MICR 420

1. MICR 420 Emerging and Re-Emerging Infectious Diseases Lecture 8: Influenza Viruses Dr. Nancy McQueen & Dr. Edith Porter

2. Overview • RNA viruses • Influenza viruses • Brief history • Nomenclature • Morphology and nature of the genome • Viral replication cycle • Genetic variability • Pathogenesis and clinical symptoms • Diagnosis • Treatment • Prevention • Threat

3. What a virus? • Not a living cell • Acellular particle • No cell membrane • Consist of nucleic acid and protein only • RNA or DNA, not both • Protein coat : capsid • Some are enveloped: membrane from host cell • Infects living host cells to replicate • All forms of cells can be infected by a virus • Bacteria, archaea, eukaryotes • Virus depends on host metabolism • Viral genome subverts host cell’s machinery to reproduce • Estimate of 1032 viruses on earth

4. Transcription and replication of RNA viruses

5. RNA viruses • Why are so many of the newly emerging infectious diseases caused by RNA viruses? • All must bring in their own RNA dependent RNA polymerases (replicases) • Are error-prone - error rate of 10-3- 10-5 • Have no proof reading function • Many quasi-species found in viral infections • Nitric oxide (NO) production by host accelerates viral mutations Rapid evolution

6. What is influenza? • “Influenza (the flu) is a contagious respiratory illness caused by influenza viruses. It can cause mild to severe illness, and at times can lead to death.” (http://www.cdc.gov/flu/about/disease/index.htm) Influenza Lung Normal Lung Figure 1. Gross and microscopic lesions from dog infected with highly pathogenic avian influenza (HPAI) H5N1. A) Severe congestion and edema in the lung. B) Lung histopathologic results showing severe pulmonary edema and hemorrhage with black-brown particles (hemosiderin) (magnification ×100). (cdc web site). http://www.biologyofhumanaging.com/slides/lng120d1.jpg

7. Brief history and epidemiology • Influenza appears to have afflicted humans since ancient times. • Hippocrates in 412 BC • Numerous epidemics in the middle ages • Epidemics of influenza • Every winter • > 20,000 deaths/year • Elderly or immunocompromised individuals. • Pandemics • Irregular 10-50 year intervals. • The Spanish flu (1918-1920) - killed 20-40 million people worldwide • The Asian flu (1956-1957) - 60,000 deaths in North America.

8. Spread of the Asian Influenza pandemic in 1957

9. Nomenclature • Family Orthomyxoviridae • Myxo = mucus - virons bind to sialic acid residues in mucoproteins • Genus: Influenza Virus • Three groups which share a common structure and mode of replication, but differ serologically based on M and NP antigens • Type A infect humans and animals; epidemics and pandemics • Type B infects humans only; epidemics • Type C infects humans and pigs; mild disease

10. Classification of human influenza viruses • Type A or B • Geographic source • Isolate number • Year of isolation • Four HA: H0, H1, H2, H3 • Two NA: N1, N2 More on the significance of HA and NA will be discussed later on

11. Hemagglutinin subtype Influenza type Year of isolation A/Panama/2007/99(H3N2) Isolate number Geographic source Neuraminidase subtype World Health OrganizationInfluenza Nomenclature

12. Morphology and nature of the genome • SS (-) RNA genome • Segmented genome (8 segments encode 11 proteins) • Virions may be spherical or filamentous

13. Influenza Virus

14. Replication of Influenza Virus

15. Why do we continue to have Influenza Virus Epidemics? • Genetic variability • Influenza virus keeps changing its structure via two different mechanisms: • Antigenic drift - changes in the antigenic determinants of the HA and NA that accumulate with time. (result in variants of the SAME NA or HA type) • Viral RNA polymerase • Error prone • No proofreading • Provides a selective advantage • Antigenic shift - major changes due to a re-assortment of genes that occurs when two different influenza viruses infect the same host. • Recombination by template switching • Between homologous segments of two different strains • More common in avian isolates Rapid evolution

16. Antigenic Shift Two different viruses infect the same pig and through re-assortment of the gene segments, a new virus is generated Human influenza virus Avian influenza virus New human influenza virus

