Purpose: To gain a better understanding of influenza, its genomes, mutations and vaccines.

Procedural overview: This guided research activity encourages the exploration of public data and information to learn more about influenza’s genes and proteins, how a flu vaccine is made and the nine human genes that predict how well a flu vaccine will work in a person.

Approximate class time: 40 to 60 minutes.

Materials:

• Activity Guide for Students: Catching the flu
• Internet access
• Recommended textbooks or other reference books

Notes to the teacher:

Depending on your preference, students can work through these questions either in class or at home, and either individually or in groups. Also, feel free to get creative with dividing up the questions or parts of questions among groups. For example, question 11 involved researching information about nine genes. This would be a good question to divide up among groups. It would allow students to present information to the rest of the class during a discussion.

The following websites and books are very good sources of information to provide your students:

• Science News for Students: “Explainer: What is a vaccine?” (https://www.sciencenewsforstudents.org/article/explainer-what-vaccine)
• Science News for Students: “Explainer: What is a virus?” (https://www.sciencenewsforstudents.org/article/explainer-what-virus)
• Uniprot Protein Data Base (http://www.uniprot.org)
• Protein Data Bank (http://www.rcsb.org/pdb/home/home.do)
• National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov)
• CDC Influenza (https://www.cdc.gov/flu/index.htm)
• ViralZone (http://viralzone.expasy.org)
• Virus Pathogen Resource (https://www.viprbrc.org)
• NCBI Influenza Virus Resource (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database)
• Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, and Jane B. Reece. 2016. Campbell Biology. 11th ed. Pearson. See especially Ch. 19 (Viruses) and Ch. 43 (Immune System).
• David M. Knipe and Peter M. Howley (eds.). 2013. Fields Virology. 6th ed. Lippincott Williams & Wilkins.
• Lauren Sompayrac. 2012. How Pathogenic Viruses Think. 2nd ed. Jones & Bartlett.
• Jane Flint, Vincent R. Racaniello, Glenn F. Rall, Anna Marie Skalka, and Lynn W. Enquist. 2015. Principles of Virology. 4th ed. 2 vols. American Society for Microbiology.
• John Carter and Venetia Saunders. 2013. Virology: Principles and Applications. 2nd ed. Wiley.
• Lauren Sompayrac. 2016. How the Immune System Works. 5th ed. Wiley Blackwell.
• Thao Doan, Roger Melvold, Susan Viselli, and Carl Waltenbaugh. 2013. Lippincott’s Illustrated Reviews: Immunology. 2nd ed. Lippincott, Williams & Wilkins.
• Judith A. Owen, Jenni Punt, and Sharon A. Stranford. 2013. Kuby Immunology. 7th ed. Macmillan.
• Abul K. Abbas, Andrew H. Lichtman, and Shiv Pillai. 2017. Cellular and Molecular Immunology. 9th ed. Elsevier.
• Kenneth Murphy and Casey Weaver. 2016. Janeway’s Immunobiology. 9th ed. Garland.
• William E. Paul (ed.). 2012. Fundamental Immunology. 7th ed. Lippincott Williams & Wilkins.

Student instructions and questions with answers:

Directions: Do research online or in books to answer the following questions. Write down your answers as you go and be prepared to discuss your findings in class.

1. Is the genome of a flu virus made of DNA or RNA, and is it single-stranded or double stranded?

Single-stranded RNA.

2. How many separate segments or pieces is the flu virus genome divided into? What genes are on each piece, what are the genes’ associated proteins and what do those proteins do?

8 segments:

The PB2 genome segment yields the PB2 component of RNA polymerase.

The PB1 genome segment yields the PB1 component of RNA polymerase, PB1-F2 proteins that fight cellular defenses and PB1 N40 protein that’s function is currently unknown.

The PA genome segment yields the PA component of RNA polymerase.

The HA genome segment yields HA hemagglutinin glycoproteins, which cover the surface of virus particles and help them enter and leave cells.

The NP genome segment yields NP proteins that are required for packaging of specific RNA segments.

The NA genome segment yields NA neuraminidase glycoproteins, which cover the surface of virus particles and help it enter and leave cells.

The M genome segment yields M1 matrix proteins, which help form the structure of virus particles, and M2 ion channels, which sense when the virus particles have gotten inside a cell.

The NS genome segment yields NS1 non-structural proteins that fight cellular defenses and NEP/NS2 non-structural proteins mediate export of viral ribonucleoprotein.

3. Different strains of influenza can have different versions of each of these genome segments. For the influenza A virus, how many known versions of HA are there?

There are at least 18 major variations, H1 through H18. There are also minor variations of each of these major variations to fool the immune system into thinking it has never seen the virus before.

