Influenza Viruses
Daniel R. Perez
Virginia-Maryland Regional College of Veterinary Medicine
University of Maryland, College Park
INFLUENZA Facts
• Influenza and pneumococcal pneumonia (the most
common complication of influenza) together are the
fifth leading cause of death in the U.S. in people ≥65
years old.
• In the U.S., 20,000-40,000 die each year from flu-related
illness.
• Worsening of chronic heart and lung disease
• Annual direct medical costs of flu are estimated at up
to $4.6 billion (over $12 billion adding indirect costs)
• Devastating pandemics
Common Symptoms
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Respiratory disease
Abrupt onset of symptoms
Fever (up to 104° F)
Chills (sometimes shaking)
Muscle aches and pains
Sweating
Dry Cough
Nasal congestion
Sore throat
Headache
Malaise
Fatigue
Influenza: Who’s at risk?
• Everybody
• People with greater risk:
– ≥ 65 years old
– Patients with chronic diseases
• Asthma/Lung chronic disease
• Chronic Heart Disease
– ≤ 5 years old
Influenza: Transmission
• Incubation period: 1-4 days, average 2 days
• Transmission may start 1 or 2 days before onset of
symptoms and last for a week
• Immunocompromised patients may transmit the virus for
up to a month after onset of symptoms
• Virus particles spread through coughing and sneezing
• One infectious particle can generate up to 1,000 virus
particles
Influenza types
Type A
Type B
Type C
Potentially severe illness
Epidemics and pandemics
Rapidly changing
Usually less severe illness
Epidemics
More uniform
Usually mild or asymptomatic
illness
Minimal public health impact
Centers for Disease Control and Prevention. Influenza Prevention and Control. Influenza.
Available at: http://www.cdc.gov/ncidod/diseases/flu/fluinfo.htm.
Naming influenza viruses
Type
16
9
Hemagglutinin
Neuraminidase
A/Hong Kong/156/97 (H5N1)
Origin
Year
isolated
Strain
ID
A/Chicken/Mexico/31381-1/94 (H5N2)
Influenza type A: prototype of
emergent disease
• Responsible for annual epidemics
– Vaccine Re-formulation
– Re-vaccination
• Responsible for devastating pandemics
– 1918 “Spanish Flu”: >20 million people died
– 1957 “Asian Flu”: 100,000 people died, 70,000 in the U.S.
– 1968 “Hong Kong Flu”: 700,000 people died, 33,000 in
the U.S.
– The emergence of a new pandemic strain is considered
imminent. Can we avert it?
INFLUENZA VIRUS
• Family: Orthomyxoviridae
• Negative sense single strand RNA genome
– Genus: Influenza A, B
• Eight segments
– Genus: Influenza C
• Seven segments
– Genus: (unnamed, Thogoto-like viruses)
• Seven segments
– Genus: (unnamed, Infectious Salmon Anemia virus)
• Seven segments
Influenza A virus
Family: Orthomyxoviridae
•Segmented negative sense single strand RNA genome
2-6Gal
2-3Gal
HA
NA
PB1, PB2, PA
NP
M1
NEP
NS1, PB1-F2
Infected cells
M2
www.cdc.gov
HA side view
HA top view
SAα2,3-gal
SAα2,6-gal
Type A influenza
cannot be eradicated
2-3Gal
2-6Gal
2-3Gal
16 HA subtypes
9 NA subtypes
2-6Gal
2-3Gal
2-6Gal
Quail Overlooked as Intermediate Host
Hypothesis: quail can act as an intermediate host for the genesis of
influenza viruses that are able to cross the species barrier.
Reassortment
1918
H1N1 “Spanish flu”
>20 million deaths
1957
H2N2 “Asian Flu”
~2 million deaths
Pandemic influenza strains contain
genes from the avian reservoir
1968
H3N2 “Hong Kong Flu”
~700,000 deaths
?
