Influenza

1. Influenza • Chapter 39

2. Seasonal Influenza • Causative Agents • Influenza A virus • Family Orthomyxoviridae • Negative-stranded RNA genome • Genome divided into 8 gene segments • Spiked envelope • H spike – hemagglutinin (subtypes H1-H16) • Mediates attachment • N spikes – neuraminidase (subtypes N1-N9) • Cleaves H protein to allow fusion of viral and cellular membranes (i.e., entry into the cell) • Requires cellular enzyme trypsin to facilitate infection • Influenza B & C viruses only circulate in humans

3. Influenza Genes • Influenza A viruses have 8 gene segments that encode 10 polypeptides • Segment 1 (2,341 nt): PB2 (RNA transcriptase) • Segment 2 (2,341 nt): PB1 (RNA transcriptase) • Segment 3 (2,233 nt): PA (RNA transcriptase) • Segment 4 (1,778 nt): HA (hemagglutinin) - 16 known subtypes • Segment 5 (1,565 nt): NP (nucleoprotein) • Segment 6 (1,413 nt): NA (neuraminidase) - 9 known subtypes • Segment 7 (1,027 nt): M1, M2 (matrix proteins) • Segment 8 (890 nt): NS1, NS2 (inhibits mRNA splicing and IFN response; nuclear export signal for viral RNPs)

4. The HA and NA Polypeptides • HA encodes hemagglutinin spike protein • Binds to any cellular protein with a α2,6-sialic acid group • Mediates penetration of viral core and RNA into target cell • It is the major target of neutralizing antibodies for vaccines and successful immune responses • NA encodes neuraminidase protein • This enzyme cleaves the HA protein upon cell binding and facilitates penetration of the viral core • Together they mediate cell infection • Influenza viruses, in part, are named by their HA and NA subtypes (e.g., H5N1)

5. Seasonal Influenza • Symptoms • Influenza Type A • Short incubation period • Averaging 2 days • Headache • Fever • Muscle pain • Dry cough • Acute symptoms abate within a week • Cough, fatigue and generalized weakness may linger

6. Adaptation/ reassortment with swine influenza viruses Transmission to humans Circulates with limited pathology Transmission to domestic fowl Influenza A Transmission Cycle

7. Seasonal Influenza • Pathogenesis • Acquired through inhalation of infected respiratory secretions • Virus attaches to host cells via hemagglutinin spikes • Once attached viral envelope fuses with host membrane, leading to viral replication within the cell • Mature viruses bud from host cell • Budding allows mature virus to pick up envelope • Infected cells die and slough off • Host immunity quickly controls viral spread • Anti-HA neutralizing IgG is protective • Mortality rate is low • However, hundreds of thousands or millions of people are infected each year in the U. S. • On average, about 30,000 Americans, mostly elderly and very young children, die from influenza each year

8. Seasonal Influenza • Epidemiology • Outbreaks occur in United States every year • Vaccines are formulated months in advance using prominent circulating strains • 2012-13 vaccine strains • A/California/7/2009 (H1N1)-like virus (same strain as 2011-2012 flu season) • A/Victoria/361/2011 (H3N2)-like virus • B/Wisconsin/1/2010-like virus • Pandemics occur periodically • Most infamous pandemic of 1918 (Spanish flu) • Spanned the globe in 9 months • Pandemics have higher than normal morbidity Type / Geographic origin / Strain/ Year isolated (H & N genes)

9. Seasonal Influenza • Epidemiology • Spread caused by major antigenic changes • Antigenic drift • Consists of amino acid changes in spikes (point mutations) • Particularly hemagglutinin • Changes minimize effectiveness of immunity to previous strains • Ensures enough susceptible people are available for continued virus survival • Antigenic shiftRepresent more dramatic changesVirus strains are drastically antigenically different from previous strains, importantly hemagglutininNew virus comes from genetic re-assortmentOccurs when two different viruses infect a cell at the same timeGenetic mixing results in new virus that is often more virulent

10. Seasonal Influenza

11. Seasonal Influenza • Prevention and Treatment • Vaccine can be 80% to 90% effective • New vaccine required every year • Due to antigenic drift • Antiviral medications are 70% to 90% effective • Include amantadine, rimantadine, and Tamiflu • Must be taken early • Not a substitute for vaccine

