Influenza viruses

1. Influenza viruses Dr Edward Wright wrighte@westminster.ac.uk 1

2. Key features of influenza viruses Orthomyxovirus ss segmented RNA genome 8 segments 6 of the 8 segments in SO-H1N1 A were from swine influenza Lipid envelope with matrix Proteins project through the envelope: M2, haemagglutinin (H or HA) and neuraminidase (N or NA) HA contains 2 polypeptides, HA1 + HA2 concerned with fusion with host cell HA1 is especially variable NA is an enzyme cleaving sialic acid, has several functions, most importantly virion release 2

3. Influenza subtypes 3 types � antigenic differences in nucleocapsid proteins Type A moderate to severe illness epidemics every 2-3 years humans and other animals (birds and pigs) affects all age groups Type B milder epidemics every 4-6 years humans only, primarily children Type C rarely reported in humans no epidemics 3

4. Influenza A + human disease WHO describes each new isolate as follows: A/Chicken/Hong Kong/317/01 (H5N1) Subtype/host of origin/geographical origin/sequential isolate number/year of isolation (H+N type) 4

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7. The key advantage of a segmented genome is that it facilitates recombination between two strains coinfecting the same cell. New strain can evade the immune system. 7

8. The flu virus has no geometric capsid. RNA genome is loosely contained by a shell of matrix proteins. 8

9. Influenza A virus 9

10. Influenza A viral structure 10

11. Classical influenza Sudden onset of fever, chills, headache, myalgia and anorexia Respiratory symptoms may be associated with both URT + LRT infection, often with dry cough Symptoms may vary with age, with adults more likely to have the systemic effects Long convalescent period of 1-2 weeks but short incubation period of 1-4 days 11

12. Complications Viral pneumonia may rapidly be fatal Bacterial pneumonia due to 2o infection Risk of such complications increases with age and with underlying disease Sometimes adults are most at risk Depending on strain: high case fatalities 12

13. Symptoms and Diagnosis Coughing and sneezing Extreme coldness and fever Fatigue Headache Nasal congestion Aches � especially joints and throat Abdominal pain and diarrhea Throat swab and vial culture - 3-10 days Serology - days PCR - hours 13

14. Influenza Sub-categorised on the basis of membrane glycoprotein subtypes: Haemagglutinin Viral binding / entry, 16 types differentiated serologically Neuraminidase Infection, viral exit, 9 types differentiated serologically Current human subtypes in circulation � H1N1, H3N2, H1N2 Host immune response is directed primarily against HA and NA moieties � limit spread and neutralise infectivity respectively 14

15. The hemagglutinin envelope protein attaches to a host cell by binding to a sialic acid receptor protein The virion is taken up by endocytosis. - acidification induces a conformational change Fusion of envelope and the host membrane - contents of the virion are released into the cytoplasm 15

16. Animation: Influenza Virus Entry into a Cell 16

17. Viral (�) strand RNA are uncoated and enter the nucleus Influenza mRNA synthesis is primed by capped RNA fragments cleaved from host mRNA Viral mRNA return to cytoplasm for translation Genomic RNA synthesis is primed by NP (+) strand RNA is synthesised by prepackaged RNA-RNA pol, which then uses it as a template for (�) RNA strands These are packaged in newly made nucleocapsid proteins (NPs) and exported to the cytoplasm. 17

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19. Capsid assembly occurs in the cytoplasm. Envelope proteins are synthesised at the ER, where they are glycosylated by host enzymes and transferred to the Golgi for export to the cell membrane. At the membrane, the packaged (�) RNA segments are enveloped by host membrane containing the envelope proteins. Mature virions then bud out of the cell membrane. 19

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21. Animation: Influenza Virus Replication 21

