1. Influenza

2. http://subwayblogger.com/wp-content/uploads/2008/10/sneeze.jpg

3. Flu Pandemics Russian Flu (1889 – 1890) –Approx. 1 million deaths –H2N2 subtype Spanish Flu (1918 – 1919) –40 – 100 million deaths –H1N1 subtype Asian Flu (1957 – 1958) –1 – 1.5 million deaths –H2N2 strain Hong Kong Flu (1968- 1969) –0.75 to 1 million deaths –H3N2 subtype 2009 H1N1 –Between 151,700 and 575,4000 with 80% of deaths in those 65 and younger –H1N1 subtype

4. Flu RNA Virus 3 Influenza types –A–A Initial host - wild aquatic birds but have now infected many different animals Divided into 2 categories –Low pathogenic and Highly Pathogenic –B–B Primary host - Humans –C–C Only hosts - Swine and Humans Types A and B cause yearly outbreaks A mutates the fastest B mutates at a rate 2-3 times lower than type A However, since it does mutate, then no lasting immunity is possible C is the slowest to mutate

5. Influenza A and B A is most virulent A can be subdivided into serotypes –Hemagglutinin 17 subtypes –Neuraminidase (Sialidase) 10 subtypes –Genome is generally divided between 8 separate gene segments B can not be broken down into subtype Both have various strains Current Vaccine help A and B but not C –an A/California/7/2009 (H1N1); –an A(H3N2) virus A/Victoria/361/2011; –a B/Massachusetts/2/2012-like. Quadrivalent vaccine this year includes a B/Brisbane/60/2008-like (B/Victoria lineage) virus. http://www.microscopy.fsu.edu/cells/viruses/images/influenzafigure1.jpg Last year a New Flu – H3N2v which had Pig to human transmission

6. Influenza A Nomenclature http://en.wikipedia.org/wiki/File:Influenza_nomenclature.svg

7. Human, Swine or Avian Flu? Most of the time they all are Influenza Type A The subtype can be the same –for example - H1N1 or H3N2 But different strains have adapted for the particular host –Seasonal flu is caused by Influenza A viruses that have changed to spread in humans (human influenza) Currently circulating in humans are H3N2 and H1N1 viruses –Avian flu is caused by Influenza A viruses that affect birds. –Swine flu is caused by Influenza A viruses that affect pigs Influenza A viruses that typically infect one species can cross over and cause illness in a different species –Until 1998 only one H1N1 circulated in pigs then a human H3N2 crossed over –Recently a H3N8 from horses moved to dogs Influenza A viruses “mutate” allowing yearly outbreaks and species jumps –Random mutation or reassortments

8. Antigenic Drift Random mutations that change the antigens in the virus These mutations allow the virus to evade the immune response Primarily concerned with mutation in hemagglutinin which is the cell surface protein responsible for entry into cells Diagram from the National Institute of Allergy and Infectious Diseases

9. Antigenic Shift Reassortment of two or more different strains Typically from two different species Often enables a flu strain to jump from one animal species to another Could be from direct or intermediate transmission with no genetic changes Diagram from the National Institute of Allergy and Infectious Diseases

10. Avian Influenza A viruses typically are transmitted to humans in two ways –Through direct contact –Through an intermediate host Avian Flu replicates less efficiently in humans Human less efficiently in birds Avian viruses preferentially bind to N-acetylneuraminic acid- α2,3 galactose Human viruses preferentially bind to N-acetylneuraminic acid- α2,6 galactose Swine have BOTH!

11. Overlaid images of α(2,6)- and α(2,3)-linked sialylpentasaccharides LSTc and LSTa, respectively, in the HA receptor binding site

12. Hurdles to Cross “Species Jump” –adaptation that allows the strain to pass to another species –Infection generally occurs via direct contact with animals who are ill. Human to Human –development of changed viruses with the ability to cause infection and spread in the human population Examples – 1918 flu pandemic which is believed to have been initially caused by an antigenic drift from an avian flu to a human flu –antigenic shifts from the reassortment of an avian flu and a human flu (e.g Asian and Hong Kong pandemics) There may be little or no immunity in the human population to these new viruses.