1977
H1N1 “Russian Flu”
2009
H1N1 “American Flu”
Pandemic Influenza 2009 - Natural history of swine influenza
North American Swine Influenza
(~last 12 years)
Eurasian Swine Influenza
trH3N2
trH3N2
cH1N1
trH1N2
H3N2
H1N1
cH1N1
PB2 = North Am. Avian
PB1 = Human
PA = North Am. Avian
HA = Human
2009 swine-like H1N1
NP = Classical swine
“American flu”
NA = Human
M = Classical swine
NS = Classical swine
trH1N1
PB2 = H3N2 swine
PB1 = H3N2 swine
PA = H3N2 swine
HA = Classical swine
NP = Classical swine
NA = Eurasian swine
M = Eurasian swine
NS = Classical swine
Surface glycoprotein genes
HA and NA
HA
Neuraminidase Inhibitors
“Tamiflu” “Relenza”
X
Release
NA
ER - Golgi
Master donor
strain genes
PB2
AAAA
AAAA
AAAA
PB1 PA
M
NP
Packaging and Budding
NS
Transfection of plasmids
into cells
AAAA
Nucleus
AAAA
Host mRNAs
Receptor binding and Entry
Uncoating
Amantadine
“Symmetrel”
“Flumadine”
H+
X
Endocytosis
X
IFN
Influenza virus Reverse Genetics
viral protein
translation
mRNA
cap
AAAA
RNA polymerase II (pol II)
pIICMV
pABGH
ATG--------- viral cDNA -------TAA
tI
pIh
RNA polymerase I (pol I)
(-) vRNA
ppp
3‘
5‘
Influenza A viruses to order reverse genetics
Pol I transcription
PB2 PB1 PA HA
NP NA
M
NS
8 plasmids
Nucleolus
Pol II transcription
C
vRNA
N
mRNA
?
Influenza A virus
Influenza type A: prototype of
emergent disease
Antigenic “Drift” and “Shift” (surface proteins)
Drift: progressive accumulation of mutations within
one subtype
Shift: acquisition of new genes or an entire new strain
Surveillance is key to understand both phenomena
Influenza A virus in poultry
• Defined in 1878 as “fowl plague”.
• Important economic losses
• Low pathogenic influenza viruses (LPAI).
– Associated with disease outbreaks in young domestic
turkeys.
– Progenitors of HPAI viruses.
• Highly Pathogenic Influenza viruses (HPAI).
– H5 and H7 subtypes.
– Polybasic amino acid region at the HA1/HA2 cleavage site.
– 23 outbreaks since 1959, 11 outbreaks since 1990
• 5 in turkeys, 16 in chickens, 1 in terns, 1 multiple
species
• 11 H7 subtype, 12 H5 subtype
2004 Human
P E N P K QAY-Q-K-RM T R
G L
P E K P K TCSPLSRCRK T R
G L
P E K P K TCSPLSRCRE T R
G L
P E I P K -
G L
-
-R R R R
H7N3 BC
H7N3 Chile
H7N7 Human
Influenza A viruses from domestic poultry can cause
disease in humans.
1997
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–
–
–
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1999
–
–
–
2003
–
H5N1 outbreak in chickens in Hong Kong.
HPAI virus, transmitted to humans.
18 people diagnosed with H5N1 virus, 6 died.
No human to human transmission.
Slaughter of > 1,000,000 poultry prevented new cases.
Recurrent outbreaks in poultry.
February 2003, 2 people infected, 1 died.
H9N2 virus in domestic poultry in Hong Kong and
Southern China
LPAI virus, highly prevalent in quail and chickens.
Transmitted to humans, respiratory disease.
Some circulating strains have human-like receptor specificity.
H7N7 outbreak in domestic poultry in the Netherlands
89 H7N7 human infections, 1 fatal.
HA subtypes in different animal species
Subtype
Human
Swine
Horse
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
H13
H14
H15
HA subtypes in Humans: H1, H2, H3, H5, H7, H9
Bird
NA subtypes in different animal species
Subtype
Human
Swine
Horse
N1
N2
N3
N4
N5
N6
N7
N8
N9
NA subtypes in Humans: N1, N2, N7
Bird
What can we do about it?