22. Haemagglutinin (HA) Responsible for infectious entry of influenza into cells by binding to sialic acid receptor on glycolipids and glycoproteins on the surface of lung epithelial cells Name from the property of agglutinating RBCs Entry: through receptor-mediated endocytosis + low-pH-induced fusion from within acidic endosomes Most important surface antigen (neutralising antibodies) It is glycosylated (may help virus to shield antigenic sites from Nabs) Cleaved by cellular protease into 2 subunits: HA1: receptor binding HA2: membrane fusion activity Avian/Human viruses: HA has different cellular receptor specificity (2,3 vs 2,6 linkage) 22

23. Cell receptors for influenza virus Receptor: sialic acid (a family of 9-carbon monosaccharide) Sialic acids are present on termini of oligosaccharides on cell surface. Ligand: haemagglutinin 23

24. NA NA: cleaves the sialic acid receptor to release progeny virus from infected cell surface NA: role in entry also? NA: target for antiviral drugs [zanamivir + oseltamivir]. Sialic acid analogues: inhibit the release of progeny virus from infected cells 24

25. Seasonal epidemics vs pandemic Seasonal epidemic: 5 to 20% of general population infected Nursing home attack rates of up to 60% 85% of flu-related deaths in ages > 65 Yearly vaccine made against circulating strains Pandemic Novel virus to which population has little or no immunity Virus that is pathogenic and virulent in humans Virus must be capable of sustained person-to-person transmission No vaccine 25

26. Antigenic properties altered by: Antigenic DRIFT: accumulation of mutations in antigenic epitopes of viral antigens Evolutionary, immunological & drug pressure Antigenic SHIFT: new HA subtype from recombination Genetic reassortment between viruses Antigenic Drift and Shift 26

27. Antigenic Drift and Shift (2) A can do both B + C can only drift Changes in HA + NA profiles, reflecting changes in coding RNA Instability of viral RNA during replication 27

28. Antigenic drift Minor changes due to infidelity in viral replication, selective pressure Partial immunity in population Antigenic shift Recombination of viral nucleic acid segments Direct infection of human by different host virus (eg. avian) Reassortment in intermediate host (eg. pigs) Re-introduction of an �old� strain into the population Change in HA and/or NA No previous immunity Pandemic potential Antigenic Drift and Shift (3) 28

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30. Avian influenza Reservoir of influenza A viruses: wild birds Avian viruses: cross species barrier to humans and pigs Result: influenza is a non-eradicable disease Pandemic preparedness: surveillance of influenza in birds, pigs and humans required 30

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32. Antigenic shift Shift results from large changes in viral genome, producing new combinations of HA + NA Genetic reassortment between 2 parental viruses has been demonstrated in the lab Coinfection might enable this to happen in a human or other animal host Might see a very different virus appearing without warning; no-one would be immune! Such a virus could spread rapidly through human populations, resulting in a PANDEMIC 32

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34. Pandemics of the 20th century (have occurred before 20thC but no reliable scientific records) 1918 Spanish flu� H1N1 1957 Asian flu� H2N2 1968 Hong Kong flu� H3N2 1977 Red (Russian) flu� H1N1 Different antigenic strains except 1977 Not clear why H1N1 reappeared 34

35. 1918 pandemic(virus has been recently investigated) Avian origin Genome has been sequenced from human tissue but virus was never isolated Geographical origin is UNKNOWN � best called 1918 influenza, not Spanish! 35

36. 1918 pandemic (2) Spread in 3 waves: Europe, Asia, N America: ~ 50 million died during 1918-1920 Clearly a sudden appearance of a new virus: no-one was immune! High mortality Combination of effective transmission and high pathogenicity: function of its HA + NA profile High mortality in the children and young adults No antibiotics to treat secondary bacterial infections 36

37. A big worry! Could an apparently novel strain suddenly appear (as in 1918) to cause a similar pandemic? Require a virus that can both readily be transmitted from man to man and able to cause severe disease 37

38. Recently, we have had 2 candidates: Avian influenza virus Swine �flu virus 38

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41. In the UK: H5N1 Bernard Matthews turkeys in Suffolk in early 2007 Association with Hungary, virus probably brought in infected meat Outbreak contained by culling 100,000 birds No prosecutions; compensation! 41