13. Viral Infection RNA dependent RNA Polymerase has no proof reading activity, so Error rate of 10 5 which results in antigenic drift 1.Binding 2.Uncoating 3.Replication/ transcription 4.Translation 5.Protein processing 6.Reassembly 7.Budding

14. Influenza Genes and Proteins Genes are on 8 separate segments of negative-sense single-stranded RNA that encode 11 known proteins General category Protein(s) encoded Protein functions Surface proteinsHemagglutinin (HA)Viral attachment and fusion protein Neuraminidase (NA)Virus release M2 ion channelFacilitates viral RNP uncoating Internal proteinsMatrix (M1) proteinViral structural protein regulates RNP nuclear import Nucleoprotein (NP)Required for viral RNA synthesis Polymerase ComponentsPAPolymerase subunit PB1Polymerase catalytic subunit endonuclease activity PB2Polymerase subunit binds caps of host cell mRNAs Non-structural proteinsNS1Evasion of interferon responses PB1-F2Proapoptotic factor, possible immune evasion function Nuclear export protein Mediates viral RNA nuclear export (NEP or NS2) Antiviral ResearchAntiviral Research Volume 79, Issue 3, September 2008, Pages 166-178Volume 79, Issue 3

15. Hemagglutinin Lectin (Glycoprotein) that mediates binding of virus to cell Viruses bind through hemagglutinin onto sialic acid sugars on the surface of epithelial cells triggering uptake of the virus via endocytosis

16. pH structure change Low pH in endosome causes conformational changes that facilitate fusion of viral and host membranes. Postulated to be similar to SNARE complexes Fusion between endosome and virus forms a pore through which the viral genetic material enter the cytosol

17. Hemagglutinin The presence of a multibasic cleavage site is not the only determinant of viral pathogencity. The 1918 Flu had a single site while H5N1 has a multibasic site HA0 must undergo proteolytic cleavage into HA1 and HA2 to allow the conformational change required for membrane fusion. LPAI have a single basic singe basic cleavage site HPAI have a multibasic cleavage site Variations in host protease determines location and effectiveness of infection LPAI is cleaved by a host trypsin like protease limited to the respiratory tract HPAI is a furin-like protease which present in virtually all cells thus allowing HPAI to spread easily throughout the body often causing fatal systemic infections

18. M2 protein Proton selective ion channel located in the viral envelope Transports protons from the endosome into the virus particle Lower pH allows dissociation of the viral matrix protein M1 from the ribonucleoprotein RNP Crucial step in “uncoating” of the RNP and exposing the content to the cytoplasm of the host cell The RNP complex is transported to nucleus where replication and transcription occurs The gene for M2 is susceptible to mutations, so strains are developing resistance

19. M2 Antiviral drugs Symmetrel and Flumadine –function by blocking the channel –effective against influenza A –Many strains are resistant too

20. Neuraminidase Glycoprotein Glycosidase Cleaves the sialic acid residue on the host cell allowing the mature virus to detach Flu treatment –neuraminidase inhibitors –effective against both influenza A and B

21. Neuraminidase Inhibitors Tamiflu Relenza Structure based design

22. Avian “Bird” Flu Only a few subtypes have been highly pathogenic in humans. –Spanish, Asia and Hong Kong Most recent are H5N1 and H7N9 in Asia Most infections were contracted after direct contact with infected bird Limited evidence of human to human transmission Expected to be the next deadly global pandemic Highly Pathogenic H5N1 which generally results in severe pneumonia has 60% mortality world wide

23. 1918 flu pandemic H1N1 subtype Initially caused by an antigenic drift High death rate was due to cytokine storm –too many immune cells are activated in a single place In lungs fluids and immune cells accumulate and eventually block off the airways More susceptible were those with healthy immune systems

24. Mortality Age Distribution for 1918 epidemic and normal epidemics (CDC is author, Taken from "1918 Influenza: the Mother of All Pandemics" Jeffery K. Taubenberger and David M. Morens, http://www.cdc.gov/ncidod/EID/vol12no01/05-0979.htm )

