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The Threat of Influenza
Damara Gebauer
Sai Jahann
Bonnie Hart
Influenza
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•
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Overview
Molecular Biology
Clinical
Bioweaponization
Influenza Overview
• Commonly called “the flu”. It is a highly
contagious disease caused by the influenza
virus.
• It is a disease of the respiratory system,
namely the throat, nose and lungs.
• Can affect people of all ages including
healthy people and symptoms are seen
suddenly.
Influenza Overview
• People most susceptible are the elderly,
small children and immuno-compromised,
although anyone can develop complications.
• Complications include pneumonia,
bronchitis, nose and ear infection.
• “Stomach Flu” Myth
• No aspirin for children or teenagers
Influenza Overview
• 10-20% of the US population come down with the
flu each year.
• ~36,000 Americans succumb to complications of
the disease. 250,000 people die world wide.
• Vaccines are the first line of defense.
• Antiviral medication is also available
• Viral receptor proteins are primary targets of
vaccines and antivirals
• Hemagglutinin (HA) and Neuraminidase (NA) are
targeted viral proteins
Influenza Strains
• Single negative-stranded RNA virus
• 3 Types: A, B, C
http://web.uct.ac.za/depts/mmi/stannard/fluvirus.html
Influenza Strains
Type Natural Reservoir Animals affected
Disease
Epidemic in Humans
A
waterfowl
humans, pigs, chickens flu and flu complications
yes
B
unknown
mostly humans only
flu and flu complications
yes
C
unknown
humans
mild respiratory illness
no
Type A
www.omedon.co.uk/.../beans/ influenza%20virus.jpg
Beware of Type A
• 15 known HA subtypes: H1-H15
• 9 known NA subtypes: N1-N9
• While all subtypes can be found in birds, only H1H3 and N1-N2 are known to circulate widely in
humans.
• H1-H3 are the only types known to have caused
pandemics in humans
– Pandemics arise from flu strains that have novel HA
and NA proteins that people have no immunity to
Beware of Type A
• New strains can form by genetic re-assortment
between animal and human strains. 1956 (Asian)
and 1968 (Hong Kong) were formed this way.
• Recently been discovered that wholly avian strains
CAN directly infect humans
• Epidemic occurred in 1997 in Hong Kong. 18
people were infected and 6 died from
complications.
Beware of Type A
• Previously it was thought that humans could not
be infected by wholly avian flu and that an
intermediary step was required
• Pigs were thought to be this intermediary step.
• Avian or some other animal flu infected pigs, reassortment occurs creating new strain which then
has potential to infect humans.
• 1997 Hong Kong incident showed pigs are not
required to be intermediary step since reassortment can occur directly in humans
Avian flu: Why such a threat?
• HA and NA surface proteins are generally
not recognized by human respiratory cells
• Wholly avian flu infects humans at a low
frequency but has huge pandemic potential.
• 1997 Hong Kong incident was first case of
wholly avian flu infecting humans.
Fortunately, flu could not spread from
person to person.
Avian flu: Why such a threat?
• If avian strain were to spread from person to
person, most of the population will have no prior
immunity to the HA and NA proteins and a
devastating pandemic could occur.
• Also, if avian strain and common human strain
infect host simultaneously, could get re-assortment
and creation of a super influenza strain.
• WHO and other organizations are watching Asia
and other countries with avian flu outbreaks very
closely
Three Pandemics in 20th Century
• 1918 Spanish Influenza– A(H1N1)
• 1957Asian Influenza—A(H2N2)
• 1968 Hong Kong Influenza—A(H3N2)
Spanish 1918 Pandemic
Influenza A H1N1
http://www.stanford.edu/group/virus/uda/
Spanish Influenza 1918
• The most devastating flu pandemic the world had
seen.
• Named Spanish influenza because of the severe
loss in Spain. 8 million people died in May 1918.
• In the U.S, first signs were seen in early spring in
military camps in Kansas, but received little
attention because of the war in Europe.
Spanish Influenza 1918
• By the fall, hospitals were overwhelmed
with patients, many of whom were dying 23 days after exhibiting symptoms.