• Surveillance and Biosecurity are the first lines of defense
against pandemic influenza
– Cooperation between Human Health and Animal Health
components are essential
• During outbreaks
– Stamping out remains the best tool available to contain outbreaks
– Antiviral prophylactic treatment of populations at risk
• Vaccine for pandemic preparedness
• Establishing the molecular basis of interspecies transmission
and pathogenesis
Influenza Prevention
• Vaccination before the start of influenza season
– Northern Hemisphere: October-November
– Southern Hemisphere: April-May
• Antiviral treatment
– Therapeutic
– Prophylactic
Viral immunity - Vaccines
• Infection: solid immunity to homologous
virus
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–
–
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Antibody to surface genes; HA and NA
CTL: peptides from internal proteins
Two circulating subtypes: H1N1 and H3N2
H3N2 more important in morbidity and mortality
• Inactivated virus vaccines
– Safe and generally efficacious
• Live attenuated vaccines (FluMist®)
– Safe and generally efficacious
Inactivated vaccines
•Reformulated
every year to provide protection against
virus strains which are prevalent and/or currently
circulating
•Prevalent
•Virus
A subtype (two) and B viruses
grown in the allantoic cavity of 10-days old chicken
embryos (reassortants derived from A/PR/8/34 and
B/Lee/1/40)
•Viruses
are inactivated with formalin and standardized for
HA content
Efficacy of inactivated vaccines
• Efficacy
– Varies with age and immunocompetence
– Depends on match between projected vs actual strains
• Children/Teens
– Stimulates high HA-inhibition antibody titers
– Prevents infection
• Elderly
– Produces lower HA-inhibition antibody titers
– May not eliminate URTI susceptibility
– May reduce LRTI morbidity/mortality
Live Attenuated Influenza Vaccine
(LAIV, Flumist®)
Cold adapted influenza viruses:
ca
A/Ann Arbor/6/60 (H2N2)
ca
B/Ann Arbor/1/66
Viruses grown at 25ºC in chicken embryos
Intranasal administration (0.5 ml allatoic fluid
diluted to contain 106.5-7.5 TCID50/ml)
Flumist®: Efficacy and limitations
•Live viruses with limited replication in the upper respiratory
tract
•Prevents (>90%) disease symptoms
•Limited use: Only approved for people 5 to 49 years old in
good health condition
•Children between 5 and 8 years old: two doses with an
interval of 60 days (if not previously vaccinated with Flumist®)
•Expensive ($70?)
Antivirals
• M2 ion channel inhibitors
– Amantadine
– Rimantidine
• Neuraminidase inhibitors
– Tamiflu™ (Roche)
– Relenza® (Glaxo-SmithKline)
endocytosis
Amantadine and Rimantidine
•
Interfere with replication by blocking M2 H+ channel pump
– Lack of acidification of the virus’ interior
• No structural change of HA
• Fusion between endosomal and viral membranes is inhibited
• Inhibition of RNP release into the cytoplasm
•
Administered within the first 48 hrs of the onset of symptoms,
decrease duration and severity of disease
•
Approved for people ≥ 1 years old
•
Prophylactic treatment of populations at risk
•
Escape mutants are infectious and common (mutations in TM)
•
Homotetramer with a 24 aa N-terminus sequence, 19 aa
transmembrane domain, and 54 aa C-terminus tail
•
Generated by splicing of mRNA encoded in segment 7
Neuraminidase inhibitors
•
Block release of virus particles from infected cells by binding
tightly to catalytic site
•
Mutants are not viable
•
Effective against A and B types
•
Tamiflu (oseltamivir phosphate)
– Treatment, people ≥ 1
– Prophylaxis, people ≥ 13
– Oral administration
•
Relenza (zanamivir)
– Treatment, people ≥ 7
– Inhalation
• NA, homotetramer, encoded in segment 6, ~490 aa.
