Influenza Viruses Influenza Viruses Daniel R. Perez Daniel R. Perez Virginia-Maryland Regional College of Virginia-Maryland Regional College of Veterinary Medicine Veterinary Medicine University of Maryland, College Park University of Maryland, College Park
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Influenza Viruses Daniel R. Perez Virginia-Maryland Regional College of Veterinary Medicine University of Maryland, College Park.
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Influenza VirusesInfluenza Viruses
Daniel R. PerezDaniel R. PerezVirginia-Maryland Regional College of Veterinary MedicineVirginia-Maryland Regional College of Veterinary Medicine
University of Maryland, College ParkUniversity of Maryland, College Park
INFLUENZA FactsINFLUENZA Facts
• Influenza and pneumococcal pneumonia (the most Influenza and pneumococcal pneumonia (the most
common complication of influenza) together are the common complication of influenza) together are the
fifth leading cause of death in the U.S. in people ≥65 fifth leading cause of death in the U.S. in people ≥65
years old.years old.
• In the U.S., 20,000-40,000 die each year from flu-related In the U.S., 20,000-40,000 die each year from flu-related
illness.illness.
• Worsening of chronic heart and lung diseaseWorsening of chronic heart and lung disease
• Annual direct medical costs of flu are estimated at up Annual direct medical costs of flu are estimated at up
to $4.6 billion (over $12 billion adding indirect costs) to $4.6 billion (over $12 billion adding indirect costs)
• Devastating pandemics Devastating pandemics
Common SymptomsCommon Symptoms
•• Respiratory diseaseRespiratory disease• Abrupt onset of symptomsAbrupt onset of symptoms• Fever (up to 104° F)Fever (up to 104° F)• • Chills (sometimes shaking)Chills (sometimes shaking)• • Muscle aches and painsMuscle aches and pains• • SweatingSweating• • Dry CoughDry Cough• • Nasal congestionNasal congestion• • Sore throatSore throat• • HeadacheHeadache• • MalaiseMalaise• • FatigueFatigue
Influenza: Who’s at risk?Influenza: Who’s at risk?
• EverybodyEverybody
• People with greater risk:People with greater risk:
– ≥ ≥ 65 years old 65 years old
– Patients with chronic diseasesPatients with chronic diseases
• Incubation period: 1-4 days, average 2 daysIncubation period: 1-4 days, average 2 days
• Transmission may start 1 or 2 days before onset of Transmission may start 1 or 2 days before onset of
symptoms and last for a weeksymptoms and last for a week
• Immunocompromised patients may transmit the virus for Immunocompromised patients may transmit the virus for
up to a month after onset of symptoms up to a month after onset of symptoms
• Virus particles spread through coughing and sneezing Virus particles spread through coughing and sneezing
• One infectious particle can generate up to 1,000 virus One infectious particle can generate up to 1,000 virus
particlesparticles
Centers for Disease Control and Prevention. Influenza Prevention and Control. Influenza. Available at: http://www.cdc.gov/ncidod/diseases/flu/fluinfo.htm.
Influenza typesInfluenza types
Type AType A Potentially severe illnessPotentially severe illness
Epidemics and pandemicsEpidemics and pandemics
Rapidly changingRapidly changing
Type BType B Usually less severe illnessUsually less severe illness
EpidemicsEpidemics
More uniformMore uniform
Type CType C Usually mild or asymptomatic Usually mild or asymptomatic illness illness
Minimal public health impactMinimal public health impact
Influenza type A: prototype of Influenza type A: prototype of emergent diseaseemergent disease
• Responsible for annual epidemicsResponsible for annual epidemics– Vaccine Re-formulationVaccine Re-formulation
– Re-vaccinationRe-vaccination
• Responsible for devastating pandemicsResponsible for devastating pandemics– 1918 “Spanish Flu”: >20 million people died1918 “Spanish Flu”: >20 million people died
– 1957 “Asian Flu”: 100,000 people died, 70,000 in the U.S.1957 “Asian Flu”: 100,000 people died, 70,000 in the U.S.
– 1968 “Hong Kong Flu”: 700,000 people died, 33,000 in 1968 “Hong Kong Flu”: 700,000 people died, 33,000 in
the U.S. the U.S.
– The emergence of a new pandemic strain is considered The emergence of a new pandemic strain is considered
imminent. Can we avert it?imminent. Can we avert it?
