from the (-) sense RNA virus genome

moves into cytoplasm of the host cell.moves into cytoplasm of the host cell.

Ready for……. Ready for…….

TranslationTranslation

24Source: http://genetics.nbii.gov/Basic1.html

5’ 3’

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 anti-codon 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.

Anti-codon = 3 bases in tRNA

Amino Acid

Codon = 3 bases in mRNA

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T C A G

T

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]

TGT Cys [C]

TGC Cys [C]

TGA Ter [end]

TGG Trp [W]

T

C

A

G

C

CTT Leu [L]

CTC Leu [L]

CTA Leu [L]

CTG Leu [L]

CCT Pro [P]

CCC Pro [P]

CCA Pro [P]

CCG Pro [P]

CAT His [H]

CAC His [H]

CAA Gln [Q]

CAG Gln [Q]

CGT Arg [R]

CGC Arg [R]

CGA Arg [R]

CGG Arg [R]

T

C

A

G

A

ATT Ile [I]

ATC Ile [I]

ATA Ile [I]

ATG Met [M]

ACT Thr [T]

ACC Thr [T]

ACA Thr [T]

ACG Thr [T]

AAT Asn [N]

AAC Asn [N]

AAA Lys [K]

AAG Lys [K]

AGT Ser [S]

AGC Ser [S]

AGA Arg [R]

AGG Arg [R]

T

C

A

G

F i r s t

P o s i t i o n

G

GTT Val [V]

GTC Val [V]

GTA Val [V]

GTG Val [V]

GCT Ala [A]

GCC Ala [A]

GCA Ala [A]

GCG Ala [A]

GAT Asp [D]

GAC Asp [D]

GAA Glu [E]

GAG Glu [E]

GGT Gly [G]

GGC Gly [G]

GGA Gly [G]

GGG Gly [G]

T

C

A

G

T h i r d

P o s i t i o n

Source: http://psyche.uthct.edu/shaun/SBlack/geneticd.html

Back to page 28

A series of three nucleotides coding for an amino acid in DNA is a A series of three nucleotides coding for an amino acid in DNA is a triplettripletNucleotides in the genetic code correspond to sense strand DNA or mRNA.Nucleotides in the genetic code correspond to sense strand DNA or mRNA.

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The Genetic CodeThe Genetic CodeSince RNA is constructed from four types of nucleotides, there are 64 Since RNA is constructed from four types of nucleotides, there are 64

possible codons (4x4x4). possible codons (4x4x4).

Three of these codons specify the termination of the polypeptide chain Three of these codons specify the termination of the polypeptide chain = STOP codons. = STOP codons.

That leaves 61 codons to specify only 20 different amino acids. That leaves 61 codons to specify only 20 different amino acids.

Most amino acids have more than one codon. Most amino acids have more than one codon. Exceptions to this rule are the START transcription amino acid Exceptions to this rule are the START transcription amino acid

Methionine (Met) and the amino acid Tryptophan (Trp)Methionine (Met) and the amino acid Tryptophan (Trp)

The genetic code is said to be The genetic code is said to be degeneratedegenerate. .

NB/ The sequences coding for the protein N in this activity are NB/ The sequences coding for the protein N in this activity are shown as shown as sensesense DNADNA rather than mRNA. rather than mRNA.

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Cracking the Flu CodeCracking the Flu Code

STUDENT ACTIVITYSTUDENT ACTIVITY

Now you will use Now you will use Comparative GenomicsComparative Genomics

toto

Look for changes or mutations in the gene for influenza N Look for changes or mutations in the gene for influenza N antigen.antigen.

Use the student instructions Use the student instructions AndAnd

Answer the questions in your student worksheet Answer the questions in your student worksheet as you complete these activities.as you complete these activities.

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N SequencesN Sequences1.1. Click on the Click on the ‘‘ii ’’ button above to open the text file button above to open the text file

containing the N sequences you will use in this activity. containing the N sequences you will use in this activity. 2.2. Save this file to your desktop for later use in Biology Save this file to your desktop for later use in Biology

Workbench. Workbench. 3.3. Click the URL below to open The Biology WorkBench to Click the URL below to open The Biology WorkBench to

run sequence alignments:run sequence alignments:

http://workbench.sdsc.edu/

Follow the student instructions to:Follow the student instructions to:

Import the N sequences from the text file on your Import the N sequences from the text file on your desktop into Biology WorkBench, and desktop into Biology WorkBench, and

Run a multiple sequence alignment.Run a multiple sequence alignment.The button on the left will take you The button on the left will take you back to the slide containing the back to the slide containing the genetic code used for translating genetic code used for translating your protein sequences.your protein sequences.