Alternative targets
A
B
153
76
B
C
55
105
A
D1
E F G
130
H 159
170
G
146
129
E
1 F
C
105
D
113
38
153
HA
•Binds to sialic acid receptors
•Acidic pH results in structural changes that lead to the
exposure of a hydrophobic peptide -> fusion of endosomal
and viral membranes
•Segment 4, ~1700 nts., ~560 aa
Replication complex-P complex
•
Replication complex or Ribonucleoprotein complex (vRNP)
– Four viral proteins
• PB1, segment 2, 2341 nts, 757 aa
• PB2, segment 1, 2341 nts, 759 aa
• PA, segment 3, 2233 nts, 716 aa
• NP, segment 5, 1565 nts, 498 aa
– vRNA segments
– Amino acid position 627 in PB2 is a marker of host restriction
• Glutamic (E) in majority of avian influenza viruses
• Lysine (K) in human influenza viruses
Replication complex - P-complex
• P-complex
– Three polymerase subunits: PB1, PB2, and PA
– Heterotrimeric complex
– PB1 binds PA and PB2 separately
– No PA binding to PB2
• PB1 is the catalytic domain, polymerization
• PB2 and PA are accessory proteins
– PB2 primer-dependent initiation
– Cleavage of host mRNA to use as primers
– PA primer-independent initiation
P complex: synthesis of RNA molecules
3’
vRNA(-)
UCGCUUUCGUCC
U
GGAACAAAGAUGAppp
mRNA(+)
5’
m7GpppXmY…AGCGAAAGCAGG
G A
(10-13 nts.)
5’
pppAGCGAAAGCAGG
A
5’
3’
A(n) (15-22 nts.)
cRNA(+)
3’
CCUUGUUUCUACU
Matrix (M1): most abundant viral protein
M1
252aa
M2
97aa
• M1
– Encoded in segment 7, 1023 nts.
– Provides structural integrity to virions
– Binds to vRNPs and promotes export of vRNPs out of the
nucleus
– Accumulation in the nucleus triggers assembly steps
– Modulates transcription and replication
NS1 and NEP (NS2):
NS1
237aa
NEP
121aa
NS1
• Segment 8, 890 nts.
• Inhibition of the nuclear export of poly(A)-containing
mRNAs, and
• Inhibition pre-mRNA splicing
• Inhibits PKR
• IFN antagonist
• Binds to p85, part of PI3K
• A single aa at position 92 (Glu) implicated in resistance to
cytokines and cytokine imbalance.
• Promotes viral mRNA translation
NS1
RNA
binding
1
Effector domain
73
142
NES
186
223 231
30kDa
PABII
CPSF
NS1 and NEP (NS2):
NS1
237aa
NEP
121aa
Nuclear Export Protein (NEP or NS2)
• Segment 8, 890 nts.
• Interacts with CRM1, implicated in export of RNPs out of the
nucleus
• Necessary for vRNP assembly and export to the cytoplasm
• Minor component of virus particles
Influenza vs. Bioterrorism
• Class C agent
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–
–
–
–
Respiratory virus easily transmitted by aerosol
Poultry
Swine
Equine
Humans
• Nature-made or Man-made?
– HPAI outbreaks
– Swine influenza outbreaks
– 1977 Russian flu (man-made?)
• How much technology is needed to create a harmful virus?
Vaccine for pandemic preparedness
• Is the circulating virus amenable for vaccine
development?
– How well does it grow in eggs?
– Does it kill the embryo?
• Surrogate virus available?
• Can we prepare a vaccine by reverse genetics?
– Asian H5N1 viruses put reverse genetics to the test
Homework
What alternatives do you envision to prevent the
emergence of novel influenza virus strains in humans
and animals?
Which additional pathways in the virus’ life cycle can be
targets for antiviral intervention? Why?
What is missing in inactivated vaccines to prevent
disease but not infection?
If an influenza pandemic strain were to emerge
tomorrow, what would you do to ameliorate the spread
of the disease?
Additional material: Fields Virology 3rd and 4th Ed.,
“Orthomyxoviruses”