• Family: OrthomyxoviridaeFamily: Orthomyxoviridae• Negative sense single strand RNA genomeNegative sense single strand RNA genome
– Genus: Influenza A, BGenus: Influenza A, B• Eight segmentsEight segments
Pandemic Influenza 2009 - Natural history of swine influenzaPandemic Influenza 2009 - Natural history of swine influenzaPandemic Influenza 2009 - Natural history of swine influenzaPandemic Influenza 2009 - Natural history of swine influenza
Influenza A viruses to order - Influenza A viruses to order - reverse geneticsreverse genetics
Pol I transcription
Pol II transcriptionvRNAvRNA
mRNAmRNA
?
8 plasmids
C
NN
Nucleolus
Influenza A virus
PB2 PB1 PA HA
NP NA NSM
Antigenic “Drift” and “Shift” (surface proteins)Antigenic “Drift” and “Shift” (surface proteins)
Drift: progressive accumulation of mutations within Drift: progressive accumulation of mutations within one subtype one subtype
Shift: acquisition of new genes or an entire new strainShift: acquisition of new genes or an entire new strain
Surveillance is key to understand both phenomena Surveillance is key to understand both phenomena
Influenza type A: prototype of Influenza type A: prototype of emergent diseaseemergent disease
Influenza A virus in poultryInfluenza A virus in poultry
• Defined in 1878 as “fowl plague”.Defined in 1878 as “fowl plague”.• Important economic losses Important economic losses • Low pathogenic influenza viruses (LPAI).Low pathogenic influenza viruses (LPAI).
– Associated with disease outbreaks in young domestic Associated with disease outbreaks in young domestic turkeys.turkeys.
– Progenitors of HPAI viruses.Progenitors of HPAI viruses.• Highly Pathogenic Influenza viruses (HPAI).Highly Pathogenic Influenza viruses (HPAI).
– H5 and H7 subtypes.H5 and H7 subtypes.– Polybasic amino acid region at the HA1/HA2 cleavage site.Polybasic amino acid region at the HA1/HA2 cleavage site.– 23 outbreaks since 1959, 11 outbreaks since 199023 outbreaks since 1959, 11 outbreaks since 1990
• 5 in turkeys, 16 in chickens, 1 in terns, 1 multiple 5 in turkeys, 16 in chickens, 1 in terns, 1 multiple speciesspecies
P E N P K QAY-Q-K-RM T R G L H7N3 BCP E K P K TCSPLSRCRE T R G L
P E K P K TCSPLSRCRK T R G L H7N3 ChileP E I P K - - -R R R R G L H7N7 Human
Influenza A viruses from domestic poultry can cause Influenza A viruses from domestic poultry can cause disease in humans.disease in humans.
1997 1997 H5N1 outbreak in chickens in Hong Kong.H5N1 outbreak in chickens in Hong Kong.– HPAI virus, transmitted to humans.HPAI virus, transmitted to humans.– 18 people diagnosed with H5N1 virus, 6 died.18 people diagnosed with H5N1 virus, 6 died.– No human to human transmission.No human to human transmission.– Slaughter of > 1,000,000 poultry prevented new cases.Slaughter of > 1,000,000 poultry prevented new cases.– Recurrent outbreaks in poultry.Recurrent outbreaks in poultry.– February 2003, 2 people infected, 1 died.February 2003, 2 people infected, 1 died.
1999 1999 H9N2 virus in domestic poultry in Hong Kong and H9N2 virus in domestic poultry in Hong Kong and Southern ChinaSouthern China
– LPAI virus, highly prevalent in quail and chickens.LPAI virus, highly prevalent in quail and chickens.– Transmitted to humans, respiratory disease. Transmitted to humans, respiratory disease. – Some circulating strains have human-like receptor specificity.Some circulating strains have human-like receptor specificity.
20032003 H7N7 outbreak in domestic poultry in the NetherlandsH7N7 outbreak in domestic poultry in the Netherlands– 89 H7N7 human infections, 1 fatal.89 H7N7 human infections, 1 fatal.
NA subtypes in different animal speciesNA subtypes in different animal species
NA subtypes in Humans: N1, N2, N7NA subtypes in Humans: N1, N2, N7
What can we do about it?What can we do about it?