4. For the influenza A virus, how many known versions of NA are there?

There are at least 11 major variations, N1 through N11. There are also minor variations of each of these major variations to fool the immune system into thinking it has never seen the virus before.

5. Including all of the known HA and NA versions, how many possible influenza A strains are there?

18 HA x 11 NA = 198 influenza A strains with different combinations of major variations of HA and NA on the surface of the virus. Including the minor variations, there are thousands or more of possible strains.

6. In terms of the benefits to the virus, why is it important for influenza to have so many possible HA and NA glycoprotein versions?

The surface of influenza virus particles are covered with HA and NA glycoproteins. If the virus particles change those glycoproteins, they can trick lymphocytes into thinking the immune system has never seen the flu virus before.

7. The annual flu vaccine usually contains HA and NA glycoproteins from three different flu strains. How many different strains could the vaccine protect against?

If a vaccine contains different HA and NA glycoproteins from three flu strains, there would be 3 possible HA types and 3 possible NA types, or 3 x 3 = 9 possible strains against which the vaccine could provide protection. That is a small fraction of the total number of possible flu strains that exist. The fraction is even smaller if you consider some of the three flu strains used to make the vaccine share an HA or NA type, or how many minor variations there can be in flu strains that could evade the vaccine.

8. Suppose that one flu virus strain has its own version of each of its genome segments, and a second flu virus strain has a different version of each of the genome segments. If both flu strains infect the same cell, the new virus particles that get assembled may end up with some genome segments from one strain and some from the other strain. Assuming that each new virus ends up with one copy of each genome segment, and that each genome segment can come from either original flu strain, how many possible flu strains could be produced by that one doubly-infected cell?

2 possibilities for each of 8 genome segments = 2⁸ = 256 possible resulting strains

9. Why does the flu virus mutate so easily?

1. Reassortment of segments between different strains that infect the same cell, which is not a problem with viruses that only have one genome segment.

2. Errors produced by RNA polymerase in copying the genome; the viral RNA polymerase is much more error-prone than human DNA polymerase.

10. In what parts of the United States is the flu currently the worst? See: The Weekly US Map: Influenza Summary Update by the U.S. Centers for Disease Control and Prevention (https://www.cdc.gov/flu/weekly/usmap.htm)

Answers will vary over time.

11. Listed below are the nine genes whose activity was found to be correlated with strong immune responses to flu vaccines. From online searches, briefly summarize what is known about the function of each gene’s corresponding protein, and speculate why the protein (and the amount of the protein produced, called its gene expression level) might be involved in strong immune responses to flu vaccines.

RAB24

Ras-related protein Rab-24. It is known to be involved in intracellular protein trafficking, like a postal worker helping the right envelopes get to the correct zip codes. Perhaps immune system cells or lymphocytes use this protein to help deliver components of antibodies and T cell receptors.

GRB2

Growth factor receptor-bound protein 2. Growth factor receptors help cells sense external signals that they should grow and divide. Perhaps the protein helps immune cells receive signals that they need to grow and multiply.

DPP3

Dipeptidyl-peptidase 3 serine protease. Serine proteases are involved in intracellular signaling. Perhaps the protein helps immune cells signal when they detect the vaccine. 

ACTB

Beta-actin cytoskeleton. This protein is part of the skeleton that helps cells keep their proper shape and even move, just as your skeleton helps you keep your shape and move. Perhaps the protein helps immune cells maintain the best shape and move toward their targets.

MVP

Major vault protein. It is involved in helping to transport things in and out of the cell nucleus. Perhaps the protein helps immune system cells by helping signal other proteins to get into the nucleus or messenger RNAs to get out so that they can better respond to the vaccine.

DPP7

Dipeptidyl peptidase 7. It is involved in maintaining the resting state of immune system cells. Perhaps changes in its gene expression are involved in helping immune cells wake up and respond to the vaccine.

ARPC4

Actin related protein 2/3 complex subunit 4. It controls actin polymerization and is involved in controlling the shape and movement of the cytoskeleton. Perhaps the protein helps immune cells adjust their shape and movements to better respond to the vaccine.

PLEKHB2

Pleckstrin homology domain-containing family B member 2. It is involved in retrograde transport of recycled endosomes. Perhaps the protein helps in carrying B cell or T cell receptors that have detected vaccine components into the cell and initiating an immune response.

ARRB1

Arrestin beta 1. It is involved in dampening cellular responses to extracellular signals. Perhaps changes in its gene expression are involved in helping immune cells be more sensitive to vaccines.

12. What have you learned about influenza that has surprised you?

Student answers will vary.