annotated-Pre Lab Tracking Influenza Fall 2023-1.docx

Pre-Lab Activity Influenza A H1N1 Fall 2023-1 1 Pre-Lab 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 Question:  What type of mutation (antigenic drift or antigenic shift) was responsible for the 2009 Influenza H1N1 outbreak?  Why did the influenza vaccine available in 2009 not provide protection agains the influenza virus in the population? Summary:  In this lab you will analyze nucleotide sequences from Influenza A viruses, specifically the nucleotide sequence that codes for the HA protein. These sequences were obtained from patients during the 2009 H1N1 Influenza pandemic. We will look at sequences of influenza prior to 2009 as well as the nucleotide sequence of the virus in the influenza vaccine. Materials Needed for Lab  Computer with access to the internet

Pre-Lab Activity Influenza A H1N1 Fall 2023-1 2 Background Influenza , commonly called the flu, is a highly contagious disease caused by influenza viruses. The flu can cause mild to severe illness, and at times can lead to death. Flu viruses spread mainly by tiny droplets made when people with flu cough, sneeze or talk. These droplets can land in the mouths or noses of people who are nearby. Less often, a person might get the flu by touching a surface or object that has flu virus on it and then Influenza, commonly called the flu is a highly contagious disease that is caused by influenza viruses. The flu can cause symptoms of headache, sore-throat, body aches, and high temperature; these symptoms are usually mild in healthy individuals. The influenza virus is responsible for 250,000 to 500,000 deaths a year. However, there have been influenza pandemics where influenza has been responsible for millions of deaths; 1918 Spanish flu, 1957 Asian flu, the 1968 Hong Kong flu, and the 2009 H1N1 flu. A pandemic is a worldwide spread of a new disease; an influenza pandemic occurs when a new influenza virus emerges and spreads around the world, and most people do not have immunity. There are three types of influenza virus; Influenza A, Influenza B and Influenza C. Influenza A can infect humans and many different types of animals including swine and avian. Influenza B and Influenza C circulate among humans. Influenza A is the most significant and most studied of the three types of viruses. Type A Influenza viruses are named after surface proteins that are present on the outside of the capsid. Influenza viruses produce 11 different surface proteins that attach to molecules located on host cells, such as those found in the respiratory system. Two of those surface proteins; hemagglutinin (H) and neuraminidase (N) are used to name the virus. For example, H1N1 has H type 1 and N type 1 surface proteins present on the viruses’ surface. Influenza A subtypes H1N1, H1N2 and H3N2 mainly infect humans.

Pre-Lab Activity Influenza A H1N1 Fall 2023-1 4 Evolution of Influenza Influenza is a virus that replicates quickly and is prone to replication errors or mutations in its RNA sequence. The influenza virus genome is a single strand of RNA. Over time, as the virus replicates, random changes to the nucleotide sequence occurs, creating many possible mutations. These mutations may be detrimental to the virus and even result in defective viruses, others will be neutral and some mutations may offer some type of advantage. In this way viruses transmitted from one person to another are related and may accumulate differences overtime. In the influenza virus genome, if mutations occur in the RNA segment that codes for the two surface proteins (H and N) on the virus, then your existing antibodies may not recognize the virus and will not bind to it. These small changes in the surface of the virus may accumulate overtime and result in the hosts’ immune system failing to recognize and respond to a virus. These small changes to the surface protein as a result of errors during RNA replication is called antigenic drift (Figure 3) Antigenic drift occurs in Influenza Type A and B viruses. Antigenic drift is ongoing and is the basis for evaluating the components of the infuenza vaccine each year. As these small changes accumulate, the effectiveness of the vaccine may decrease. Another type of mutation, antigenic shift (Figure 3) in influenza virus occurs when genes of a single strain, or genes of two different strains exchange genetic material. This may produce a novel combination of genes that has a survival advantage over other strains. Viruses that originate from different species (avian and swine) may take part in this process. Reassortment events, from human and avian influenza A viruses have been the source of pandemics. Antigenic shift happens less frequently than antigenic drift and only occurs in Influenza Type A viruses. When a new version of a virus emerges as a result of antigenic shift, it may cause major illness if it is not recognized by the hosts’ immune system. Additionally, the influenza vaccine that is available at the time, may not be effective and the manufacture of a second vaccine required. Figure 3. Illustration of antigenic shift and antigenic drift. Source: http://www.influenzacentre.org/aboutinfluenza.htm

