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BLAST BLAST ((Basic Local Alignment Search Tool)Basic Local Alignment Search Tool)

It!It!Disease Diagnosis Disease Diagnosis

using bioinformaticsusing bioinformatics

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The Case The Case StudyStudy

You are a General Practitioner You are a General Practitioner working in Peabody, America.working in Peabody, America.

It is the first of October, the It is the first of October, the beginning of flu season.beginning of flu season.

A male patient 30 years of age walks A male patient 30 years of age walks

into your office…..into your office…..

Whenever you see this Whenever you see this diagram, make a diagram, make a

diagnosis and diagnosis and recommend medication recommend medication

to your patient.to your patient.

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Symptoms that began 24 Symptoms that began 24 hours agohours ago

During consultation, he tells you that he has During consultation, he tells you that he has just arrived home from working in China, just arrived home from working in China, mainly in Hong Kong. mainly in Hong Kong.

Your diagnosis (record this in Your diagnosis (record this in Table 1Table 1 in in your your qq worksheet). worksheet).

Recommended treatment.Recommended treatment.

Advise to patient.Advise to patient.

Source: http://www.sla.purdue.edu/academic/fll/JapanProj/FLClipart/Medical.html

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After 47 After 47 Hours…Hours…

The patient returns with a severe fever. You The patient returns with a severe fever. You take his temperature and it is 40take his temperature and it is 40ooC! C!

He has a severe cough and complains of He has a severe cough and complains of difficulty breathing. You listen to his difficulty breathing. You listen to his chest and alarmingly, it sounds like there chest and alarmingly, it sounds like there is a huge build up of fluid in the lungs.is a huge build up of fluid in the lungs.

Has your diagnosis Has your diagnosis changed? changed?

Recommended Recommended medication.medication.

Advise to patient.Advise to patient.

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After 48 After 48 Hours…Hours…

Outside your office, he collapses and you are Outside your office, he collapses and you are called to the scene.called to the scene.

What is your recommended treatment What is your recommended treatment forfor

this patient?this patient?

You take a blood sample and send it You take a blood sample and send it to pathology marked URGENT!to pathology marked URGENT!

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After 60 After 60 Hours…Hours…

Blood test results:Blood test results: Isolation of an Isolation of an Influenza virusInfluenza virus Isolation of the bacteria Isolation of the bacteria Haemophilus InfluenzaeHaemophilus Influenzae What medication do you recommend for What medication do you recommend for

treatment?treatment?

You are worried about the speed and extent of infection by You are worried about the speed and extent of infection by the influenza virus, given your patient was young and fit. the influenza virus, given your patient was young and fit. You decide to do some research into flu viruses. You decide to do some research into flu viruses.

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3 Flu 3 Flu Types: Types: Influenza A

Spreads fast

Influenza B Influenza C

Mild infections

What type of influenza do you think your patient is infected with?

Answer question 1 in your worksheet.

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To identify the To identify the Flu virus…Flu virus…

You mark a sample of the virus You mark a sample of the virus ‘URGENT!’ and courier it to a ‘URGENT!’ and courier it to a laboratory for laboratory for Gene SequencingGene Sequencing. This . This is an accurate way to find out what is an accurate way to find out what type of flu virus it is. type of flu virus it is.

What is DNA/Gene sequencing?What is DNA/Gene sequencing?

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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 2 in your worksheet.

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Back in the sequencing Back in the sequencing laboratory…laboratory…

Scientists are busy sequencing the Scientists are busy sequencing the gene that codes for the production of a gene that codes for the production of a particular protein found on the surface particular protein found on the surface of the virus, the N protein.of the virus, the N protein.

This surface protein is an antigen. This surface protein is an antigen. Influenza antigens are used to identify Influenza antigens are used to identify specific strains of this virus. specific strains of this 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.

Answer Question 3 (a) + (b)

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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 your patient How your patient responds to responds to antigens…antigens…The H and N antigens are like the ‘face’ of a flu virus. The H and N antigens are like the ‘face’ of a flu virus.

If the virus strain has infected you before, the virus If the virus strain has infected you before, the virus ‘face’ is recognised and your immune system goes to ‘face’ 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 don’t even get sick.don’t even get sick.

If the virus ‘face’ changes slightly (genetic drift), it If the virus ‘face’ changes slightly (genetic drift), it can still be recognised quite quickly and your 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 ‘face’ changes radically (genetic shift), it If the virus ‘face’ changes radically (genetic shift), it is not recognised. it takes longer for your immune is not recognised. it takes longer for your immune system to prepare for war. The virus takes hold and system to prepare for war. The virus takes hold and can make you very sick.can make you very sick.

Answer question 3(c) in your worksheet.Answer question 3(c) in your worksheet.Major changes to the shape of the virus ‘face’ can Major changes to the shape of the virus ‘face’ can cause a Pandemiccause a Pandemic

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PandemicPandemicss

Outbreak in Kansas and Africa, 1918

The Hong Kong Flu in 1968 evolved into H3N2. 750,000 people died of the virus worldwide

The Spanish Flu in 1918, killed approximately 50 million people. It was

caused by the H1N1 strain of influenza A.

The Asian Flu in 1957 was the H2N2 influenza A strain.

Worldwide it is estimated that at least one million people died

from this virus.

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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

Complete Complete Table 2Table 2 in your worksheet 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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AfterAfter 70 Hours…You 70 Hours…You have your Sequencing have your Sequencing

ResultsResults1.1. Right click on the ‘i’ button and choose “Open Right click on the ‘i’ button and choose “Open

hyperlink” to view the DNA sequence for the N hyperlink” to view the DNA sequence for the N antigen from your patient.antigen from your patient.

