C hair of M icrobiology, V irology, and I mmunology

1. Chair of Microbiology, Virology, and Immunology Paramyxoviruses: Mumps virus, Measles virus.

2. Orthomyxovirus (Influenza) Family The name myxovirus was originally applied to influenza viruses. It meant virus with an affinity for mucins. Now there are 2 main groups – the orthomyxoviruses and the paramyxoviruses Differences between orthomyxoviruses and paramyxoviruses

3. Influenza • “La malatia per l’influenza della stella”(the disease caused by the influence of the stars) • In French: grippe, from French verb “agripper” (clinging)

4. INFLUENZA • severe respiratory disease • 20-50 million respiratory illnesses each year in the U.S. • 30 million visits to physicians, 200,000 hospitalizations • 20,000 deaths • new influenza virus strains associated with severe pandemics and high mortality.

5. ORTHOMYXOVIRUSES (INFLUENZA VIRUSES) Classification • Type A viruses cause the most cases of influenza in humans and undergo mutations more frequently than the other type viruses • Type B viruses are endemic in USA and associated with local epidemics • Type C viruses rarely cause disease

6. Orthomyxoviruses. Nomenclature Human influenza virus Influenza A/Bangkok/1/79(H3N2) Influenza A/Singapore/1/57(H2N2) Influenza B/Ann Arbor/1/86 Influenza type Hemagglutininsubtype Year of isolation A/Sydney/5/97 (H3N2) Geographic source Neuraminidase subtype Isolate number

7. Influenza virus A Orthomyxoviruses: medium-sized, enveloped, (-) sense that vary in shape from spherical to helical. Their genome is segmented into eight pieces

8. Influenza viral genome • (-) ssRNA • 8 segments (pieces) • One gene per segment • nucleoprotein • matrix proteins • NS (nonstructural proteins, that are not incorporated into viral particles) gene encodes two different non-structural proteins • subunits of RNA polymerase • spikes (about 500) Flu viruses are named by the type of surface proteins • Hemagglutinin - trimer (HA) • Helps virus enter cell • Type A infects humans, birds and pigs • Type A has ~ 20 different sub types • Neuraminidase - tetramer (NA) • Helps virus exit cell • 9 subtypes Influenza Virus Structure • Flu Viruses Currently infecting... • Humans: H1N1, H1N2, and H3N2 • Avian Flu Virus: H5N1

9. ORTHOMYXOVIRUSES HA - hemagglutinin NA - neuraminidase helical nucleocapsid (RNA plus NP protein) lipid bilayer membrane polymerase complex M1 protein type A, B, C : NP, M1 protein sub-types: HA or NA protein

12. Influenzavirus B • Virions enveloped • About 500 spikes • Nucleocapsid enclosed within lipoprotein membrane • Virions contain 8 segments of linear negative-sense single stranded RNA • Total genome length is 13588 nt • The largest segment 2341 nt • Infect much man and birds. • Cause human disease but generally not as severe as A types. • Believed to be epidemiologically important - reassortment with type A leads to epidemics.

13. Influenzavirus C • Virions enveloped • Many spikes • Nucleocapsid enclosed within lipoprotein membrane • Virions contain 7 segments of linear negative-sense single stranded RNA • Total genome length is 12900 nt • Glycoprotein • -hemagglutinin esterase fusion (HEF) • esterase -> receptor destroyingenzyme

14. Antigen Influenza viruses are divided into 3 groups determined by the ribonucleoprotein (RNP) antigen and M antigen • Soluble antigens: include ribonucleoproteinandM protein which are much stable in antigenicity. • Surface antigens: include HAandNA which are much variable in antigenicity.

15. Features of viral genera severity of illness animal reservoir human pandemics human epidemics antigenic changes segmented genome amantadine, rimantidine zanamivir surface glycoproteins TYPE A ++++ yes yes yes shift, drift yes sensitive sensitive 2 TYPE B ++ no no yes drift yes no effect sensitive 2 TYPE C + no no no (sporadic) drift yes no effect (1)

16. Animal Susceptibility and Growth of Virus Human strains of the virus can infect different animals; ferrets are most susceptible. Serial passage in mice increases its virulence, producing extensive pulmonary consolidation and death The developing chick embryo readily supports the growth of virus, but there are no gross lesions.

