Transcript BLAST It!

BLAST
It!
(Basic Local Alignment Search Tool)
Disease Diagnosis
using bioinformatics
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The Case Study
Whenever you see this
diagram, make a
diagnosis and
recommend medication
to your patient.
You are a General Practitioner working in
Peabody, America.
It is the first of October, the beginning of flu
season.
A male patient 30 years of age walks into your
office…..
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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.
q
Your diagnosis (record this in Table 1 in your
worksheet).
Recommended treatment.
Advise to patient.
Source: http://www.sla.purdue.edu/academic/fll/JapanProj/FLClipart/Medical.html
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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.
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After 48 Hours…
Outside your office, he collapses and you are called to the
scene.
What is your recommended treatment for
this patient?
You take a blood sample and send it
to pathology marked URGENT!
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After 60 Hours…
Blood test results:
Isolation of an Influenza virus
 Isolation of the bacteria Haemophilus Influenzae
 What medication do you recommend for treatment?

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.
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3 Flu Types:
What type of influenza do you think
your patient is infected with?
Answer question 1 in your worksheet.
Influenza A
Spreads fast
Influenza B
Influenza C
Mild infections
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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 DNA/Gene sequencing?
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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 2 in your worksheet.
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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.
(N)
There are 9 known N
and 16 known H types.
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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 3 (a) + (b)
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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 virus
Human Lung Cell
particles.
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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
don’t 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 3(c) in your worksheet.
Major changes to the shape of the virus ‘face’ can
cause a Pandemic
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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
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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
Complete Table 2 in your worksheet
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After 70 Hours…You have your
Sequencing Results
1.
Right click on the ‘i’ button and choose “Open
hyperlink” to view the DNA sequence for the N
antigen from your patient.
2.
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.
3.
To do this right click on the link below and choose
“Open hyperlink” or copy and paste the link into
your browser:
http://www.ncbi.nlm.nih.gov/
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Run an NCBI Search
4.
5.
6.
7.
8.
Locate and click on BLAST in the menu
Locate and click on the “nucleotide blast” link
Under the heading “Enter Query Search” paste the sequence into the
BLAST Search field
Under the heading “Choose Search Set” select “others (nr etc)” from the
database options
Click on the BLAST button at
the bottom of the page.
9. Wait for your results….
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Your Results
10.
Scroll down past the “Graphic Summary” and “Descriptions” headings for
now and look at the “Alignments” for the first result. It looks like this:
The “Query” sequences is part of your original input.
The “Sbjct” sequences are the resulting matches found in the most likely strain of
flu in the database.
Compare the two Sequences: Answer questions 4 & 5 on your worksheet.
11. Next, Scroll back up the page until you see a list of ‘Sequences producing
significant alignments’ under the “Descriptions” heading.
Answer questions 6 – 10 on your worksheet.
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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
blue “WHO” link button above).
Look at the latest report of human cases
Answer question 12 in your worksheet.
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Could H5N1 Cause a Pandemic? For this section enlarge the PowerPoint to full screen. There are some animations in the
following slides that will not work unless the PPT is full screen.
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.
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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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.
Q16. How do people contract this disease?
Q17. Is it possible for this disease to spread from our son to those people he
was in contact with?
Q18. How did he become infected with this strain of Influenza virus?
Q19. What available treatment might save his life?
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After 130 Hours:
The Outcome
You 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,
the causal agent of pneumonia
Answer question 20 in your worksheet.
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EMERGENCY MEETING
Emergency influenza convention to discuss the
H5N1 pending pandemic. Formulate
recommendations for the American government
and report back to the American Advisory
Committee for Infectious Diseases.
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AMERICAN 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 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
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
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