• The pandemic was extremely sudden. No
one was prepared. In the US, the average
life span was reduced by 10 years.
• ~675,000 American deaths.
Spanish Influenza 1918
• 40 million people worldwide were dead from the
flu.
• Most striking was the high morbidity of young
people (20-40 years old).
• Influenza's full impact: millions of
hospitalizations, secondary bacterial pneumonias,
and middle ear infections in infants and young
children.
• Caused by H1N1 strain that resembled most
closely swine origin.
1957 Asian Influenza—A(H2N2)
• Re-assortment of avian and human strains.
• Re-assortment thought to have occurred in
pigs.
• 70,000 deaths in America. First identified in
China in February 1957, it spread to the US
by June 1957
1968 Hong Kong Influenza—A(H3N2)
• Also re-assortment of avian and human
strains.
• 34,000 deaths in America. Started in Hong
Kong in early 1968 and spread to America
by the end of the year.
• A(H3N2) is still circulating in human
population today.
Influenza Today
• Kills an average of 36,000 Americans
every year and 250,000 around the
world.
• ~115,000 Americans are hospitalized for
the flu each year
http://www.nlm.nih.gov/medlineplus/news/fullstory_16132.html
Recent News: Revenge of the Birds
• 1997 Hong Kong A(H5N1)- First reported case of
direct transmission from bird to human.
• 1999 Hong Kong A(H9N2)- 2 children infected
with avian flu and transmission was believed to be
direct bird to human. Both children recuperated.
• 2003 Hong Kong A(H5N1)- a father and son
traveling to mainland came down with flu, father
did not survive. Source of infection remains
unknown.
Recent News
• 2003 Netherlands A(H7N7)- Outbreak of avian flu
in farmed poultry. 80 poultry workers and their
families became ill. There seemed to be some
human to human transmission. One patient died.
• 2003 Hong Kong A(H9N2)- One child became ill
with avian flu but recovered.
• Present day- Several Asian countries including
China, Thailand, Vietnam, Indonesia and others
are having outbreaks of avian flu among farmed
poultry.
Influenza: Molecular Biology
• What differentiates
influenza from other
viruses?
• How does an influenza
virus particle interact
with a host cell?
• Why is it so contagious?
So dangerous?
Characteristics of viruses
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Genome enclosed in protein shell
Can only reproduce within host cell
Each type of virus has specific “host range”
Reprogramming of cell
– Copy viral genes
– Manufacture viral proteins
Structure of Influenza
• Viral envelope
– Surface studded with spikes
• Matrix protein (M1)
• 8 RNA segments
• Non-structural proteins
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–
–
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Nuclear export protein (NEP)
NS1 protein
Nucleocapsid protein (NP)
Polymerase components
Surface proteins of influenza A/B
• Hemagglutinin (HA)
– Rod-shaped
– Binds virus to host cell to
initiate infection
– Brings about fusion
– 15 types
• Neuraminidase (NA)
– Mushroom-shaped
– Prevents viral aggregation
upon release
– 9 types
Genome segments of influenza A
Genome segments – encoding
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•
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•
•
4: HA
6: NA
7: M1/M2 Matrix proteins
5: Nucleoprotein (NP)
1,2,3: Polymerase machinery (PB-2, PB-1,
PA)
• 8: Non-structural proteins: NS1, NEP
Genetic variation of influenza A
• Causes introduction of new, pandemic strains
• Mutational frequency comparable to other
viruses
– Can’t be the only explanation
• Unique ability to undergo antigenic variation
– Antigen: interacts with cell and antibody
– Antigenic drift: minor changes
– Antigenic shift: major differences; new strains
Genetic Variation: Antigenic Drift
• Accumulation of point mutations eventually
result in amino acid substitutions
• HA glycoprotein:
– Results in differences in key antigenic sites at
which the host antibody binds
– Prevent binding of antibodies induced by
previous infection
• Also occurs in NA glycoprotein
Genetic Variation: Antigenic Shift
• Involves replacement of entire gene
segments
– Results in novel viruses
• Occurs suddenly in association with
pandemics
• Through dual infection: different influenza
viruses infect a single cell
• Does not occur in NA glycoprotein
Genetic Variation: Antigenic Shift
• One cell is infected by two
different influenza A viruses
– Not necessarily human flu
• Inside the cell, spontaneous
self-assembly can produce
recombinant viruses
• Those viruses bud out of cell
and infect other host cells
• A new flu strain is born!
