These slides are available at http://jbloom.github.io/FluPhylogeneticsModule/slides.html
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These slides are for a high-school level class, and were created by Greg Ballog, Jesse Bloom, and Trevor Bedford.
On April 15, 2009, doctors in California detect a new strain of influenza in a 10-year old child. A few days later, a similar virus is found in another child in California.
A week later, the Centers for Disease Control (CDC) publishes an article in Morbidity and Mortality Weekly Report describing these infections:
On April 17, 2009, CDC determined that two cases of febrile respiratory illness occurring in children who resided in adjacent counties in southern California were caused by infection with a swine influenza A (H1N1) virus. The viruses from the two cases are closely related genetically... and contain a unique combination of gene segments that previously has not been reported among swine or human influenza viruses in the United States or elsewhere. Neither child had contact with pigs; the source of the infection is unknown.
On April 23, 2009, it is discovered that the virus has also infected people in Texas, as described in this New York Times article.
It quickly becomes apparent that the virus is already circulating fairly extensively in Mexico, as described in this New York Times article.
A few days later, the U.S. declares a health emergency, as described in this New York Times article.
Within six weeks, the virus has spread to at least 74 countries. The WHO issues a declaration that the virus is a pandemic, as reported in this New York Times article.
You will use techniques from computational biology to address two questions that scientists needed to answer when faced with this infection:
"Flu" is the common name for influenza, which is a virus that infects many species including birds, pigs, horses, humans, and dogs.
Watch this video for some background on influenza pandemics.
You will use techniques from computational biology to address two questions that scientists needed to answer when faced with this infection:
One of the first questions scientists wanted to know: was the virus was resistant to Tamiflu (also known by the chemical name oseltamivir) (which is the most commonly used drug against influenza)?
Earlier studies had shown that influenza of this type was resistant if it had a mutation of from His to Tyr at position 275 of the virus's neuraminidase protein (this mutation is called His275Tyr).
So did the new 2009 pandemic virus have the His275Tyr mutation? To answer this question, we need to learn a little bit about influenza's genes.
Influenza is an RNA virus that encodes its genes in the negative sense. This means that its genes are made of RNA (rather than the DNA used to make our genes) that needs to be copied into mRNA (positive sense) before it can be translated into protein:
viral RNA -> mRNA -> protein
Using this knowledge, we will look at the neuraminidase gene for the strain A/California/07/2009 to see if it contains the Tamiflu-resistance mutation His275Tyr.
Use the portion of the sequence below for neuraminidase to see whether virus A/California/07/2009 has His275 (Tamiflu will work) or the resistance mutation His275Tyr (Tamiflu will not work). To go from viral RNA to mRNA, using the base-pair rules (A - U and C - G). To go from mRNA to protein, use the genetic code:
viral RNA: 3'-... cgg gga uua aua gug aua cuc cuu acg agg ...-5'
mRNA: 5'-... gcc ccu ??? ??? ??? ??? ??? ??? ??? ??? ...-3'
protein: ... Ala ??? ??? ??? ??? ??? ??? ??? ??? ??? ...
number: ... 271 272 273 274 275 276 277 278 279 280 ...
Exercise 1: Is this virus resistant to Tamiflu? If yes, what is a single-nucleotide mutation that would make it non-resistant? If no, what is a single-nucleotide mutation that would make it resistant?
To answer this question, we will use phylogenetics.
A phylogenetic tree represents evolutionary histories.
Geneology of the royal family.
Haeckel's tree of life (1879).
Tree of eutherian mammals from this paper.
Tree of influenza viruses taken from here.
You will build a phylogenetic tree to determine the origins of the 2009 pandemic virus.
You will do this using the Influenza Research Database.
After signing in, go to the homepage of the Influenza Research Database.
Click on the option under Search that says Sequences & Strains.
Click on the ADVANCED OPTIONS button at lower left.
Select the option that says Keyword search.
In the box on the right that says in All, change this to in Unique Sample Identifier.
Enter the following list identifiers (separated by commas) in the empty white search box:
FJ966082, CY121680, EU604689, CY022477, CY022333, CY036799,
CY026283, CY021725, AB671289, CY014968, EU735802
Click on the Workbench button at the top of the Influenza Research Database.
On the list of analyses, click on View next to the flu sequences sequence set that you saved.
Look at the names. Flu sequences from humans are named like this:
A/Location/Number/Date (such as A/California/04/2009)
while sequences from other species are named like this:
A/Species/Location/Number/Date (such as A/swine/Iowa/17672/1988)
We will now look at the composition of the sequence set that you have saved.
Exercise 2: How many of the sequences come from humans? How many from pigs? How many from birds? What is the date of the earliest sequence?
Click to Select all 11 segments. Then click on the Download button near the top of the page to download the sequence set. Select the Gene FASTA format. Then click on the option to custom format the the Definition Line and add only Strain Name (this ensure the sequences are named by strain). Then click Download and save the file GeneFastaResults.fasta.
This downloaded file has all of the sequences along with the strain name in FASTA format.
Shortcut: If you have problems with these steps, you can Download the file by clicking here.
In your sequence set, there are two sequences from the 2009 outbreak in California. These sequences are A/California/04/2009 and A/California/07/2009.
The rest of the sequences are from pigs, from birds, or from another earlier strain of flu that was already infecting humans.
Exercise 3: Based on the news articles we discussed earlier, make a hypothesis about the phylogenetic tree. To which sequence(s) do you think the 2009 sequences from California will be most closely related?
Exercise 4: Answer the following questions:
The exercises that you have just performed are a simpler version of the studies that scientists actually performed to understand the 2009 swine-origin H1N1 influenza pandemic.
If you are interested, you can read about the similar but more complex analyses that scientists actually performed to study this virus outbreak by looking at this paper.