Assignment about Using Blast and Expasy

Using BLAST and ExPASy for Genetic and Protein analysis of H1N1 variability, including
mutations that confer resistance to antiviral medications.
Introduction: Influenza is an infection of the upper respiratory tract that causes sickness and
death and widespread outbreaks also cause a significant economic impact as well. The H1N1
influenza A pandemic of 2009 caused millions of people to become sick, hundreds of thousands
to be hospitalized and thousands of deaths in the United States alone1. The new flu strain
contained genes from influenza viruses from avian (bird), swine (pig) and human. New strains
often cause more infection because our immune systems have been conditioned to respond to
previously encountered strains. When strains mutate it allows them to more easily slip by our
body’s defenses. In addition, mutation can make previous vaccines and antiviral medications
ineffective against them.
Background: Most of us are familiar now with the nomenclature “H1N1” but few of us know
the meaning of these letters and numbers. Scientists label flu viruses based on the presence of
two antigens on their surface. The first letter, “H” refers to the type of Hemagglutinin present on
the virus’ surface. This molecule is primarily responsible for the virus’ ability to infect cells.
The second letter, “N”, refers to the type of Neuraminidase present. This molecule helps the
replicated virus leave from the cell in which is has replicated, allowing the many copies to infect
more cells. These two proteins together determine much about the virus, including, in general,
which species the virus can infect and the virulence of the particular strain. Even though there
are many different strains of flu virus, they infect mammals and birds and there remains a lot of
similarity between them. These similarities can be exploited to develop vaccines and/or antiviral
drug therapies that could be used against a wide array of similar viruses. Between 1999 and
2002 two new antiviral drugs were introduced into the population: zanamivir and oseltamivir.
These same drugs were used to treat patients with H1N1. In just about 10 years it is possible for
flu viruses to evolve resistance to these drugs. In order to monitor the potential development of
resistant strains the Neuraminidase Inhibitor Susceptibility Network was established.
Directions: You are a member of the Neuraminidase Inhibitor Susceptibility Network and the
World Health Organization has contacted you with information about a new viral strain that
might have resistance to the antiviral drugs zanamivir and oseltamivir (Tamiflu). In this case
study you will:
•Use BLAST to compare H1N1 sequences to observe conserved and variable regions
•Align nucleotide sequences of normal strains of H1N1
•Align multiple nucleotide and protein sequences to look for mutations.
•Compare the newly identified strain and hypothesize its resistance based on its mutations
Part A: Introduction to blastn. Listed below are the identifying numbers for several “normal”
(non-resistant) strains of H1N1 that were sequenced during the 2009 pandemic. In part A of this
activity you will use online database tools to look at virus variation that does not confer antiviral
resistance.
Step 1: Go to the National Center for Biotechnology Information’s website at:
http://www.ncbi.nlm.nih.gov. From the menu at the top right, click on BLAST. BLAST stands
for “Basic Local Alignment Search Tool” and it is one of the many web-based applications

available to investigate, analyze, research and identify genes and proteins of interest. The
applications of BLAST and other tools are almost limitless! All of these resources are free and
open to the public. This particular site is maintained by the National Institutes of Health (NIH).
Step 2: Choose nucleotide blast from the menu of choices under the heading “Basic BLAST”.
To become familiar with BLAST output we will compare the nucleotide sequences of two strains
of H1N1. Enter this accession number in the box, CY056295. Give your job the title, “align two
non-resistant H1N1 strains”.
Step 3: Click on the box in front of “Align two or more sequences”. This will open a new box.
Enter accession number CY062530 in this box.
Step 4: At the bottom section called “Program Selection” select the choice “Highly similar
sequences (megablast). Then click “show results in a new window” so we can refer back to our
search criteria if needed.
Step 5: Click the “BLAST” button. Your search will show up in a new window or tab and if
you scroll down you can see the aligned nucleotides for the two neuraminidase genes in our two
strains.
Answer the questions for Part A once the BLASTN search is finished.
Part A Questions:
1. What are the lengths of the two sequences used in this comparison?
Query: _____1410____ nucleotides Subject: __1421_______ nucleotides
2. Now that the sequences are aligned for best fit, how many differences can be found between
the two aligned regions? (count them!)
____11_____ nucleotides
3. Since every 3 bases codes for 1 amino acid, what is the maximum number of amino acids in
the protein that would be made from the shorter of the two sequences?
____470_____ amino acids
4. Will every nucleotide difference result in an amino acid difference? Explain your answer.
Yes, nucleotide difference leads to an amino acid difference because 3 nucleotides codes for one
amino acid. Hence, if there is a deletion of nucleotide would cause a difference between
nucleotide and amino acids.
5. Since neither of these strains is resistant to anti-viral therapies, what can be concluded about
the variation in the gene for neuraminidase between these two strains?

It can be concluded that Neuraminidase (NA) is a surface proteins of influenza A virus, and a
variation in the gene is observed that plays an essential role in inoculation beside influenza
infection in addition to is predictable as imperative therapeutic objective. Hence, the genetic in
addition to antigenic variations besides exchanges can stimulates the effectiveness of vaccine in
addition to modification viral understanding to NA inhibitors (NAIs) (Göktepe & Kodaz, 2018).
Part B: Collecting Info on your sequences. There are a lot of details recorded about the
sequences that are uploaded to the GenBank database. In Part B of this activity we will collect
information about our strains, including a reference number to their translated amino acid
(protein) sequence. We will use these sequences in Pact C of this activity.
Step 1: From the top of the BLAST results page, click on the link after “Query ID”. This will
bring up all of the information about this sequenced gene. Look over the information and answer
the questions:
Part B Questions:
1. When was this sample collected?
The sample was collect on 7th March 2010.
2. Who was the host of this virus? Be as specific as possible.
The host virus is Influenza A virus (A/Henan/1/2010(H1N1))
3. From which country was this host?
The country China was its host.
4. If you scroll down to the bottom you can find the entire sequence and translated amino acid
sequence. See appendix A or http://www.bio.davidson.edu/courses/genomics/jmol/aatable.html
for a list of amino acid abbreviations. Write down the protein_id
number: ____ ADD52538.1" _______________
Step 2: Use the back button of the web browser to return to your BLAST results. Scroll down
and click on the accession number of our subject sequence, CY062530.1. Answer the same 4
questions about this viral strain.
1. When was this sample collected?
The sample was collected on 4th May 2010.
2. Who was the host of this virus? Be as specific as possible.
The host of this virus was Guadalajara, Jalisco, Mexico