2. Introduction Humans acquire more viruses by the respiratory route than any other. Most common: mumps, measles, chicken pox, German measles, common cold, and influenza. Coughs/Sneezes contain large numbers of virus so some get past the defense of mucus in nasal passage.

3. Flu: The virus Influenza viruses that cause serious human disease are divided into two groups: A and B. Influenza A -- responsible for major worldwide epidemics (H1N1) – Divided into subgroups based on two proteins on the surface of the virus hemagglutinin antigen (H) (15 subgroups) neuraminidase antigen (N) (9 subgroups) H1N1 and H3N2 (associated with more deaths) are the current subtypes of flu virus A in humans Data suggests that all influenza A type viruses were in fact a bird virus originally. Electron micrographs of regular particles of influenza virus after growth in the laboratory. Micrographs prepared by Ross Hamilton.

4. Influenza and RBC’s: How did Hemagglutinin get its name? Hemagglutinin got its name because of the affect it has on RBC’s, which carry hemoglobin. If influenza infects RBC, it is possible for the hemagglutinin receptors (~500 on one virus), to bind to more than one RBC at a time. This causes clumping of many RBC’s. If you’re blood is clotting….death is possible.

5. Epidemiology: how the Flu spreads Pandemics - influenza A pandemics arise when a virus with a new haemagglutinin (antigen) subtype emerges as a result of antigenic shift. As a result, the population has no immunity against the new strain. (unusual flu type) An antigenic shift: Caused from two or more viruses recombining their genetic material to create new viral surface antigen. Epidemics - epidemics of influenza A and B arise through more minor antigenic drifts as a result of mutation. (yearly flu types) Either can cause death in individuals. Due to global travel, Pandemics are a real possibility as seen in 2009 with the H1N1 Mexico (Swine) Flu

6. Past Antigenic Shifts: cause pandemics 1918H1N1 “Spanish Influenza” 20-40 million deaths 1957H2N2 “Asian Flu” 1-2 million deaths 1968H3N2 “Hong Kong Flu” 700,000 deaths 1977H1N1 Re-emergenceNo pandemic 1997 H5N1 Bird FluEpidemic in Asia 2009H1N1 Re-emergencePandemic (swine flu) At least 15 H subtypes and 9 N subtypes occur in nature. Up until 1997, only viruses of H1, H2, and H3 are known to infect and cause disease in humans.

7. How Does Seasonal Flu Differ From Pandemic Flu? U.S. Department of Health and Human Services

8. Avian Influenzas: The Viruses Birds are infected only by influenza A viruses (avian influenza A viruses). – Many forms do not cause symptoms. – Occur naturally in bird populations. – All Human Influenza viruses are part Avian Highly virulent – Humans do not have pre-existing immunity. First human infection in 1997 – Acquired from poultry – No direct human-to-human transmission documented. Evolution of H5N1 since 1997 – Evidence suggests H5N1 has acquired the ability to replicate in mammals (possibly pigs). – H5N1 has caused the death of large numbers of wild migratory birds. 1997 was the first year that H5N1, a strain of avian influenza A virus, was found in humans. Photo: CDC

9. Avian Influenzas: Change Mechanisms (part 1) Antigenic “drift” – Small errors (mutations) occur during the copying of genetic information. – Flu A viruses are unable to repair errors. – Small changes make the virus look new to the immune system. – Immunity against previous strains does not protect against the new version. NIAID/NIH

10. Avian Influenzas: Change Mechanisms (part 2) Antigenic “shift” – Drastic change in the composition of a virus. – Influenza A viruses can exchange genetic material with other subtypes. – This process results in new combinations of H and N subtypes. – Hong Kong flu resulted from the emergence of a new H3N2 combination. NIAID/NIH

11. Human Influenza Virus Type of Nucleic Acid: Negative sense RNA Genome: consists of 8 gene segments + RNA polymerase protein. Viral Structure: Enveloped virus (with part of human membrane from budding), with hemagglutinin and neuraminidase spikes. Spikes are antigens. 3 types of Virus: A (most dangerous), B (seasonal), and C (mild) Type A undergoes antigenic shift due to the inability to proofread its new RNA strands. RNA polymerase makes a mistake nearly every 10,000 new nucleotide copies, so every virus is a mutant. Type B undergoes antigenic drift only (not Type C is relatively stable, and mild (Courtesy of Linda Stannard, University of Cape Town, S.A.)

12. Influenza A viral reproduction Human cells have a receptor called: Sialic Acid Viruses have a receptor called: Hemagglutinin How does a virus connect to the human cell? Answer: by the lock/key method: Sialic Acid is the lock, Hemagglutinin is the key. When the key is in the lock, a conformational change happens to the sialic acid receptor, causing the cell to engulf(eat) the virus and bring it inside the cell in an endosome. An endosome is a small membrane “bubble” that travels.