Infection cycle of influenza
1.
2.
3.
4.
5.
6.
7.
Binding of virus to cell
Cell engulfs virus via
endocytosis
Membrane of virus fuses with
endosome; RNA released into
cell
Viral polymerase produces
mRNA from viral RNA
Protein, new RNA produced
Self-assembly produces virions
Virions bud off cell membrane
Infection cycle:
Binding and endocytosis
• HA contains receptor binding
site for virus
– Binds to sialic acid residue on
cell surface glycoprotein
• Binding triggers receptormediated endocytosis
– Virus is taken into endosome
– Low pH of endosome causes
fusion of viral and endosome
membranes
Infection cycle:
Endocytosis and membrane fusion
Infection cycle:
Fusion of viral and cell membranes
• Cleavage of HA is necessary
– This allows fusion of membranes
• HA cleaved at arginine in cleavage site
• More arginines = more proteases cleaving
– Mutated viruses can be cleaved by more proteases
• Virus is more infective
– More of the cells it attaches to will get exposed to viral
genome.
Infection cycle:
Viral replication
• Negative-sense viral RNA is transcribed to
mRNA by viral polymerase (PB1, PB2, PA)
– mRNA complements made for incorporation
into new virions
– mRNA translated to produce viral proteins
• For incorporation into virions
• For use in infected cell (NEP, NS1)
Infection cycle:
Viral budding
• HA and NA incorporated
into host cell membrane
• M1 matrix protein forms
shell, bound to:
– Cytoplasmic tails of HA,
NA
– Viral ribonucleoproteins
– Other M1 molecules
• Virus buds off via
exocytosis
Host cell v. influenza A virus
• Human cell has initial antiviral defenses
– Interferon-α/β-independent response
– Protein kinase R
– Interferon-α/β response
• Virus needs to counteract these to reproduce
– NS1A protein
Host cell v. influenza A virus
• Interferon-α/β-independent protection
– Upon infection by virus, activate transcription
factors that control expression of antiviral genes
• Interferon regulatory factor-3 (IRF-3), IRF-7
• Combine with coactivators to form virus-activated
factor (VAF)
• VAF induces transcription of genes that code for
antiviral proteins
Host cell v. influenza A virus
• Protein Kinase-R (PKR) protection
– Activated by presence of double-stranded RNA
• Consequence of RNA virus presence in cell
– Activated PKR inhibits protein synthesis and
therefore viral replication
• Phosphorylates translation initiation factor eIF2
Host cell v. influenza A virus
• Interferon-α/β response
– Infected cell produces IFN-α/β
• Signal travels to neighboring uninfected cells
– In infected and neighboring cells, induces
transcription of antiviral proteins
• Protects infected cell against current infection
• Prepares at-risk cells to withstand infection
– Induces production of MxA protein
• Largely responsible for inhibition of influenza A
Host cell v. influenza A virus
• Influenza NS1A protein
inhibits 3’-end processing of
cell mRNA
– Prevents addition of poly-A tail
– Doesn’t affect viral mRNA
processing
• Inhibits activation of PKR
– Mechanism not clearly
understood
• Enough IFN-β produced to
protect neighboring cells
Influenza effect on host cells
• Turns off protein function
– NS1A protein
• Causes cell death
– Induces apoptosis
– Dead cells shed off respiratory
tract lining
– Can cause shedding down to
basement membrane layer
• Infects respiratory tract;
causes clinical symptoms
Flu-Clinical
Influenza Virus Types
• Type A
– humans and other animals
– all age groups
– moderate to severe illness
• Type B
– milder epidemics
– humans only
– primarily affects children
• Type C - uncommon strain, no epidemic
Common Flu Symptoms
• High Fever
• Headache
• Extremetiredness/weakness
• Dry cough
• Sore throat
• Stuffy/runny nose
• Muscle aches
• Diarrhea and vomiting
Cold v. Flu
• Flu is worse than common cold
• Symptoms more intense in flu (fever, body
aches, tiredness, and dry cough)
• Colds- more likely to have runny or stuffy
nose