12. Avian Influenza • There are hundreds, if not thousands, of influenza A viruses circulating in nature • Seasonal influenza occurs from mammalian viruses • Pigs in SE Asia are frequently a source of these viruses • New reassortants arise every year, but most are not pathogenic to humans • HA attaches to α2,6-sialic acid receptor • Avian influenza viruses routinely circulate among wild birds • Some species can be infected without conspicuous pathology • These species often carry the viruses along migratory routes, exposing other birds • HA attaches to α2,3-sialic acid receptor

13. Avian Influenza • Most avian influenza viruses are highly inefficient at infecting humans • However, some cultures have domestic birds and pigs in close periodomestic proximity • This practice increases the chance of • Reassortment with mammalian influenza viruses • 1957, 1967 pandemic strains were reassortant mammalian viruses with avian segments (antigenic shift) • Emergence of mutant avian strains that can infect humans • 1918 pandemic strain was an avian virus that adapted to efficient human to human transmission (antigenic drift)

14. Recovery of the Spanish Flu Virus Using Reverse Genetics • Spanish Flu pandemic: 1918-1919 • Estimated 20 to 50 million dead • High death rate among young, health people • Played a role in ending World War 1 • No viable virus was saved

15. Spanish Influenza • Rescue of the 1918 pandemic strain • Virology did not exist in 1918 • The virus could not be isolated, thus went extinct when the pandemic ended • In 2005 a group resurrected the 1918 strain from bodies buried in Alaskan permafrost • Viral genome sequencing indicated it was an avian influenza A virus

16. Molecular Recovery of Gene Segments • Collect infected tissues from victims of the virus buried in permafrost or formalin-fixed tissues • Use reverse transcription-PCR to amplify gene segments • Clone into plasmids • Transfect MDCK (canine kidney cell line) • Recover infectious viruses

17. Characteristics of the Spanish Flu Virus • It is an avian influenza virus (i.e., directly jumped from birds to humans) • In infects cells in culture without trypsin (an indicator of pathogenicity in mammals) • It was an H1N1 virus • Kills chicken eggs in 4.5 days • Pathology was largely confined to the lungs of experimentally-infected mice (similar to humans) • Mice died in as little as 3 days post infection • Substantial damage occurred in the lungs (similar to humans) • Very high titers of virus were obtained from the lungs

18. Tumpey et al., 310:77. 2005 Avian Influenza Lungs from Mice Infected with Rescued 1918 H1N1 Pandemic Virus 1918 Strain 1918 Strain 1918 Strain(HN)/Texas 1991 Strain hybrid 1918 Strain 1918 Strain with Texas 1991 H segment Texas 1991 strain (control)

19. Is Another Avian Influenza Pandemic Upon Us? • The current avian influenza (H5N1) virus that has been circulating since 1997 has reached Europe and Africa • All human cases thus far are confined to SE Asian countries • Sporadic person to person transmission has occurred • Will this virus become efficient at person to person transmission?

20. Avian Influenza 1 Seasonal influenza 2 Currently circulating H5N1 avian influenza virus 3 Rescued 1918 pandemic H1N1 avian influenza virus 4 Cytopathic effect (damage directly caused by the virus) Red - more pathogenic feature Yellow - less pathogenic feature

21. Will A Vaccine Be Easy to Produce? • For seasonal influenza, viruses are grown in chicken eggs for 7 days to produce high-enough titers for vaccines • Avian influenza viruses kills eggs in 4.5 days • An insufficient titer is generated for vaccines • The world’s annual vaccine capacity is 300 million doses • If the vaccine can be produced by cell culture, then adequate supplies should be able to be produced • A reverse genetics vaccine may be the only way to prevent an avian influenza pandemic • A DNA vaccine has recently been described that protects mice from lethal challenge to the 1918 strain • DNA vaccines are plasmids that express viral antigens under the control of a mammalian promoter • Although many have been shown to be effective, none have been approved for use with the general public

22. Will A Vaccine Be Easy to Produce? • For seasonal influenza, viruses are grown in chicken eggs for 7 days to produce high-enough titers for vaccines • Avian influenza viruses kills eggs in 4.5 days • An insufficient titer is generated for vaccines • The world’s annual vaccine capacity is 300 million doses • If the vaccine can be produced by cell culture, then adequate supplies should be able to be produced • A reverse genetics vaccine may be the only way to prevent an avian influenza pandemic • A DNA vaccine has recently been described that protects mice from lethal challenge to the 1918 strain • DNA vaccines are plasmids that express viral antigens under the control of a mammalian promoter • Although many have been shown to be effective, none have been approved for use with the general public