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43. Avian vaccines Only inactivated ones can be legally used Activated ones exist and are known to be used illegally Risk of using activated forms � reassortment to produce a novel and highly pathogenic virus in vaccinated birds 43

44. H5N1 human vaccine Prepandemic & pandemic preparedness Undergoing clinical trials Various inactivated vaccines stockpiled Baxter, Novartis, Sanofi Pasteur, GSK� Current status and progress of prepandemic and pandemic influenza vaccine development. Leroux-Roels I, Leroux-Roels G Expert Rev Vaccines 2009;8:401-23 44

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46. �Swine �flu�Pandemic Influenza A H1N1(v) www.who.org www.hpa.org.uk/publications/infectious diseases/influenza/ 46

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48. Pandemic H1N1 in UK Imported from Mexico or US in 2009 UK and Ireland were hard hit: Mexico is an important holiday destination Began to appear in late April/early May Then transmission in households + schools � some school closures First death reported 14 June 2009 This was the first wave of transmission and its peak in July was associated with schools 48

49. Pandemic H1N1 in UK (2) Second wave started when schools returned in autumn and peaked in October This is the start of the typical flu season It was the prevailing strain of influenza being transmitted and replaced the predicted seasonal strains 49

50. Younger people were most likely to be affected Severe disease and deaths most common in those aged UNDER 65 years old Those with underlying disease were no more likely to catch disease but were more likely to be hospitalised or die Overall case fatality rate: 0.4% NOT A SEVERE DISEASE in the healthy! 50

51. 2 vaccines licensed for Europe Pandemrix Not a live vaccine Usually only one dose needed Prepared (as with seasonal vaccine) in hen�s eggs Celvapan Not a live vaccine 2 doses, three weeks apart Not prepared in eggs- may be used for those with egg allergy 51

52. Epidemics These are continuously occurring and surveillance both nationally and by WHO can help predict the most likely new strain(s) responsible Can do this by monitoring disease and the RNA of isolates Such predictions enable protective measures to be put in place 52

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55. Vaccine strain selection: seasonal Vaccine components have to match those of the circulating strains in target season Current vaccines contain 3 virus strains: 2 A strains [H3N2/H1N1] and 1 B strain Northern hemisphere recommendations [due Feb 2011] for November 2011-April 2012 Northern hemisphere 2011-12 vaccine strain selections: An A/California/7/2009 (H1N1)-like virus An A/Perth/16/2009 (H3N2)-like virus A B/Brisbane/60/2008-like virus 55

56. Vaccine strategies against H5N1 influenza 56

57. Avian influenza vaccine strain selections [H5N1] 57

58. Serology/Vaccine Evaluation The influenza virus surface glycoprotein hemagglutinin (HA) is the most important antigenic determinant for virus-neutralising antibodies generated during natural infection or elicited by immunisation. Hemagglutination inhibition (HI) assays are employed for the detection of antibody in serum, with HI titres correlating with protection from influenza in humans. Neutralisation assays allow for more sensitive detection of H5 antibodies, but these are laborious and require Biosafety Level 3 laboratory facilities or higher which are not always available at the front line of an outbreak, especially in resource-limited regions. 58

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61. Microneutralisation Virus + cells (MDCK) -> CPE (cytopathic effect) The presence of neutralising antibody in the plasma sample inhibits viral infection of permissive cells and hence no CPE. It measures protection. 61

62. New assays for neutralising antibodies In order to make neutralization assays more widely applicable there are two realistic options for rapid development: 1. To use reverse genetics to engineer a safer, attenuated virus by deletion of the polybasic cleavage site in HA as is done for the development of inactivated vaccines for pandemic influenza. 2. The construction of viral pseudotypes bearing the influenza HA glycoproteins as surrogate viruses for use in neutralization assays. The first option has its inherent problems, namely the issue of possible reversion to the wild type virus via recombination. The level of attenuation of such mutants may differ between strains/isolates and careful risk assessment is required. With the pseudotype system however, only the HA from influenza is required, with no possibility of recombination or virus escape. 62

63. Reverse genetics 63

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