• Colds don’t result in serious health
problems (pneumonia, bacterial infections,
or hospitalizations)
Increased Risk
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Age 65 and older
Any age with chronic medical conditions
Pregnant women
Children 6-23 months
Emergency Warning Signs- In
children
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High or prolonged fever
Fast breathing or trouble breathing
Bluish skin color
Not drinking enough fluids
Changes in mental status
Flu-like symptoms improve but then return
Worsening of underlying chronic medical
conditions
Emergency Warning Signs- In
adults
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High or prolonged fever
Difficulty breathing or shortness of breath
Pain or pressure in chest
Near-fainting or fainting
Confusion
Severe or persistent vomiting
Peak Months for Flu Activity
(over the past 21 years)
40
35
30
25
20
15
10
5
0
Dec
Jan
Feb
Mar
Apr
May
How the Flu Spreads
Spread of Flu
• Droplet Spread
– from a person’s cough or sneeze
– person touches respiratory droplets on another
person or object and then touches their own
mouth or nose
• Incubation period = 1-4 days (avg. = 2 days)
• Adults infectious from day before
symptoms begin to 5 days after illness onset
• Children- infectious for > 10 days
Symptoms
• Adults- shed virus 1 day before developing
symptoms to 7 days after getting sick
• Young children- can shed virus for longer
than 7 days
Complications
• Bacterial pneumonia
• Dehydration
• Worsening chronic conditions
– congestive heart failure
– asthma
– diabetes
• Children can develop sinus problems and
ear infections
Complications-cont.
• Lead to pulmonary or cardiac disease
• Lead to 2ndary bacterial pneumonia or
primary influenza viral pneumonia
• Children
– 20% hospitalized can have febrile seizures
• Influenza is associated w/ encephalopathy,
transverse myelitis, Reye syndrome,
myositis, myocarditis, and pericarditis
Influenza and Complications
Among Nursing Home Residents
9
8
7
6
5
Vaccinated
Unvaccinated
4
3
2
1
0
Hosp
Pneu
Death
Hospitalization from Influenza
• Highest rate among young children and
persons >65 yrs
• 1969-70 through 1994-95 influenza
epidemic: 16,000-220,000 hosp./epidemic
• 114,000 hospitalizations/yr with 57%
occurring in ages < 65 yrs
• Highest # caused by type A (H3N2) viruses
Deaths
• Result from pneumonia and/or worsening of
cardiopulmonary conditions and other
chronic diseases
• 5th leading cause of death for adults > 65
• 1976-90 epidemic  19,000 deaths
• 1990-99 epidemic  36,000 deaths
• In 23 epidemics (‘72-’95): 20,000 excess
deaths in 11 epidemics and 40,000 deaths in
6 epidemics
Death rates from influenza-associated
pulmonary and circulation
deaths/100,000 persons
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•
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0-44 yr: 0.4 - 0.6
50-64yr: 7.5
 65yrs: 98.3
Reasons:
– more older people has inc.
– Influenza A associated with higher mortality
– Influenza A predominates in 90% of seasons from
1990-99 compared w/57% of seasons 1976-90
Laboratory Diagnosis
• Can determine circulating types, subtypes,
and strains
• Tests include
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viral culture
serology
rapid antigen testing
PCR
immunofluorescence
Commercial rapid diagnosis tests
• Detect viruses in 30 minutes
• Specimens used are either throat swabs,
nasal wash or nasal swab
• Sensitivity of rapid tests are lower than that
of viral cultures  confirm (-) tests with
viral culture
• Does not provide specific info on
circulating subtypes and strains
Preventing the Flu
• Good Health Habits
• Vaccination
• Antiviral Medications
Good Health Habits
• Avoid close contact
• Stay home when you
are sick
• Cover your mouth
• Clean your hand
• Avoid touching your
eyes, nose or mouth
• Get plenty of rest
• Drink plenty of liquids
• The simplest way
to avoid the flu is
to avoid crowds.