17. Influenza virus reproduction Insert figure 25.1 Influenza cycle

18. Single-cell reproductive cycle 1. Attachment to the epithalial cells of the host through hemagglutinin. 2. Endocytosis 3. Uncoating - > This exposes the contents of the virus to the cytosol. 4.The RNA enter the nucleus of the cell where fresh copies are made. 5. These copies return to the cytosol where some serve as mRNA molecules to be translated into the proteins of fresh virus particles. 6. Progeny virions are formed and released by buddingfrom the plasma membrane of the cell (aided by the neuraminidase) thus spreading the infection to new cells.

19. AAA Need to make mRNA MINUS (NEGATIVE) SENSE RNA GENOMES proteins (+ve) sense mRNA (-ve) sense genomic RNA

20. AAA Need to make mRNA MINUS (NEGATIVE) SENSE RNA GENOMES RNA polymerase must be packaged in virion. proteins (+ve) sense mRNA If used, RNA modifying enzymes are packaged in virion. (-ve) sense genomic RNA

22. Influenza Type A Viruses: antigenic Shift 1889-1977

23. 1918 Influenza epidemic > 20 million died of the flu during WW I A new influenza vaccine must be developed yearly

24. DIRECT Mechanisms of Influenza Virus Antigenic “Shift” 15 HAs 9 NAs Non-human virus Human virus Reassortant virus

25. Antigenic changes of Influenza A • Viruses can undergo frequent changes due to recombination, reassortment, insertions and point mutations • Antigenic drift • Antigenic shift occurs every 8-10 yrs • Minor antigenic changes favor persistence of the viruses in the population and allow recombination that can eventually lead to severe epidemics and/or pandemics

26. ANTIGENIC DRIFT • Gradual accumulation of mutations that allow the hemagglutinin to escape neutralizing antibodies • Epidemic strains thought to have changes in three or more antigenic sites GRADUAL ANTIGENIC CHANGE WITHOUT A CHANGE IN SUBTYPE H3N2 1975 VICTORIA H3N2 1993 BEJING H3N2 1968 HONG KONG H3N2 2004 FUJIAN

27. Antigenic drift • Antigenic differences can result from changes in one amino acid • Can involve any antigenic protein • Can occurs every year • RNA replication is error prone • New HA types are created frequently • Requires new vaccine every “season”

28. Antigenic shift • Occurs every 8-10 yrs • Major antigenic change of either H or N antigens or both H and N • Occurs by gene reassortment after simultaneous infection of a cell with two different viruses • Three different H proteins and 2 major N proteins have evolved HA HA

29. What is an Epidemic? • The occurrence of more cases of disease than expected in a given area or among a specific group of people over a particular period of time*. Epidemic What is a Pandemic? Pandemic • An epidemic occurring over a very wide area (several countries or continents) and usually affecting a large proportion of the population. • Examples: • Cholera • AIDS • Pandemic Influenza

30. Where does influenza come from? Type A constantly circulates in natural reservoirs • Birds are the natural reservoir of all subtypes of Influenza A viruses • Migratory waterfowl • Chickens, turkeys, ducks, geese • Humans • Pigs • Horses • Other

31. Why do we not have influenza B pandemics? • so far no shifts have been recorded • no animal reservoir known

32. Epidemiology • Source of infection: patients and carriers. • AEROSOL • 100,000 TO 1,000,000 VIRIONS PER DROPLET • Common: large droplets (sneezing, coughing, contact with saliva) • Probably common: contact • Direct • Fomite • Rare: airborne over long distance • 18-72 HR INCUBATION

33. SYMPTOMS • FEVER • HEADACHE • MYALGIA • COUGH • RHINITIS • OCULAR SYMPTOMS • CHILLS and/or SWEATS Infection may be very mild, even asymptomatic, moderate or very severe

34. Clinical Responses • Acute Symptoms last one week • Abrupt onset of fever, myalgia, headache and non-productive cough • Fatigue and weakness can last 2-3 weeks. • Infected individual predisposed to bacterial infections – Staphylococcus, Streptococcus, Hempohilus • Other complications - Reyes Syndrome • Immunity dependent upon localized anti-viral secretory IgA ( strain specific) • Develop long lasting circulating anti-viral IgG

35. NORMAL TRACHEAL MUCOSA 3 DAYS POST-INFECTION 7 DAYS POST-INFECTION

36. Immunity to influenza • Antibody to HA ->protective • Antibody to NA -> decreasesevereity • Serum antibody - > years • Secretory antibody -> months