• Surveillance and Biosecurity are the first lines of defense Surveillance and Biosecurity are the first lines of defense
against pandemic influenzaagainst pandemic influenza
– Cooperation between Human Health and Animal Health Cooperation between Human Health and Animal Health
components are essentialcomponents are essential
• During outbreaksDuring outbreaks
– Stamping out remains the best tool available to contain outbreaksStamping out remains the best tool available to contain outbreaks
– Antiviral prophylactic treatment of populations at riskAntiviral prophylactic treatment of populations at risk
• Vaccine for pandemic preparednessVaccine for pandemic preparedness
• Establishing the molecular basis of interspecies transmission Establishing the molecular basis of interspecies transmission
and pathogenesisand pathogenesis
Influenza PreventionInfluenza Prevention
• Vaccination before the start of influenza seasonVaccination before the start of influenza season– Northern Hemisphere: October-NovemberNorthern Hemisphere: October-November
• Infection: solid immunity to homologous Infection: solid immunity to homologous virusvirus
– Antibody to surface genes; HA and NAAntibody to surface genes; HA and NA
– CTL: peptides from internal proteinsCTL: peptides from internal proteins
– Two circulating subtypes: H1N1 and H3N2Two circulating subtypes: H1N1 and H3N2
– H3N2 more important in morbidity and mortalityH3N2 more important in morbidity and mortality
• Inactivated virus vaccinesInactivated virus vaccines– Safe and generally efficaciousSafe and generally efficacious
• Live attenuated vaccines (FluMistLive attenuated vaccines (FluMist®®))– Safe and generally efficaciousSafe and generally efficacious
•Reformulated every year to provide protection against Reformulated every year to provide protection against
virus strains which are prevalent and/or currently virus strains which are prevalent and/or currently
circulatingcirculating
•Prevalent A subtype (two) and B virusesPrevalent A subtype (two) and B viruses
•Virus grown in the allantoic cavity of 10-days old chicken Virus grown in the allantoic cavity of 10-days old chicken
embryos (reassortants derived from A/PR/8/34 and embryos (reassortants derived from A/PR/8/34 and
B/Lee/1/40)B/Lee/1/40)
•Viruses are inactivated with formalin and standardized for Viruses are inactivated with formalin and standardized for
HA contentHA content
Inactivated vaccinesInactivated vaccines
Efficacy of inactivated vaccinesEfficacy of inactivated vaccines
• Efficacy Efficacy – Varies with age and immunocompetenceVaries with age and immunocompetence– Depends on match between projected vs actual strainsDepends on match between projected vs actual strains
• Children/TeensChildren/Teens– Stimulates high HA-inhibition antibody titersStimulates high HA-inhibition antibody titers– Prevents infectionPrevents infection
• ElderlyElderly– Produces lower HA-inhibition antibody titers Produces lower HA-inhibition antibody titers – May not eliminate URTI susceptibilityMay not eliminate URTI susceptibility– May reduce LRTI morbidity/mortalityMay reduce LRTI morbidity/mortality
Amantadine and RimantidineAmantadine and Rimantidineendocytosis
• Interfere with replication by blocking M2 HInterfere with replication by blocking M2 H++ channel pump channel pump
– Lack of acidification of the virus’ interior Lack of acidification of the virus’ interior
• No structural change of HANo structural change of HA
• Fusion between endosomal and viral membranes is inhibitedFusion between endosomal and viral membranes is inhibited
• Inhibition of RNP release into the cytoplasm Inhibition of RNP release into the cytoplasm
• Administered within the first 48 hrs of the onset of symptoms, Administered within the first 48 hrs of the onset of symptoms,
decrease duration and severity of diseasedecrease duration and severity of disease
• Approved for people ≥ 1 years oldApproved for people ≥ 1 years old
• Prophylactic treatment of populations at riskProphylactic treatment of populations at risk
• Escape mutants are infectious and common (mutations in TM)Escape mutants are infectious and common (mutations in TM)
• Homotetramer with a 24 aa N-terminus sequence, 19 aa Homotetramer with a 24 aa N-terminus sequence, 19 aa
transmembrane domain, and 54 aa C-terminus tailtransmembrane domain, and 54 aa C-terminus tail
• Generated by splicing of mRNA encoded in segment 7Generated by splicing of mRNA encoded in segment 7
Neuraminidase inhibitorsNeuraminidase inhibitors• Block release of virus particles from infected cells by binding Block release of virus particles from infected cells by binding
tightly to catalytic sitetightly to catalytic site
• Mutants are not viableMutants are not viable
• Effective against A and B types Effective against A and B types
– Prophylaxis, people ≥ 13Prophylaxis, people ≥ 13
– Oral administrationOral administration
• Relenza (zanamivir)Relenza (zanamivir)
– Treatment, people ≥ 7Treatment, people ≥ 7
– InhalationInhalation
• NA, homotetramer, encoded in segment 6, ~490 aa.NA, homotetramer, encoded in segment 6, ~490 aa.