Can’t keep you
kids cooped up?
Frequent hand
washing is the
next best thing
Vaccination
Vaccination
• Best way to prevent flu
• Selection of virus for manufactured vaccine
made in Feb and April each year
• Get vaccinated each fall
• People at high risk should get vaccinated
• 2 kinds of vaccines
– inactivated
– live attenuates (LAIV) (for ages 5 - 49)
Who Should Not Get Vaccine
• Have severe allergy to hen’s eggs
(anaphylactic allergic rxn)
• People who previously developed GuillianBarre syndrome (GBS) w/in 6 weeks after
getting a flu shot
Live Attentuated Intranasal
Influenza (LAIV)
• Contains weakened live influenza vs killed
viruses
• Administered by nasal spray
• Contains 3 different live (but weakened)
viruses, which stimulate body to make
antibodies
Live Attentuated Intranasal
Influenza (LAIV)
• Attenuated, producing mild or no signs or
symptoms
• Temperature-sensitive-limits the replication
of vaccine viruses at 38-39°C  restricts
LAIV viruses from replicating efficiently in
human lower airways
• Cold-adapted, replicating efficiently at 25°C
 restrictive for replication of different
wild-type viruses
Dosage-LAIV
• 0.5 mL of vaccine: 0.25 mL for each nostril
• Children aged 5-8 previously unvaccinated:
receive 2 doses separated by 6-10 weeks
• Children aged 5-8 previously vaccinated:
receive 1 dose (do not require a 2nd dose)
• Persons aged 9-49: receive 1 dose
Efficacy & Effectiveness of LAIVchildren
• Season 1:
– 93% efficiency for those who received 2 doses
– 91% for those those who received 1 dose
• Season 2:
– 86% overall efficiency
– A (H2N2) component of vaccine was not well
matched for circulating virus strains
Efficacy & Effectiveness of LAIVadults
• 85% overall efficiency
• Vaccination reduced severe febrile illnesses
by 19% and upper respiratory tract illnesses
by 24%
• Fewer days of illness
• 15-42% fewer health care provider visits
• 43-47% less use of antibiotics
LIAV Side Effects
• Children
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–
–
–
–
runny nose
headache
vomiting
muscle aches
fever
• Adults
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–
–
–
–
runny nose
headache
sore throat
cough
fever
Inactivated Influenza Vaccine
• Contains two type A and one type B
• Made from purified, egg grown viruses that
have been inactivated or killed
• Antibiotics can be added to prevent
bacterial contamination
• Vaccinated people develop high
postvaccination hemagglutination inhibition
antibody titers
Dosage-Inactivated
• 15g/dose of H1N1 virus and H3N2 type A
virus plus a type B strain
• Children previously unvaccinated: 2 doses one
month apart (2nd dose should be administered
before December)
• Vaccinated in the deltoid muscle ( needle
length > 1 inch in order to penetrate muscle
tissue in adults;7/8-1 inch for children)
• Infants should be vaccinated in the
anterolateral aspect of the thigh
Effectiveness of Inactivated
Vaccine- Children
• 77% - 91% effective against influenza
respiratory illness
• Effective against influenza seroconversion:
– age 1-5
– age 6-10
– age 11-15
 44- 49%
 74-76%
 70- 80%
• Another study: > 89% overall efficiency for
6-24 months old
Effectiveness of Inactivated
Vaccine-Adults
• Aged < 65 yrs old:
– 70-90% efficient
–  work absenteeism,  health-care resources
• Aged > 65 yrs old:
– 50-60% effective in preventing hospitalization
for pneumonia and influenza
– 80% effective in preventing death
Side Effects to Inactivated Vaccine
• Soreness at vaccination site
• Fever, malaise, myalgia
• Guillain Barre Syndrome: 1 additional case
per 1 million people
– Body's immune system attacks part of the
nervous system and results in weakness or
tingling sensations in the legs that can spread to
the arms and upper body.
