BLAST It

1
BLAST It!

• Disease Diagnosis using bioinformatics

2
The Case Study
Whenever you see this diagram, make a diagnosis
and recommend medication to your patient.

• You are a General Practitioner working in
  Melbourne, Australia.
• It is the first of July, the beginning of the
  winter season.
• A male patient 30 years of age walks into your
  office..

3
Symptoms that began 24 hours ago

• During consultation, he tells you that he has
  just arrived home from working in China, mainly
  in Hong Kong.
• Your diagnosis (record this in your
  worksheet).
• Recommended medication.
• Advise to patient.

Source http//www.sla.purdue.edu/academic/fll/Ja
panProj/FLClipart/Medical.html
4
After 47 Hours

• The patient returns with a severe fever. You
  take his temperature and it is 40oC!
• He has a severe cough and complains of difficulty
  breathing. You listen to his chest and
  alarmingly, it sounds like there is a huge build
  up of fluid in the lungs.

Has your diagnosis changed?
Recommended medication. Advise to
patient.
5
After 48 Hours

• Outside your office, he collapses and you are
  called to the scene.
• What is your recommendation for this
  patient?
• You take a blood sample and send it to
  pathology marked URGENT!

6
After 60 Hours

• Blood test results
• Isolation of an Influenza virus
• Isolation of the bacteria Haemophilus Influenzae
• What medication do you recommend?
• You are worried about the speed and extent of
  infection by the influenza virus, given your
  patient was young and fit. You decide to do some
  research into flu viruses.

7
3 Flu Types
What type of influenza do you think your patient
is infected with?
Influenza B
Influenza A Spreads fast
Influenza C Mild infections
8
To identify the Flu virus

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

9
What is Gene Sequencing?
Gene sequencing is identifying and determining
the order of the base pairs in a segment of RNA
or DNA
.
.
.
T
A
T
G
A
T
G
A
A
A
G
G
G
A
A
G
A
A
C
T
T
T
Answer question 1 in your worksheet.
10
Back in the sequencing laboratory

• Scientists are busy sequencing the gene that
  codes for the production of a particular protein
  found on the surface of the virus, the N protein.
• This surface protein is an antigen. Influenza
  antigens are used to identify specific strains of
  this 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. There are 9 known N and 16 known
H types.
(N)
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The role of flu virus 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 2 (a)
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H attaches to cell surface proteins so virus can
enter cell
N cuts the links between the viruses and the cell
surface so virus particles are free to go and
infect more cells.
Virus
Proteins on cell surface
Virus genes are released into the cell. The lung
cell is tricked into using these genes to make
new virus particles.
Human Lung Cell
14
How your patient responds 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 dont even get
sick. If the virus face changes slightly
(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), 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. Answer question 2(b)
in your worksheet. Major changes to the shape of
the virus face can cause a Pandemic
15
Pandemics
Outbreak in Kansas and Africa, 1918
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.
The Hong Kong Flu in 1968 evolved into H3N2.
750,000 people died of the virus worldwide
16
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

17
After 70 HoursYou have your Sequencing Results

• Click on the i button to view the DNA sequence
  for the N antigen from your patient.
• Save this sequence to your desktop for later use.
• You can now use this sequence to find out the
  type of Influenza infecting your patient.
• To do this launch internet explorer and type in
  the following URL (or click on this link)
• http//www.ncbi.nlm.nih.gov/

18
Run an NCBI Search

• Click on BLAST in the menu
• Click on the Nucleotide-nucleotide BLAST (blastn)
• Paste the sequence into the BLAST Search box
• Select others (nr etc) from the options beside
  choose database
• Click on the BLAST! button

• When the next page appears, Click Format and Wait
  for your results.

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Your Results

• Scroll down and look at the alignment for the
  first result. It looks like this
• Compare the two Sequences Answer questions 4 5
  on your worksheet.
• Next, Scroll back up the page until you see a
  list of Sequences producing significant
  alignments Click on the first one.
• Answer questions 6 9 on your worksheet.

20
Your Results cont

• Now use the back arrow on the toolbar to return
  to the previous screen. Look down the list of
  matches for your gene to find other animals
  infected with H5N1.
• Answer questions 10 11 on your worksheet.

21
After 80 Hours
WHO

• Your patient lost consciousness overnight and is
  on life support
• You look up patient mortality for this virus by
  visiting the World Health Organisation (WHO)
  website (click on link button)
• Look at the latest report of human cases
• Answer question 12 in your worksheet.

22
Could H5N1 Cause a Pandemic?

• The epidemic of flu caused by H5N1 began in Korea
  in December 2003. Since then it has spread to
  other Asian 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 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.
• 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.
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!
24
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.
25
Talking to the Parents
WHO site on Avian Influenza

• The parents of the patient would like some
  information on the disease contracted by their
  son. How would you answer the following
  questions for them?
• Answer these questions in your worksheet.
• Q13. How do people contract this disease?
• Q14. Is it possible for this disease to spread
  from our son to those people he was in contact
  with?
• Q15. How did he become infected with this strain
  of Influenza virus?
• Q16. What available treatment might save his life?

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After 130 HoursThe Outcome

• You diagnosed correctly and saved your patient by
  prescribing the following medication
• Relenza a drug designed by Australian
  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 Australian Scientists. Used
  to kill off the secondary bacterial infection in
  your patients lungs caused by Hemophilus
  Influenzae, the causal agent of pneumonia
• Answer question 17 in your worksheet.

27
EMERGENCY MEETING

• Emergency influenza convention to discuss the
  H5N1 pending pandemic. Formulate recommendations
  for the Australian government and report back to
  the Australian Advisory Committee for Infectious
  Diseases.

28
AUSTRALIAN ADVISORY COMMITTEE

• Avoiding 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 Australian Government to
  citizens travelling to Asia.
• Recommend strategies that the Government should
  employ to ensure H5N1 does not enter Australia.
• Recommend precautions that the Government should
  take to ensure Australia is prepared for a H5N1
  influenza outbreak
• Discuss strategies that should be employed to
  stop H5N1 becoming the next Influenza pandemic

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