Alternative targetsAlternative targets
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
– Amino acid position 627 in PB2 is a marker of host restrictionAmino acid position 627 in PB2 is a marker of host restriction• Glutamic (E) in majority of avian influenza virusesGlutamic (E) in majority of avian influenza viruses
• Lysine (K) in human influenza virusesLysine (K) in human influenza viruses
• P-complexP-complex– Three polymerase subunits: PB1, PB2, and PAThree polymerase subunits: PB1, PB2, and PA– Heterotrimeric complex Heterotrimeric complex – PB1 binds PA and PB2 separatelyPB1 binds PA and PB2 separately– No PA binding to PB2 No PA binding to PB2
• PB1 is the catalytic domain, polymerizationPB1 is the catalytic domain, polymerization
• PB2 and PA are accessory proteinsPB2 and PA are accessory proteins– PB2 primer-dependent initiation PB2 primer-dependent initiation – Cleavage of host mRNA to use as primersCleavage of host mRNA to use as primers– PA primer-independent initiationPA primer-independent initiation
UCGCUUUCGUCC GGAACAAAGAUGAppp
vRNA(-)
U
3’ 5’
m7GpppXmY…AGCGAAAGCAGG A(n)
mRNA(+)
(10-13 nts.)
(15-22 nts.)G A
5’ 3’
pppAGCGAAAGCAGG CCUUGUUUCUACUA
5’ 3’cRNA(+)
P complex: synthesis of RNA moleculesP complex: synthesis of RNA molecules
M1 252aa
M2 97aa
Matrix (M1): most abundant viral proteinMatrix (M1): most abundant viral protein
• M1M1– Encoded in segment 7, 1023 nts.Encoded in segment 7, 1023 nts.– Provides structural integrity to virionsProvides structural integrity to virions– Binds to vRNPs and promotes export of vRNPs out of the Binds to vRNPs and promotes export of vRNPs out of the
nucleusnucleus– Accumulation in the nucleus triggers assembly steps Accumulation in the nucleus triggers assembly steps – Modulates transcription and replicationModulates transcription and replication
NS1 237aa
NEP 121aa
NS1 and NEP (NS2):NS1 and NEP (NS2):
NS1NS1• Segment 8, 890 nts.Segment 8, 890 nts.• Inhibition of the nuclear export of poly(A)-containing Inhibition of the nuclear export of poly(A)-containing
mRNAs, and mRNAs, and • Inhibition pre-mRNA splicingInhibition pre-mRNA splicing• Inhibits PKRInhibits PKR• IFN antagonistIFN antagonist• Binds to p85Binds to p85, part of PI3K, part of PI3K• A single aa at position 92 (Glu) implicated in resistance to A single aa at position 92 (Glu) implicated in resistance to
cytokines and cytokine imbalance.cytokines and cytokine imbalance.• Promotes viral mRNA translationPromotes viral mRNA translation
RNAbinding
1 73
30kDa30kDaCPSFCPSF
142 186 223 231
Effector domain
NESNES
PABIIPABII
NS1NS1
NS1 237aa
NEP 121aa
NS1 and NEP (NS2):NS1 and NEP (NS2):
NNuclear uclear EExport xport PProtein (NEP or NS2)rotein (NEP or NS2)• Segment 8, 890 nts.Segment 8, 890 nts.• Interacts with CRM1, implicated in export of RNPs out of Interacts with CRM1, implicated in export of RNPs out of
the nucleus the nucleus • Necessary for vRNP assembly and export to the cytoplasmNecessary for vRNP assembly and export to the cytoplasm• Minor component of virus particlesMinor component of virus particles
Influenza vs. BioterrorismInfluenza vs. Bioterrorism
• Class C agentClass C agent– Respiratory virus easily transmitted by aerosolRespiratory virus easily transmitted by aerosol
– PoultryPoultry
– SwineSwine
– Equine Equine
– HumansHumans
• Nature-made or Man-made?Nature-made or Man-made?– HPAI outbreaksHPAI outbreaks