37. Laboratory Diagnosis VIROLOGICAL Respiratory secretions (direct aspirate , gargle , nasal washings) • Virus isolation and growth in embryonated eggs • Cell culture in primary monkey kidney or madindarby canine kidney cells • Hemagglutination (inhibition) • Hemadsorption (inhibition) • IFA/ ELISA • Direct immunofluorescence

38. Laboratory Diagnosis Serodiagnosis Four-fold or greater increase in hemagglutination inhibition antibody titers between acute and convalescent specimens • Hemagglutination inhibition • Hemadsorption inhibition • ELISA • Complement fixation test • NT

39. Prophylaxis Masks and Hand Washing • To be Continued… • Hand washing • Generally perceived to be useful • No studies specifically performed for influenza • Easy to recommend • Masks • Effectiveness not shown for influenza • However, could reduce transmission associated with large droplets

40. Types of Vaccine • Killed Whole Virusinactivated virus vaccine grown in embryonated eggs; 70-90% effective in healthy persons <65 years of age, 30-70% in persons ≥65 years • Live VirusAttenuated strains were widely used in Russia but not elsewhere. • Virus SubunitHA extracted from recombinant virus forms the basis of today's vaccines. • SyntheticMuch research is being done to try and find a neutralising epitope that is more stable, and can therefore be used for a universal vaccine.

41. Trivalent Influenza virus vaccines • 1999-2000 • A/Sydney/05/97 (H3N2) • A/Beijing/262/95 (H1N1) • B/Yamanashi/166/98 • 2000-2001 • A/Moscow/10/99(H3N2)-like • A/New Caledonia/20/99(H1N1)-like • B/Beijing/184/93-like • To day • A/Brisben/59/2007 (H1N1) • A/Brisben/10/2007 (H3N2) • B/Florida/4/2006

42. Prevention and Treatment 70-90% effective in preventing illness • RIMANTADINE(blocks the M2 ion channel)(M2) • type A only, needs to be given early • AMANTADINE(blocks the M2 ion channel)(M2) • type A only, needs to be given early • ZANAMIVIR(neuraminidase inhibitors) (NA) • types A and B, needs to be given early • OSELTAMIVIR(neuraminidase inhibitors) (NA) • types A and B, needs to be given early

43. Antivirals: Adamantanes and Neuraminidase Inhibitors NA inhibitors HA blockers

44. Avian Influenza Poultry Outbreaks, Asia, 2003-06 Hong Kong 1997: 18 cases of influenza in humans caused by a highly pathogenic avian influenza virus (H5N1); 30% fatality rate • Has spread from E Asia, SE Asia and Pacific to Eurasia, Near East, Europe and to Africa • H5N1 has been identified in migratory water birds and /or poultry in 55 countries as of 5/29/06 • H9N2 subtype also detected among infected poultry • It has infected humans in 10 countries. In the future: reassortment between H9N2 or H5N1 avian viruses and H1N1 or H3N2 human viruses???

45. Avian Flu • Avian influenza, or “bird flu”, is a contagious disease of animals caused by viruses that normally infect only birds and, less commonly, pigs. Avian influenza viruses are highly species-specific, but have, on rare occasions, crossed the species barrier to infect humans. • Pandemic viruses appear as the result of antigenic shift, which causes new combinations of proteins on the surface of the virus. If the new virus spreads easily from person to person a pandemic can result.

46. Emergence of New Influenza Subtypes: H5N1 Antigenic shiftdue to genome reassortment within intermediate hosts drives flu epidemics and pandemics Solid lines: transmission demonstrated; Dotted lines: transmission postulated but not demonstrated. • Nonpathogenic H5 influenza virus: Wild fowl  domestic ducks and geese,  domestic chickens. • H5 virus became highly pathogenic in chickens  domestic ducks and geese. • Highly Pathogenic H5 virus reassorted its genome with those of other influenza viruses in aquatic birds,  spread to poultry farms, humans, and occasionally to pigs.

47. Avian influenza • Wild birds are the reservoir. • Circulation of low pathogenic avian flu in domestic poultry leads to mutations to highly pathogenic forms over time. • Co-infection with swine or humans infected with human influenza can result in genetic reassortment and highly pathogenic strains.

48. Avian influenza

49. Why do new strains of influenza and bird flu arise in Asia? In 2003, an outbreak of “chicken flu” necessitated killing tens of millions of birds

50. H5N1 in wild birds, poultry & humans – 5/19/06