Lab Activity Tracking Influenza Spring 2022

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Apr 3, 2024

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Lab Activity Influenza A H1N1 Spring 2022-1 1 Lab Activity (Data Analysis and Conclusions) Tracking the Evolution of Influenza A H1N1 Virus Learning Objectives • Explain how evolution can occur rapidly in viruses through mechanisms of antigenic drift and antigenic shift. • Explain how mutations or changes in nucleotide sequence of viruses means hosts may not recognize the virus. • Use online databases, such as GenBank to analyze and interpret nucleotide sequence data. Materials Needed for Lab Computer

Lab Activity Influenza A H1N1 Spring 2022-1 2 Tracking Evolution of Influenza A In the pre-lab activity, we looked at a portion of the nucleotide sequences for four Influenza A virus strains and compared them to the influenza A vaccine component. To test our predictions we will be using GenBank to compare the longer nucleotide sequences of segment 4 for the hemagglutinin (H) protein for the four influenza A virus strains. Activity - Nucleotide Sequence Comparison – Hemagglutinin (H) In this section we will compare the sequence of the Hemagglutinin (HA) protein in the H1N1 virus strains using BLAST. While we are using BLAST to align and compare the sequences, we could manually align the sequences if we printed the full nucleotide sequences from GenBank. The advantage of BLAST is that it will align and compare the sequences of the two strains of viruses and calculate the degree of similarity or likeness. In BLAST the % similarity is reported as the % Identities. Remember when comparing two nucleotide sequences, a larger % similarity means a larger number of nucleotides that are identical in the segment we are comparing. A 100% similarity means that there are no differences in nucleotide sequence between the strains (perfect alignment). Proceed through the steps below to find the % similarity between the different strain and the vaccine component of Influenza A H1N1. The virus strain name and the accession number for use in this procedure are given in Table 1. After finding the % similarity, we will record it in Table 2. Refer to Figures 1 – 4 and the video guide in Canvas, as needed. Step 1. Open the link to the BLAST website using the link. The website should look very similar to the screen shown in Figure 1. https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC =blasthome Step 2. Confirm that the database you are using is the blastn , as shown in Figure 1.

Lab Activity Influenza A H1N1 Spring 2022-1 3 Figure 1. BLAST website – initial screen Step 3. Enter the accession number of the first strain of H1N1 virus that you want to compare in the field Enter Accession number(s) . This field is located near the top of the webpage. Step 4. Click on the box next to Align two or more sequences . Step 5 . Under Subject Sequence enter the accession number of the second strain of H1N1 virus for comparison. Step 6. Under Program Selection the database should be automatically set to use Highly similar sequences (megablast). Step 7 . Click the BLAST button at the bottom of the screen. You will need to wait a few minutes for your results to appear in a new window.

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Lab Activity Influenza A H1N1 Spring 2022-1 4 Figure 2. Inputting Data into the BLASTN Program. Step 8. In the new window locate Descriptions. You may need to scroll down. Step 9. Click on the alignments tab. The information in the window will change and will be as shown in Figure 4. This report shows the entire sequence of each nucleotide sequence that was being compared.

Lab Activity Influenza A H1N1 Spring 2022-1 5 Figure 3. Report Generated as a Result of Running BLAST Step 10. Near the top of the report, locate Identities . This gives the percentage of the nucleotide sequence that is identical between the two sequences. The percent identical for A/Solomon Islands/3/2006 and A/New Caledonia/20/99 is 97% and has been recorded in Table 2. Confirm that this is correct.

Lab Activity Influenza A H1N1 Spring 2022-1 6 Figure 4. Alignment of Two Nucleotide Sequences using BLAST Step 11. Repeat these steps until you have compared the hemagglutinin protein of each common strain of Influenza A H1N1 that you have listed in data Table 1. Be sure to compare each possible combination.

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Lab Activity Influenza A H1N1 Spring 2022-1 7 Assignment – Exit Ticket Directions: 1. Complete Activities 1 - 2 2. Submit your completed assignment in Canvas; submitted assignment must be in PDF format. Activity 1 - Nucleotide Sequence Comparison – Hemagglutinin (H) Table 1. The most commonly subtyped (characterized) strains of H1N1 virus from influenza seasons 2006-2010. Season Most common Strains of H1N1 Subtyped that Season Accession Number 2006-07 A/New Caledonia/20/99 AJ344014.1 2007- 2008 A/Solomon Islands/3/2006 EU100724.1 2008- 2009 A/Brisbane/59/2007 KP458398.1 2009- 2010 A/California/07/2009 NC_026433.1 Table 2. Percentage similarity of Hemagglutinin (HA) nucleotide sequences between strains of H1N1. Strain/ season 2006-07 2006-07 2008-09 2009-2010 A/New Caledonia/ 99 A/Solomon Islands/2006 A/Brisbane/ 59/ 2007 A/California/ 07/ 2009 2006-07 A/New Caledonia/99 -- 98% 97% 78% 2007-08 A/Solomon Islands/3/2006 98% -- 98% 77% 2008-09 A/Brisbane/59/ 2007 97% 98% -- 77% 2009- 2010 A/California/07/ 2009 78% 77% 77% --

Lab Activity Influenza A H1N1 Spring 2022-1 8 Activity 2 - Conclusion Answer the questions below using complete sentences. Include supporting data from your results (% similarities from Table 2) to support your answer. 1. Referring to the circled section of the image of the comparison of two different single strand nucleotide sequences below, what does the blank space indicate? The above sequence depicts a base substitution in the sequence. This mutation can code for an amino acid that can result in the premature termination of transcription or translation. 2. Using the percentage similarity data in Table 2, list the strains of virus and the years they were circulating that are most closely related. Are these small changes in the nucleotide sequence a result of antigenic drift or antigenic shift? Explain your reasoning. A/New Caledonia/99 from 2006-07, A/Solomon Islands/3/2006 from 2007-08, and A/Brisbane/59/ 2007 from 2008-09, are all closely related, all with either a 97% or 98% similarity. These small changes are the result of antigenetic drift, since they are very small changes that occurred over several years. 3. Using the percentage similarity data in Table 2, list the strains of virus and the years they were circulating that are least related. Is there one strain of Influenza A H1N1 that is different from the rest. Are these changes in the nucleotide sequence a results of antigenic drift or antigenic shift? Explain your reasoning. A/California/07/ 2009, from 2009-2010 is the strain of influenza A H1N1 that is vastly different than the rest of the strains. The changes in the nucleotide sequence are the result of antigentic shift, since there is such a large difference between it and the rest of the strains that occurred in such a short time, with only 77%-78% similarity to the rest of the strains.

Lab Activity Influenza A H1N1 Spring 2022-1 9 4. As we learned in the Pre-Lab, in the year 2009 influenza cases significantly increased from previous years. The yearly influenza vaccine in 2009 contained the virus strain A/Brisbane/59/2007. Knowing that the virus strain circulating in 2009 was A/California/07/2009, do you believe the influenza vaccine containing A/Brisbane/59/2007 was effective in protecting against infection? I do not believe the influenza vaccine containing A/Brisbane/59/2007 was very effective in protecting against infection, since it only has a 77% similarity to A/California/07/2009. I ’ m sure that it was still better to have the vaccine than to not have it, but it was most certainly not as effective as it would have been if it was a similar strain.

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