2.2. Save this sequence to your desktop for later Save this sequence to your desktop for later use.use.

You can now use this sequence to find out the You can now use this sequence to find out the type of Influenza infecting your patient.type of Influenza infecting your patient.

3.3. To do this right click on the link below and To do this right click on the link below and choose “Open hyperlink” or copy and paste the choose “Open hyperlink” or copy and paste the link into your browser:link into your browser:

http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/

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Run an NCBI SearchRun an NCBI Search4.4. Locate and click on Locate and click on BLASTBLAST in the menu in the menu5.5. Locate and click on the “Locate and click on the “nucleotide blast” linknucleotide blast” link6.6. Under the heading “Enter Query Search” paste the sequence into the BLAST Under the heading “Enter Query Search” paste the sequence into the BLAST

SearchSearch field field7.7. Under the heading “Under the heading “Choose Search SetChoose Search Set” select “others (nr etc)” from the ” select “others (nr etc)” from the

database optionsdatabase options8.8. Click on the Click on the BLASTBLAST button at button at

the bottom of the page.the bottom of the page.

9.9. Wait for your results….Wait for your results….

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Your ResultsYour Results10.10. Scroll down past the “Graphic Summary” and “Descriptions” Scroll down past the “Graphic Summary” and “Descriptions”

headings for now and look at the “Alignments” for the first headings for now and look at the “Alignments” for the first result. It looks like this:result. It looks like this:

The “Query” sequences is part of your original input. The “Query” sequences is part of your original input. The “Sbjct” sequences are the resulting matches found in the most The “Sbjct” sequences are the resulting matches found in the most

likely strain of flu in the database. likely strain of flu in the database. Compare the two Sequences: Answer questions 4 & 5 on your Compare the two Sequences: Answer questions 4 & 5 on your

worksheet.worksheet.11.11. Next, Scroll back up the page until you see a list of ‘Sequences Next, Scroll back up the page until you see a list of ‘Sequences

producing significant alignments’ under the “Descriptions” producing significant alignments’ under the “Descriptions” heading.heading.

Answer questions 6 – 10 on your worksheet.Answer questions 6 – 10 on your worksheet.

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After 80 After 80 HoursHours

Your patient lost consciousness overnight and Your patient lost consciousness overnight and is on life supportis on life support

You look up patient mortality for this virus by You look up patient mortality for this virus by visiting the World Health Organisation visiting the World Health Organisation (WHO) website (click on blue “WHO” link (WHO) website (click on blue “WHO” link button above). button above).

Look at the latest report of human casesLook at the latest report of human cases

Answer question 12 in your worksheet.Answer question 12 in your worksheet.

WHO

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Could H5N1 Cause a Could H5N1 Cause a Pandemic?Pandemic?

The epidemic of flu caused by H5N1 began in Korea in The epidemic of flu caused by H5N1 began in Korea in December 2003. Since then it has spread to other Asian December 2003. Since then it has spread to other Asian countries.countries.

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.occur.

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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Talking to the ParentsTalking to the Parents

The parents of the patient would like some The parents of the patient would like some information on the disease contracted by their information on the disease contracted by their son. How would you answer the following son. How would you answer the following questions for them? questions for them? Answer these questions in your worksheet.Answer these questions in your worksheet.

Q16.Q16. How do people contract this disease?How do people contract this disease?

Q17.Q17. Is it possible for this disease to spread from our son Is it possible for this disease to spread from our son to those people he was in contact with?to those people he was in contact with?

Q18.Q18. How did he become infected with this strain of How did he become infected with this strain of Influenza virus?Influenza virus?

Q19.Q19. What available treatment might save his life?What available treatment might save his life?

WHO site on Avian Influenza

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After 130 After 130 Hours:Hours:

The OutcomeThe OutcomeYou diagnosed correctly and saved your patient by

prescribing the following medication: • Relenza – a drug designed by American scientists

which stops ‘N’ from doing its job. The virus cannot spread from cell to cell.

• Antibiotics – Anti-bacterial drugs first made available for use by American Scientists. Used to kill off the secondary bacterial infection in your patients lungs caused by Hemophilus Influenzae, Hemophilus Influenzae, the causal agent of pneumonia

Answer question 20 in your worksheet.

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EMERGENCY MEETINGEMERGENCY MEETING

Emergency influenza convention to Emergency influenza convention to discuss the H5N1 pending pandemic. discuss the H5N1 pending pandemic. Formulate recommendations for the Formulate recommendations for the

American government and report American government and report back to the American Advisory back to the American Advisory

Committee for Infectious Diseases.Committee for Infectious Diseases.

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AMERICAN AMERICAN ADVISORY ADVISORY

COMMITTEECOMMITTEE

Source for photograph: http://i.cnn.net/cnn/2003/HEALTH/03/27/illness.qa/story.hk.mystery.illness.af.jpgwww.who.int/.../ 2002/illness.htmlwww.who.int/.../ 2002/illness.html

Avoiding an H5N1 Influenza PandemicAvoiding an H5N1 Influenza Pandemic Your class consists of delegates attending a convention on influenza. Together you will:

•Report on travel warnings that you believe should be issued by the American Government to citizens travelling to Asia.

•Recommend strategies that the Government should employ to ensure H5N1 does not enter America.

•Recommend precautions that the Government should take to ensure America is prepared for a H5N1 influenza outbreak

•Discuss strategies that should be employed to stop H5N1 becoming the next Influenza pandemic