BLAST It Basic Local Alignment Search Tool Disease
BLAST It! (Basic Local Alignment Search Tool) Disease Diagnosis using bioinformatics 1
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…. . 2
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/Japan. Proj/FLClipart/Medical. html 3
After 47 Hours… The patient returns with a severe fever. You take his temperature and it is 40 o. C! 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. 4
After 48 Hours… Outside your office, he collapses and you are called to the scene. for What is your recommended treatment this patient? You take a blood sample and send it to pathology marked URGENT! 5
After 60 Hours… Blood test results: n Isolation of an Influenza virus n Isolation of the bacteria Haemophilus Influenzae n 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. 6
3 Flu Types: Influenza A Spreads fast What type of influenza do you think your patient is infected with? Answer question 1 in your worksheet. Influenza B Influenza C Mild infections 7
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? 8
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 AT G AG T A A T GG AGAAGAAC T T T. . . Answer question 2 in your worksheet. 9
Back in the sequencing laboratory… n 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. n This surface protein is an antigen. Influenza antigens are used to identify 10
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. 11
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. 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. Bird Flu H allows the virus to infect bird intestinal cells. Human Flu H allows the virus to infect human lung cells. Answer Question 3 (a) + 12
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 are free to go and infect more cells. cell 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. 13
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 14
Pandemics Outbreak in Kansas and Africa, 1918 The Spanish Flu in 1918, killed approximately 50 million people. It was caused by the H 1 N 1 strain of influenza A. The Asian Flu in 1957 was the H 2 N 2 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 H 3 N 2. 750, 000 people died of the virus worldwide 15
Naming the Flu A/chicken/Korea/01(H 9 N 2) Influenza A virus was isolated from a chicken in Korea in 2001. The antigen types were H 9 and N 2 Try some yourself: A/swine/Ehime/80(H 1 N 1) A/Tokyo/67(H 2 N 2) A/duck/Hainan/2004(H 6 N 2) 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 16
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: 17
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…. 18
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 19
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. 20
Could H 5 N 1 Cause a Pandemic? - For this section enlarge the Power. Point 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 H 5 N 1 began in Korea in December 2003. Since then it has spread to other Asian countries. H 5 N 1 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 H 5 N 1 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. 21
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. 22
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. 23
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. Q 16. How do people contract this disease? Q 17. Is it possible for this disease to spread from our son to those people he was in contact with? Q 18. How did he become infected with this strain of Influenza virus? Q 19. What available treatment might save his life? 24
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. 25
EMERGENCY MEETING Emergency influenza convention to discuss the H 5 N 1 pending pandemic. Formulate recommendations for the American government and report back to the American Advisory Committee for Infectious Diseases. 26
AMERICAN ADVISORY COMMITTEE Avoiding an H 5 N 1 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 H 5 N 1 does not enter America. • Recommend precautions that the Government should take to ensure America is prepared for a H 5 N 1 influenza outbreak • Discuss strategies that should be employed to stop H 5 N 1 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/infectious-disease-report/2002/illness. html 27
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