2. • 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
Seasonal Influenza
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. • 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
Seasonal Influenza
6. Influenza A Transmission Cycle
Circulates with
limited pathology
Transmission to
domestic fowl
Adaptation/
reassortment
with swine
influenza viruses
Transmission
to humans
7. • 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
Seasonal Influenza
8. • 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)
Seasonal Influenza
9. • 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
Seasonal Influenza
11. • 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
Seasonal Influenza
12. • 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 re-assortants arise every year, but most are not
pathogenic to humans
• HA attaches to 2,6-sialic acid receptorα
• 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 2,3-sialic acid receptorα
Avian Influenza
13. • 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
• Re-assortment 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)
Avian Influenza
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 endingWorld War 1
• No viable virus was saved
15. • 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
Spanish Influenza
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. Lungs from Mice Infected with Rescued 1918 H1N1 Pandemic Virus
1918 Strain 1918 Strain
1918 Strain
1918
Strain(HN)/Texas
1991 Strain hybrid
1918 Strain with
Texas 1991 H
segment
Texas 1991 strain
(control)
Tumpey et al.,
310:77. 2005
Avian Influenza
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. Feature SI1
H5N1 AI2
1918 H1N13
Transmission efficiency High Very low/none High
Replication site
Upper and lower
respiratory tract
Lower RT only
Likely upper &
lower RT
Viral CPE4
Limited Substantial Substantial
Immunopathology Limited Substantial Substantial
Kills embyronated chicken eggs? No Yes Yes
Requires trypsin for infection of cell
cultures?
Yes No No
Vaccine Yes No N/A
Fatality rate 0.03% (U.S.) 57% (global) About 1-2% (U.S)
Demographic
Young children,
elderly
Young adults Young adults
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
Avian Influenza
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
