Case 3: The Case of the Variable Virus

Tran was excited to be going home to Viet Nam. It had been several years since he had seen his family, and he enjoyed being back in Ho Chi Minh City, roaming the large markets and smelling all the scents of home. Members of Tran's family came from the surrounding countryside to see him, bringing the chickens and ducks they raised to cook a feaast for the extended family. All too soon, it was time to head back to Arizona.

On his first morning back, Tran awoke with a fever of 102°F, a headache, and a sore throat. He felt very tired and his muscles ached. Tran decided he didn't have the energy to go to class; he would email his professors that he was sick and go tomorrow. He dozed all day, but by evening his fever was 104°F and he was coughing. By morning he was short of breath, and his roommate took Tran to the ER. The ER physician examined Tran and ordered a chest x-ray, which showed pneumonia (fluid in Tran's lungs). From the high fever and sudden onset, the physician diagnosed influenza, but Tran said he had had a flu shot about three weeks before. When the physician learned Tran had just returned from Viet Nam, where he had had contact with poultry, the physician ordered Tran put in an isolation room and called the local public health department to report a possible case of imported avian influenza. Later laboratory tests confirmed that Tran had the H5N1 strain of influenza A (avian flu).

Influenza virus is spread through aerosols (tiny droplets in the air) and infects epithelial cells lining the upper respiratory tract. The virus spike proteins bind to the host cell surface glycoproteins and influenza virus enters the host cell, where it uses host ribosomes and enzymes (modified by viral non-structural proteins and supplemented with viral RNA-dependent RNA polymerase) to make thousands of copies of itself. The new virions bud from the host plasma membrane at sites containing virus spike proteins; the membrane becomes the viral envelope.

Influenza virus has an RNA genome made up of 8 pieces of RNA associated with nucleoprotein (NP). The genome is surrounded by a matrix protein underlying the lipid bilayer envelope acquired as the virus buds from the host cell plasma membrane. See left-hand figure below: purple squiggles are the RNA, green balls are the matrix protein, dark pink line is the lipid envelope, and light blue trapezoids are the envelope glycoprotein "spikes".

Influenza virus mutates easily, and different antigenic types of influenza usually appear each flu season. Influenza virus has two envelope (surface) glycoproteins by which different variants are characterized. One is hemagglutinin (H = HA, shown all in blue below), which is the molecule that allows influenza to bind to and infect its host cells by binding its receptor site to the host cell glycoproteins. The second is neuraminidase (N = NA, shown as purple with pink boxes), which helps the newly formed virus particles leave the host cells to infect new cells. Influenza viruses are categorized by H and N into two major groups, Influenza A and Influenza B, and into many antigenic types within each group (examples: H1N1, H2N1, H3N2, H5N1, etc.). Influenza A usually causes a more serious disease than Influenza B. Avian influenza, H5N1, is currently causing outbreaks in birds in several countries in southeast Asia and has spread west as far as Turkey. Sporadic human cases have occurred in 2003-2006, generally in people directly exposed to infected domestic birds. WHO [World Health Organization] reports that since 2003 there have been 93 confirmed cases of avian influenza A (H5N1) virus infection and 42 deaths in Viet Nam; worldwide there have been 241 cases and 141 deaths (WHO 23 August 2006).

Influenza virus structure (left) and details of hemagglutinin (H) structure.

1. REVIEW What is the cause of Tran's fever, sore throat, and cough?

2. What is the role of Natural Killer cells in an influenza infection?

3. Consider the influenza antigens recognized by the immune system.

• a. List from the information above the antigens on the Influenza envelope and what kind of molecules they are (chemically). What other antigens would influenza have?
• b. What is an antigen epitope? Do protein antigens have one or more than one epitope per molecule?
• c. How is it that influenza can change its antigens and still bind to and infect human epithelial cells? (HINT: see diagram above right)
• d. Why didn't the flu vaccine Tran received before he traveled protect him from avian influenza?

Antibodies are named for the species in which they are produced and the antigen that they recognize. For example, a human anti-influenza serum can be obtained from humans who have been infected with influenza virus. Serum from Tran would be human anti-avian influenza virus. Within that serum would be individual antibody molecules that could bind different epitopes of influenza virus. This is called polyclonal antibody, because several different naïve B cells were stimulated to produce clones of activated B cells, plasma cells and memory B cells.

• e. Would humans ever make a monoclonal antibody response to an antigen?

4. Draw and label an IgG molecule, including the specific names for the H and L chains, the V and C regions, the Fab and Fc regions, the antigen-binding sites, and the locations of the interchain disulfide bonds.

5. Explain the relationship of the hypervariable regions (CDR) to the antigen-combining region and discuss the possible consequences of amino acid changes in either the framework or CDR to the antigen-binding affinity of the antibody.

6. Describe the characteristics of immunoglobulin that define the immunoglobulin superfamily of molecules and name some other members.

9.

• a. Compare and contrast the structure and location of TCR molecules with antibody molecules. [Table is OK]
• b. Contrast the antigens "seen" (bound) by B cell receptor (BCR = Ig) and those "seen" (bound) by T cell receptor (TCR) on Tc and Th cells. [Table is OK]

10. Compare the structure and function of Class I and Class II MHC with BCR (Ig) and TCR.

11. For each of the following cell types, list whether it will express (have on its plasma membrane) membrane Ig, TCR, CD4, CD8, Class I MHC, and/or Class II MHC.

• B cells
• Helper T cells (Th)
• Cytotoxic T cells (Tc)
• Macrophages (Mf)
• Dendritic Cells (DC)
• Respiratory epithelial cells

12. Infected epithelial cells have H protein in their plasma membranes (which will become part of the virus envelope) and H peptide on MHC (which will be recognized by cytotoxic T cells). To which of these can antibody bind? What will be the consequences of antibody binding to infected epithelial cells (will this activate any immune system effector functions? (HINT: think about complement!)

Supplementary Materials: Antigen,  Antibody, MHC,  TCR

What you should Know About Avian Flu (CDC)   http://www.cdc.gov/flu/avian/index.htm

WHO statistics on Avian Flu cases http://www.who.int/csr/disease/avian_influenza/country/cases_table_2006_08_23/en/index.html