17. How do we acquire viruses from pigs?

18. Genetic variability • The 1957 Asian influenza pandemic- antigenic shift (new HA, NA, and PB1) • The 1968 Hong Kong pandemic - antigenic shift (new HA and PB1) • 1918 Spanish influenza pandemic was not due to shift- new studies indicate that it arose from an avian virus by drift. • Enhanced cleavability of HA due to NA changes! • Changes in NS1 made it a potent inducer of pro-inflammatory responses • A single mutation in HA resulted in a virus that had gained the ability to bind to sialic acid residues present in the human respiratory tract. • Changes in PB1 enhanced viral replication • PB1-F2 - promotes apoptosis

19. Pathogenesis and clinical symptoms • Aerosol transmission • 3 day incubation (influenza A) • Virus initially infects epithelial cells in the upper respiratory tract • Loss of the ciliated epithelium • Direct effect of virus multiplication and release • Due to toxic oxygen radical formation (host cell response) • Due to apoptosis • dsRNA and NA may trigger host cell responses that contribute to apoptosis • PB1-F2 sensitizes cells to apoptosis (not produced by all strains)

20. Pathogenesis and clinical symptoms • With loss of ciliated epithelium: • Loss in the ability of the respiratory tract to clear viruses or bacteria by mucociliary flow • Secondary bacterial infections • Virus replication induces interferons and other cytokines (IL-6, IL-8, TNF-) leading to local and systemic inflammatory responses. • This results in the symptoms that define the “flu” syndrome: Death

21. Pathogenesis and clinical symptoms • Fever • Headache • Chills • Malaise • Muscle aches • As the fever declines • runny nose • coughing

22. Selected virulence characteristics • HA for attachment • Inhibition of host mRNA translation (establishing control of the host) • Cap snatching • Viral mRNAs compete more effectively for initiation factors. • Inactivation of the cap binding reaction by removing the required phosphate from eIF-4E, reducing available initiation factors • NS1 interferes with host cell mRNA splicing, polyadenylation, and transport to the cytoplasm

23. Inactivation of eIF-4

24. Summary of virulence characteristics • Evasion of host defenses • NS1 binds to dsRNA to inhibit activation of IFN • Damage • Induction of apoptosis - dsRNA, NA, and Pb1-F2 all play a role • NO and O2- • NO enhances development of more quasi-species • Induction of cytokines

25. Strain dependent differences in pathogenesis • Strain differences may result in differences in the severity of the disease for both human and avian viruses. • Aquatic birds are the natural reservoir for avian influenza A viruses • Is usually asymptomatic in feral birds • Highly pathogenic strains may cause serious systemic infections in domestic poultry • Due to the presence of a polybasic cleavage site in HA (Cleavage of HA at a basic residue by host cell proteases is required for viral infectivity) • For human viruses, systemic spread has not been documented. This may be due to:

26. Strain dependent differences in pathogenesis • Lack, in other organs, of proteases that capable of cleaving the HA • Interferon activity • In humans variations in pathogenicity may be due to • Differences in the effectiveness of NS1 to antagonize IFN / production • Differences in NA that allow binding of host proteases that assist in HA cleavage activation or activation of apoptosis • Those most likely to succumb to the disease are usually the elderly and the very young. Why? • The 1918 strain was an exception to this rule - it caused more severe symptoms in those who were the most immunocompetent! • Due to an overdeveloped immune response (“cytokine storm”) of the host against the virus!

27. Cytokine storm

28. Diagnosis • Nasopharyngeal swabs, washes, or aspirates taken early in the course of the disease are the best specimens • The virus can be grown in the amniotic or allantoic cavity of embryonated chicken eggs, or in tissue culture cells with trypsin added to cleave HA.

29. Diagnosis • May assay directly for the virus (direct assay) • May assay for antibodies, produced in the host, against the virus (indirect assay) • Hemagglutination assay-a direct method to identify the presence of the virus and to get a rough titer of the virus. • Is based on the ability of influenza viruses to agglutinate RBCs. • Virus is titered by making serial two-fold dilutions of the virus and determining the highest dilution of virus that causes agglutination of the RBCs.

