1. The Threat of Influenza
Damara Gebauer
Sai Jahann
Bonnie Hart

2. Influenza
Overview
Molecular Biology
Clinical
Bioweaponization

3. 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.

4. 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

5. 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

6. Influenza Strains Single negative-stranded RNA virus 3 Types: A, B, C

7. Influenza Strains

8. Type C: Least Virulent
Type C- causes mild respiratory illness, generally not concerned with this type.
Vaccines are designed to protect against Type A and B, not type C.

9. Type B Infects humans, but has recently been seen in pigs No subtypes
Used in making vaccines

10. Type A
influenza%20virus.jpg

11. 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 H1-H3 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

12. Beware of Type A
New strains can form by genetic re-assortment between animal and human strains (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.

13. 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, re-assortment 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 re-assortment can occur directly in humans

14. 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.

15. 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

16. Three Pandemics in 20th Century
1918 Spanish Influenza– A(H1N1)
1957Asian Influenza—A(H2N2)
1968 Hong Kong Influenza—A(H3N2)

17. Spanish 1918 Pandemic Influenza A H1N1

18. 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.

19. Spanish Influenza 1918
By the fall, hospitals were overwhelmed with patients, many of whom were dying 2-3 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.

20. 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.
Under 65 accounted for 99% of deaths
20-40 were affected most severely and accounted for 50% of all deaths

21. 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

22. 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.

23. 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
WHO Global Influenza Surveillance Network

24. 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.
Since mid-December 2003, a growing number of Asian countries have reported outbreaks of highly pathogenic avian influenza in chickens and ducks. Infections in several species of wild birds and in pigs have also been reported.

25. 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.

26. 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?

27. Characteristics of viruses
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

28. Structure of Influenza
Viral envelope
Surface studded with spikes
Matrix protein (M1)
8 RNA segments
Non-structural proteins
Nuclear export protein (NEP)
NS1 protein
Nucleocapsid protein (NP)
Polymerase components

29. 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

30. Genome segments of influenza A

31. Genome segments – encoding
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

32. 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

33. 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

34. 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

35. 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!

36. Infection cycle of influenza
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

37. Infection cycle: Binding and endocytosis
HA contains receptor binding site for virus
Binds to sialic acid residue on cell surface glycoprotein
Binding triggers receptor-mediated endocytosis
Virus is taken into endosome
Low pH of endosome causes fusion of viral and endosome membranes

38. Infection cycle: Endocytosis and membrane fusion
Fusion steps:
HA binds to sialic acid residue on cell surface glycoprotein
Virus is taken into cell into endosome
HA is cleaved by cellular proteases into HA1 and HA2
Low pH of endosome causes conformational change in HA2
HA2 causes fusion of virus and cell membranes
This allows entry of viral gene segments into cell for viral replication.

39. 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.

40. 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)

41. 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

42. 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

43. 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

44. 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

45. 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

46. 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

47. 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

48. Flu-Clinical

49. Influenza Virus Types Type A Type B
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

50. Common Flu Symptoms High Fever Headache Extreme-tiredness/weakness
Dry cough
Sore throat
Stuffy/runny nose
Muscle aches
Diarrhea and vomiting

51. 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)

52. Increased Risk Age 65 and older
Any age with chronic medical conditions
Pregnant women
Children 6-23 months

53. Emergency Warning Signs- In children
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

54. Emergency Warning Signs- In adults
High or prolonged fever
Difficulty breathing or shortness of breath
Pain or pressure in chest
Near-fainting or fainting
Confusion
Severe or persistent vomiting

55. Peak Months for Flu Activity (over the past 21 years)

56. How the Flu Spreads

57. 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

58. 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

59. Complications Bacterial pneumonia Dehydration
Worsening chronic conditions
congestive heart failure
asthma
diabetes
Children can develop sinus problems and ear infections

60. 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

61. Influenza and Complications Among Nursing Home Residents

62. Hospitalization from Influenza
Highest rate among young children and persons >65 yrs
through influenza epidemic: 16, ,000 hosp./epidemic
114,000 hospitalizations/yr with 57% occurring in ages < 65 yrs
Highest # caused by type A (H3N2) viruses

63. Deaths
Result from pneumonia and/or worsening of cardiopulmonary conditions and other chronic diseases
5th leading cause of death for adults > 65
epidemic  19,000 deaths
epidemic  36,000 deaths
In 23 epidemics (‘72-’95): 20,000 excess deaths in 11 epidemics and 40,000 deaths in 6 epidemics

64. Death rates from influenza-associated pulmonary and circulation deaths/100,000 persons
0-44 yr:
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 compared w/57% of seasons

65. Laboratory Diagnosis
Can determine circulating types, subtypes, and strains
Tests include
viral culture
serology
rapid antigen testing
PCR
immunofluorescence

66. 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

67. Preventing the Flu Good Health Habits Vaccination
Antiviral Medications

68. 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

69. Vaccination

70. 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 )

71. Who Should Not Get Vaccine
Have severe allergy to hen’s eggs (anaphylactic allergic rxn)
People who previously developed Guillian-Barre syndrome (GBS) w/in 6 weeks after getting a flu shot

72. 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

73. 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

74. 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

75. Efficacy & Effectiveness of LAIV-children
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

76. Efficacy & Effectiveness of LAIV-adults
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

77. LIAV Side Effects Children Adults runny nose headache vomiting
muscle aches
fever
Adults
runny nose
headache
sore throat
cough
fever

78. 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

79. 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

80. Effectiveness of Inactivated Vaccine- Children
77% - 91% effective against influenza respiratory illness
Effective against influenza seroconversion:
age 1-5  %
age  74-76%
age  %
Another study: > 89% overall efficiency for 6-24 months old

81. 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

82. 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

83. 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

84. Antiviral 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

85. 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

86. 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

87. 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

88. Oseltamivir Side Effects
Gastrointestinal side effects
nausea
vomiting
Less severe if taken with food

89. 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.

90. Is a pandemic imminent? Experts (WHO, CDC) are saying yes.
It will occur as a natural disaster or as a bio-terrorist attack.
Either way we are not ready to respond to a flu pandemic

91. Influenza as a Bioweapon?
Suicide bio-bomber:a terrorist infects himself with super flu (e.g. avian and human influenza cross), spreads infection.
Now possible to make infectious flu virus from cloned DNA of the 8 RNA segments. This could bring back the 1918 Spanish Influenza.
Genetically modify avian flu strain so that can recognize human hosts’ receptors. This could be deadly.
http mirabye.com/the/19/numbers

92. 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.

93. 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.

94. Bioweapon Pros
Greater threat to world leaders because they are generally older and more susceptible to the virus.

95. 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.

96. 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.

97. 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