– Can result in paralysis
Inactivated v. Live Vaccines
• Similarities
– contain one influenza A
(H3N2) virus, one A
(H1N1) virus, and one
B virus
– vaccines grown in eggs
– administered annually
• Differences
– Inactivated has killed
virus, LAIV contains
attentuated viruses
– Cost: LAIV more
expensive
– who gets what vaccine
– Administration
• LAIV: intranasally
• dead: intermuscularly
Antiviral Medications
• 4 medications:
– Amantadine: orally administered, treats type A
– Rimantadine: orally administered, treats type A
only for adults
– Oseltamivir-capsule, treats type A & type B
– Zanamiv-inhaled powered drug, treats type A&
type B
• Last for 5 days and must be started w/in the
1st 2 days of illness
• Used to control outbreaks in institutions
Antiviral Medications
• Drugs are 70-90% effective for prevention
• If taken w/in 2 days of getting sick, drugs
reduce symptoms and shorten time of
sickness by 1-2 days
• Have side effects
Amantadine and Rimantadine
Side Effects
• CNS side effects: nervousness, anxiety,
difficulty concentrating, light headedness
(occur more often w/amantadine)
• Gastrointestinal side effects: nausea, loss of
appetite
• People w/ long-term illnesses: delirium,
hallucinations, agitation, and seizures
• Side effects disappear after 1 week
Zanamivir Side Effects
• Drug is inhaled and can effect those
w/asthma or other chronic lung diseases
• Decreased respiratory function,
bronchospasm
• Less than 5% reported diarrhea, nausea,
sinusitis, nasal infection, bronchitis, cough,
headache, and dizziness
Oseltamivir Side Effects
• Gastrointestinal side effects
– nausea
– vomiting
• Less severe if taken with food
Economics
• Annual direct medical cost = $4.6 billion
• Total direct and indirect costs = $12 billion
(indirect includes work days lost, school
days lost, etc.)
• Vaccination can reduce these costs from
hospitalizations, lost work days and
antibiotic use.
Is a pandemic imminent?
• Experts (WHO, CDC) are saying yes.
• It will occur as a natural disaster or as a bioterrorist attack.
• Either way we are not ready to respond to a
flu pandemic
Influenza as a Bioweapon?
1. Suicide bio-bomber:a terrorist infects himself
with super flu (e.g. avian and human influenza
cross), spreads infection.
2. Now possible to make infectious flu virus from
cloned DNA of the 8 RNA segments. This could
bring back the 1918 Spanish Influenza.
3. Genetically modify avian flu strain so that can
recognize human hosts’ receptors. This could be
deadly.
Bioweapon Pros
• Highly contagious
• Difficult to contain people (as opposed to
animals)
• Hard to determine strain for vaccine defense
• Mostly likely will not have appropriate
vaccine(s) at time of attack.
• Relatively easy to obtain.
Bioweapon Pros
• Infect farmed poultry and damage poultry industry
and the US economically.
• Flu strain need not be lethal since it’s so
contagious already. If infect enough people can
cause social and economic strain by wiping out
work force.
• First cluster of cases would probably not alert
officials. This could give pandemic head start.
• Difficult to eradicate because of bird and other
animal reservoirs.
Bioweapon Pros
• Greater threat to world leaders because they
are generally older and more susceptible to
the virus.
Bioweapon Cons
• Strain may not be lethal, people could
recuperate
• So far, wholly avian strain can not get
human to human transmission. This would
not be contagious.
• Highly unlikely that terrorists would have
expertise to conduct recombinant DNA
technology research or have the resources.
Lines of Defense
• Stockpiles of vaccines, may have to replenish to
account for strain shifts
• Increase security and monitor laboratories
conducting influenza research and manufacturers
that are distributing vaccines and antivirals.
• Healthcare workers should increase immunization
for people who need it now.
• Equip healthcare workers and possibly pharmacies
with proper flu assay kits so they can identify
disease quickly.
Therefore, Beware!
• Influenza is more than “just a cold”
• A pandemic can have drastic social,
economic, and health consequences
• Influenza is a potential and effective
bioweapon that we should be prepared to
see