30. Hemagglutination assay

31. Hemagglutination assay www.medicine.mcgill.ca/.../hemag_plate.jpg

32. Serological/Immunological Methods • Hemagglutination-Inhibition Assay – an indirect test for antibody against specific influenza virus types -

33. Hemagglutination Inhibition www.cdc.gov/.../16/2/images/09-1733-Ft.gif

34. Serological/Immunological Methods • Immunofluorescence • Enzyme immunoassay (EIA) • Optical immunoassay

35. Treatment • Amantidine and rimantidine – targets the M2 protein, blocking the ion channel it forms and preventing uncoating of the virus. • Only effective against Group A influenza viruses

36. Treatment • Zanamivar (Relenza) and Oseltamivar (Tamiflu) – target the neuraminidase, inhibiting its activity and, therefore, inhibiting release of the virus. • Effective against both Groups A and B

37. Prevention by vaccination • Need a new vaccine every year because of shift and drift of the virus • Whole inactivated virus - flu shot • Live, attenuated cold adapted virus (LAIV or FluMist) • Made by combining the HA and NA genes of the targeted virus strain with the six other gene segments from mutant viruses known to have restricted growth at 370C • Nasal-spray inoculation • The reassortment viruses cannot replicate in the lung at core body temperature, but grow well in the cooler nasal mucosa where they stimulate an excellent immune response.

38. PB2 PB2 PB1 PB1 PA PA HA HA NA NA PB2 NP NP PB1 M M PA NS NS HA NA Attenuated Influenza Vaccine Virus Virulent Wild Type Influenza Virus NP M NS Attenuated Vaccine Virus To New Virus Type

39. Vaccination in development • Subunit vaccines • Poxvirus recombinants expressing single viral proteins • Oligopeptides corresponding to the antigenic components of the HA protein • DNA-based vaccines • Target epitopes that are highly conserved in all influenza A viruses

40. WINTER - 2007Should we be afraid of the avian (bird) flu? • Starting in 1997, a highly virulent avian form of influenza (H5N1) spread through the commercial poultry farms in Hong Kong. • It has now been found in many sites in Southeast Asia and a number of humans have been infected, with several resulting deaths • Due to apoptosis of alveolar epithelial cells and leukocytes • Due to enhanced proinflammatory cytokine response • Due to pneumonia that progresses to ARDS and multi-organ failure • Fortunately, these strains have not yet shown signs of spreading efficiently among humans……….

41. Avian influenza Cycle of Avian Influenza Viruses in Animals & Humans Direct bird to human transmission is also possible. Domestic birds Natural avian influenza cycle Pandemic disease cycle Shore birds Water fowl Mammals (primarily swine)

42. No human-human spread • Avian HA proteins preferentially recognize and bind to sialoligosaccharides terminated by N-acetylsialic acid linked to galactose by an α2,3 linkage. • found on the respiratory epithelium of birds • found on lower respiratory tract of humans along with α2,6 linkage • only α2,6 linkage is found on the upper respiratory epithelium of humans • Pneumonia more common • Shift of drift could change this…….

43. Attachment of influenza viruses

44. Genes involved in H5N1 pathogenesis

45. Avian Influenza pathogenesis • Changes in polymerase allow for replication at lower temperatures of humans • The avian viruses normally do not replicate well in the upper respiratory tract of humans (33º C); normally replicate in intestinal tract of birds (41º C) • PB1-F2 causes apoptosis in macrophages which delays immune response; also enhances pro-inflammatory response • NS1 is a potent inducer of pro-inflammatory cytokines (TNF) through PDZ domain • HA binds to sialic acid residues with α2,3 linkage

46. Nations With Confirmed Cases H5N1 Avian Influenza (February 2007)

49. The pandemic H1N1 influenza virus • In 2009 a new H1N1 strain was passed from pigs to humans • The virus rapidly dispersed worldwide in a few months • Ultimately found to not be more virulent in humans than the seasonal strains