Genetic Code

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TEACHER LED DISCUSSIONTEACHER LED DISCUSSION

INVESTIGATING INVESTIGATING MUTATIONSMUTATIONS

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MutationsMutationsMutations are events which change the sequence of DNA. They can Mutations are events which change the sequence of DNA. They can be very small changes (1 base pair) or very large changes (hundreds be very small changes (1 base pair) or very large changes (hundreds of base pairs). Mutations can involve base substitutions, deletions or of base pairs). Mutations can involve base substitutions, deletions or insertions. They can also involve sequence inversions.insertions. They can also involve sequence inversions.

Mutations can: Mutations can: • affect how genes are expressedaffect how genes are expressed• how RNAs foldhow RNAs fold• how mRNAs are spliced (introns how mRNAs are spliced (introns removed), andremoved), and• how chromosomes segregate. how chromosomes segregate.

We will discuss the impact of We will discuss the impact of mutations on proteins:mutations on proteins:

SubstitutionSubstitution, , deletiondeletion, , insertioninsertion and and inversioninversion events events

Diagram showing different mutation events in DNA.

Source: http://www.uoguelph.ca/mbgwww/courses/94200/Toxicology3.html

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Point MutationsPoint MutationsSingle nucleotide mutations or Point Mutations and their Consequences on the Single nucleotide mutations or Point Mutations and their Consequences on the

genetic code:genetic code:

--Synonymous mutationSynonymous mutation: code for same amino acid (i.e. the genetic code is degenerate): code for same amino acid (i.e. the genetic code is degenerate)

GCGCUU – Alanine – Alanine GCGCCC - Alanine - Alanine

--Missense mutationMissense mutation: codes for different amino acid: codes for different amino acid

- - Conservative: Conservative: chemically similar amino acid (Lys Arg)chemically similar amino acid (Lys Arg)

AAAAG – lysineG – lysine AAGGG – ArginineG – Arginine

- - Nonconservative: Nonconservative: chemically different amino acid (Phe Ser)chemically different amino acid (Phe Ser)

UUUUC – PhenylalanineC – Phenylalanine UUCCC - SerineC - Serine

-Nonsense mutation-Nonsense mutation: stop codon: stop codon

UUGGG – TryptophanG – Tryptophan UUAAG - STOPG - STOP

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Frameshift MutationsFrameshift Mutations

Frameshift mutations occur Frameshift mutations occur when nucleotides are when nucleotides are inserted into or deleted inserted into or deleted from a coding sequence. from a coding sequence.

The worst mutations occur The worst mutations occur when insertions and when insertions and deletions are not in deletions are not in multiples of three. multiples of three.

The reading frame changes The reading frame changes and the amino acids being and the amino acids being added downstream of the added downstream of the mutation alter drastically. mutation alter drastically. This changes the shape of This changes the shape of a protein and generally a protein and generally stops it from doing its job.stops it from doing its job.

A frameshift mutation in the sequence A frameshift mutation in the sequence of the fluof the flu’’s NA gene would alter the s NA gene would alter the active site of this enzyme drastically. active site of this enzyme drastically. Sialic acid residues would no longer Sialic acid residues would no longer bind with the active site. The virus bind with the active site. The virus would stick to the host cell (see would stick to the host cell (see diagram below) so it could no longer diagram below) so it could no longer spread.spread.

Source: http://www.roche.fr/rochefr/planete/internet/internet.jhtml?ssRubrique=1700014

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Investigating MutationsInvestigating MutationsSTUDENT ACTIVITYSTUDENT ACTIVITY

Go back into Biology Workbench to:Go back into Biology Workbench to: Run a multiple sequence alignment using H3N2, H1N1 Run a multiple sequence alignment using H3N2, H1N1

and H5N1 influenza A viral sub-strains. and H5N1 influenza A viral sub-strains. Use the sequence alignment to construct a phylogenetic Use the sequence alignment to construct a phylogenetic

tree.tree.

Follow the student instructionsFollow the student instructions

AndAnd

Answer questions in the student worksheetAnswer questions in the student worksheet