Pre-Lab Activity Influenza A H1N1 Fall 2023-1 5 Tracking the Evolution of the Influenza Virus By identifying differences in viral genomes, scientists can reconstruct the history of how the virus spreads and mutates. Scientists isolate virus RNA (letters A, C, G and U) from patient samples and then using genetic sequencing techniques (Next Generation Sequencing) to obtain the nucleotide sequence of the virus. Remember, that viruses have a single strand of DNA or RNA. To compare sequences using GenBank and other large genetic databases, if the genetic material of the virus is RNA, the scientists translate the RNA to DNA (letters A, C, G, and T). They then identify where the nucleotide bases are different between the two genetic sequences. This single nucleotide difference is called Single Nucloetide Polymorphisms (SNP’s) and is a result of a mutation in the genetic code. To compare the nucleotide sequences, the two sequences are arranged, in rows, vertically above each other. Remember, that viruses have a single strand of DNA or RNA. Looking at the nucleotide sequence comparison in Figure 4, we see that we are comparing the nucleotide sequence of Query 1 and Subject 33; these are two different virus strains. When the nucleotides (A, G, T and C) are the same, there is a vertical line drawn between the two sequences. When the nucleotides are different between the two sequences, we see that there is no vertical line or a gap. The presence or absence of the vertical line, makes it easy to identify differences between the two nucleotide sequences. In this example, the two locations in the genome, where the nucleotides are different are highlighted. Figure 4. Example of nucleotide sequence comparison to highlight the difference in single nucleotides. Aligning viral genome sequence information provides information about the viral strains and allows one to determine the number of nucleotides that are different or have changed. When reporting this information in published work, scientists use degree of similarity or degree of likeness. The degree of similarity is expressed as thee % of nucleotide bases that are the same between two virus strains over a given length of alignment. The degree of similarity can be easily calculated. The higher the % similarity the more closely related the viral strains. The % similariy between the two nucleotide sequences in Figure 4 is 96%. Scientists use the measure of the degree of similarity between the virus in the vaccine and the strains of influenza virus that are circulating in the population as an indicator of how well the vaccine will work. If the degree of similarity between the strain in the vaccine and the strain circulating is high, then the vaccine will have a greater level of effectiveness. If the degree of similarity between the strain in the vaccine is low, then the vaccine will have a lower level of effectiveness in protecting against infections. In this case if the circulating virus has mutated If the degree of similarity between the virus in the vaccine and the circulating virus decreases after the vaccine is made then a second vaccine may be required. Procedure – Tracking the Evolution of Influenza A H1N1

Pre-Lab Activity Influenza A H1N1 Fall 2023-1 7 2009- 2010 A/California/07/2009 NC_026433.1 Procedure: 1. To compare the sequences of the strains of H1N1 that we identified in Table 1, we will use the NCBI GenBank Website. Copy or Click on the link below to open GenBank; you should see a screen as shown in Figure 5. https://www.ncbi.nlm.nih.gov/genbank/ Figure 5. Image of GenBank home page. 2. On the top of the webpage next to the search bar, select nucleotide in the drop-down bar; circled in Figure 5. 3. Enter in the accession number AJ344014.1 which is for A/New Caledonia/99. Click on the search button (to the right of the screen) A new window will appear as shown below in Figure 6.

Pre-Lab Activity Influenza A H1N1 Fall 2023-1 8 Figure 6. Image of the results obtained when conducting a nucleotide search for a given accession number in GenBank. 4. On the same window,scroll down to the section titled ‘ ORIGIN ’. This section contains the nucleotide sequence for the section of the HA genome that we will use for comparison of the viruses. 5. In row one (highlighted in the Figure) copy the first 16 nucleotide bases to Table 2 (located at end of document). You will notice that a reference as been entered into Table 2. 6. Repeat for each of the Influenza A H1N1 viral strains listed in Table 1. Activity 2. Analyzing Influenza A H1N1 Nucleotide Sequences

Pre-Lab Activity Influenza A H1N1 Fall 2023-1 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 # of changes Reference Sequence – strain in vaccine A T G A A A G T A A A A C T A C ---- A/New Caledonia/99 A T G A A A G C A A A A C T A C 1 A/Solomon Islands/2006 A T G A A A G T A A A A C T A C 0 A/Brisbane/59/2007 A T G A A A G T A A A A C T A C 0 A/California/07/2009 A T G A A G G C A A T A C T A G 4 Activity 3: Predictions Between 2008-09 and 2009-2010, we saw a dramatic increase in Influenza A H1N1 cases. Why do you think there was such a huge increase in influenza cases? (think antigenic drift vs antigenic shift). There was such dramatic increase in Influenza A H1N1 cases because the strand went through a lot mutations compared to the vaccine. This caused the immune cells to not recognize the cell and therefore the vaccine didn’t help as the immune system did not have the antibodys to fight off this virus infection. This is caused by the antigenic drift, as the effectiveness of the vaccine dramaticlly lowered, as the virus changed so much.