13. Endosomes Endosomes are bubbles of the cell membrane inside out, carry something into the cell. So, pH pumps are “backward” and pump hydrogen protons into the endosome. This increases the H+ inside of the endosome, causing a lower pH. Therefore, if you decrease the pH of the environment of a cell, you destroy the membrane…. Gets to a pH of ~5 Question: The influenza virus is membraned. How would this process benefit the virus?

14. Inside the acid bath… When the viral membrane is broken, it fuses with the endosome itself. Thus, the protein structure is destroyed and the RNA and polymerase is released into the cell to make its way to the nucleus.

15. Inside the nucleus (-) sense RNA can not produce proteins. It’s backward. A complementary copy of RNA is made in the (+) direction using the RNA polymerase molecule found associated with the viral strand. Viral (+) strand RNA is now viral mRNA and ready to leave the nucleus and complete translation into viral proteins.

16. Being Nasty mRNA can not start translation without a starting code called the 5’cap. Viral mRNA takes the cap off of normal cellular mRNA to work. This effectively stops cellular mRNA from working, and allows viral mRNA to make its proteins and not work has hard to do it!

17. Viral Budding: How to get out Some (+) strand mRNA copy into more (-)strand RNA for viral packaging. Remember, the virus comes with (-) strand RNA. The viral RNA also made hemagglutinin proteins that were inserted into the cellular membrane (MHC). The (-)vRNA moves to the cell membrane with the viral receptors and the membrane is pinched off into a new enveloped virus. The virus bud needs to escape the cell by not being re- trapped by sailic acid.

18. Being an escape artist During viral replication, a viral protein called neuraminidase was produced and sent to the surface of the cell. New viruses have both hemagglutinin, sailic acid, and neuraminidase receptors from the original cell membrane. Neuraminidase protein cuts off the sialic acid residues from the surface of the infected cell as it buds out severing the possibility of getting stuck at the surface.

19. Viral Spread Influenza infects epithelial cells lining the upper and lower respiratory tract. Every cell in the respiratory tract has sialic acid receptors so one infected cell spreads the virus through budding throughout the entire respiratory tract. The virus can also survive on nonporous surfaces (phones, desks, chairs) for days! Can kill virus with soap or lysol.

20. Inflammation in the lungs due to infection Influenza virus has a cytolysic cycle, which means it will pop the cell it has infected causing cell death. Extra cell death causes inflammation, and triggers a cough reflex. It only takes about 10 viruses to cause an infection and about 100 are released with 1 cough

21. Evading Host Defenses A virus making (+) mRNA from (-) mRNA will at some point have a lot of double stranded RNA while the copies of these two are being made. dsRNA induces the interferon system. Normally, interferons bind to infected cell’s receptors and tell them to produce a protein kinase called PKR. PKR is a sensor for dsRNA and activates phosphate molecules.

22. What does PKR do normally? PKR phosphorylates a protein called eIF2. (Phosphorylation means that phosphate groups are added to the molecule so that it can’t work any longer, or turns it on.) eIF2 can’t work to participate in protein synthesis if it is phosphorylated. This should help to shut down infected cells, but……

23. Influenza Virus has a counter attack on PKR! NS1 is produced by the virus. NS1 attaches to the dsRNA so that PKR isn’t activated. This buys the virus time to finish replication even though the interferon system will eventually work.

24. Immune system response Time it takes to eradicate Influenza=2wks Most useful worker- Killer T cell which responds to infected cells by killing them. Circulating memory B antibodies attach to hemagglutinin and interrupt replication Antibodies also target the neuraminidase “razor” to stop it from cutting off sialic acid receptors. This causes the virus to clog up in an area and time for macrophages to find it.

25. Influenza only causes acute infections. Acute means short term. Since the virus shows a drift of the receptor make up, our antibodies can’t always recognize the same virus so the infection occurs. This is the bait and switch. Looks different on the outside, but the same virus on the inside. Often, a virus (bird) can use a secondary animal (pig) to mutate and infect humans. This can results in dramatic mutations.

26. Influenza-Associated Pathology Interferon system (innate) is responsible for most symptoms. Remember: interferons cause cell death, which triggers a response because of too much cell waste by macrophages. This action leads to headaches, muscle aches, fever, and fatigue. Cell death causes sore throat, cough, and hoarseness. Death of ciliated cells causes a loss of function in the lungs (no mucus) so other pathogens have chance to invade. (bacteria causing pneumonia) A good immune response will keep the infection to a minimal annoyance.

27. Prevention Minor genetic changes in the virus help it to escape the immune system’s memory. This is antigenic shift. The surface receptors are very prone to mutations during RNA replication in the host cell. To keep up with these changes, influenza vaccines must be reformulated annually. Inactivated split/subunit vaccines are available against influenza A and B. The vaccine is normally trivalent, consisting of 1- A H3N2 strain, 1- A H1N1 strain, and 1- B strain. The strains used are reviewed by the WHO each year. The vaccine should be given to debilitated, asthmatics, and elderly individuals who are at risk of severe influenza infection.