25. Asian and Hong Kong Flu H2N2 A mutation in wild ducks combed with a pre-existing human strain H3N2 evolved via antigenic shift and caused a smaller outbreak

26. Protein Changes in function that could affect virulence Increased virulence of the 1918 H1N1 and H5N1 Bird flu has been linked to: the HA and NA proteins and the polymerase proteins (PB1,PB2, and PA) and the Non-structural proteins HA –Altered cleavage site allowing change in tropism; –altered receptor specificity allowing increased transmissibility and altered tropism in the airway NA –Ability to promote HA cleavage M2 –Mutations that confer resistance to amantadine and rimantadine PB1 –Enhanced virus RNA synthesis PB2 –Enhanced virus RNA synthesis in mammalian cells, enhanced polymerase function at high temperature NS1 –Enhanced ability to suppress innate immunity PB1-F2 –Enhanced pro-apoptotic activity None suggest so far for –M1, NP, PA or NS2

27. 1918 Influenza Avian HA consensus sequence –Six residues are conserved in the HA1 domains of most avian H1 HAs –implicating these residues in the ability to bind to the avian 2,3Gal receptor Comparison of the avian HA consensus sequence with the 1918 Virus shows that only one or two of these conserved residues are different –190 and 225 For H1 1918 –Residue 190 alone determined shift Know already that H5N1 found in humans have mutations at several positions which allow better recognition of the human receptor

28. H5N1 Might only require a singe BASE change of switch to human receptor Cell, Volume 153, Issue 7, 1475-1485, 06 June 2013Volume 153, Issue 7

29. H7N9 The hemagglutinin of H7N9 virus does not efficiently bind human receptors A single residue change in receptor binding site increases binding to human receptors Mutations on hemagglutinin may reduce the effectiveness of current H7 vaccines Cell, Volume 153, Issue 7, 1486-1493, 06 June 2013Volume 153, Issue 7

30. Minimum Changes Needed 1 st better binding to mammalian receptor Hemagglutinin –Only 2-4 amino acid substitutions are required to switch binding from α2,3 to α2,6 2 nd Better uptake and release of the virus Neuraminidase –In the 1918 virus, Neuraminidase plays an unusual role in binding and activating the host protease that cleaves HA rendering the virus infectious Cleavage of the Hemagglutinin precedes the conformation change which leads to cell fusion Cleavage is generally dependent on the type and distribution of the host trypsin like protease –H5N1 Neuraminidase is rather slow in cleaving the sialic acid to release the new virus particle –A few mutations will take the H5N1 closer to the 1918 neuraminidase

31. Minimum Changes Needed Polymerase proteins (PB1,PB2, and PA) –The 1918 virus contains only 10 aa changes in the sequences of the polymerase proteins –H5N1 already has 7 of the 10 –Important because avian viruses replicate around 41C but effective replication in mammals is 37C –Both 1918 and H5N1 have a single mutation, N66S, which has been linked to increased virulence

32. 2009 H1 N1:Genetics of New strain What is special about this H1N1 outbreak? Reassortment of: –One human strain – Two pig strains –One bird strain Hemagglutinin gene is similar to current swine flu viruses present in U.S. pigs Neuraminidase and matrix protein genes resembled versions present in European swine flu isolates This is different from the previous seasonal H1N1 strain High rate of Human to Human transmission But mild symptoms so far N Engl J Med 361:115-119

33. Infectivity and Virulence H5N1 and H7N9 currently has low Human to Human transmission but are highly virulent The 2009 H1N1 quickly became a pandemic (highly pathogenic) but was not very virulent What will be the next pandemic Will we see a reassortment (antigenic shift) of 2009 H1N1 and H5N1 or H7N(? Will H5N1 have a minor antigenic drift like 1918 H1N1 allowing it to be transmitted human to human? Are we prepared? Pathogenic – cause disease Virulent – extend of pathology Infectivity – the ability of a organism to invade and replicate