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

• Asthma/Lung chronic diseaseAsthma/Lung chronic disease

• Chronic Heart DiseaseChronic Heart Disease

– ≤ ≤ 5 years old5 years old

Influenza: TransmissionInfluenza: Transmission

• 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

Naming influenza virusesNaming influenza viruses

A/Hong Kong/156/97 (H5N1)A/Hong Kong/156/97 (H5N1)

StrainStrainID ID

HemagglutininHemagglutinin NeuraminidaseNeuraminidaseTypeType

OriginOrigin YearYearisolatedisolated

A/A/ChickenChicken/Mexico//Mexico/31381-1/94 (H5N2)/94 (H5N2)

1616 99

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

– Genus: Influenza CGenus: Influenza C• Seven segmentsSeven segments

– Genus: (unnamed, Thogoto-like viruses)Genus: (unnamed, Thogoto-like viruses)• Seven segmentsSeven segments

– Genus: (unnamed, Infectious Salmon Anemia virus)Genus: (unnamed, Infectious Salmon Anemia virus)• Seven segmentsSeven segments

INFLUENZA VIRUSINFLUENZA VIRUS

Influenza A virusFamily: Orthomyxoviridae

•Segmented negative sense single strand RNA genome

HA

PB1, PB2, PANP

NA

M2

M1

2-3Gal2-6Gal

NS1, PB1-F2Infected cells

NEP www.cdc.govwww.cdc.govwww.cdc.govwww.cdc.gov

SAα2,3-gal SAα2,6-gal

HA top view

HA side view

Type A influenza cannot be eradicated

2-3Gal2-6Gal

2-3Gal

2-6Gal

2-3Gal2-6Gal

16 HA subtypes 9 NA subtypes

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.

ReassortmentReassortmentReassortmentReassortment

19181918H1N1 “Spanish flu”H1N1 “Spanish flu”>20 million deaths>20 million deaths

19771977H1N1 “Russian Flu”H1N1 “Russian Flu”19771977H1N1 “Russian Flu”H1N1 “Russian Flu”????

19571957H2N2 “Asian Flu”H2N2 “Asian Flu”~2 million deaths~2 million deaths

19681968H3N2 “Hong Kong Flu”H3N2 “Hong Kong Flu”~700,000 deaths~700,000 deaths

20092009H1N1 “American Flu”H1N1 “American Flu”20092009H1N1 “American Flu”H1N1 “American Flu”

Pandemic influenza strains contain genes from the avian reservoir

North American Swine Influenza North American Swine Influenza (~last 12 years)(~last 12 years)

North American Swine Influenza North American Swine Influenza (~last 12 years)(~last 12 years)

PB2 = North Am. AvianPB2 = North Am. AvianPB1 = HumanPB1 = HumanPA = North Am. AvianPA = North Am. AvianHA = HumanHA = HumanNP = Classical swineNP = Classical swineNA = HumanNA = HumanM = Classical swineM = Classical swineNS = Classical NS = Classical swineswine

PB2 = North Am. AvianPB2 = North Am. AvianPB1 = HumanPB1 = HumanPA = North Am. AvianPA = North Am. AvianHA = HumanHA = HumanNP = Classical swineNP = Classical swineNA = HumanNA = HumanM = Classical swineM = Classical swineNS = Classical NS = Classical swineswine

2009 swine-like H1N12009 swine-like H1N1““American flu”American flu”

2009 swine-like H1N12009 swine-like H1N1““American flu”American flu”

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

PB2 = H3N2 swinePB2 = H3N2 swinePB1 = H3N2 swinePB1 = H3N2 swinePA = H3N2 swinePA = H3N2 swineHA = Classical swineHA = Classical swineNP = Classical swineNP = Classical swineNA = Eurasian swineNA = Eurasian swineM = Eurasian swineM = Eurasian swineNS = Classical swineNS = Classical swine

PB2 = H3N2 swinePB2 = H3N2 swinePB1 = H3N2 swinePB1 = H3N2 swinePA = H3N2 swinePA = H3N2 swineHA = Classical swineHA = Classical swineNP = Classical swineNP = Classical swineNA = Eurasian swineNA = Eurasian swineM = Eurasian swineM = Eurasian swineNS = Classical swineNS = Classical swine

cH1N1cH1N1cH1N1cH1N1

cH1N1cH1N1cH1N1cH1N1

Receptor binding and EntryReceptor binding and Entry

EndocytosisEndocytosis

NucleusNucleus

ReleaseRelease

UncoatingUncoating

Packaging and BuddingPackaging and Budding

AAAAAAAA

AAAAAAAA

AAAAAAAA

ER - GolgiER - Golgi

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AAAAAAAA

Host mRNAsHost mRNAs

IFNIFNXXXX

Surface glycoprotein genes HA and NASurface glycoprotein genes HA and NA

HAHA NANA

PB2PB2 PB1PB1 PAPA

NPNP

NSNS

MM

Master donor strain genes

Master donor strain genes

Transfection of plasmidsinto cells

Transfection of plasmidsinto cells

HH++HH++Amantadine

“Symmetrel” “Flumadine”

XXXX

XX

Neuraminidase Inhibitors Neuraminidase Inhibitors “Tamiflu” “Relenza”“Tamiflu” “Relenza”

Influenza virus Reverse GeneticsInfluenza virus Reverse Genetics

RNA polymerase II (pol II)

ppp(-) vRNA

RNA polymerase I (pol I)

translation

viral protein

mRNAAAAAcapcap

pIICMV ATG--------- viral cDNA -------TAA

pABGH

pIhtI

5‘3‘

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

• 11 H7 subtype, 12 H5 subtype11 H7 subtype, 12 H5 subtype

2004 Human

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.

H1H2

H4

H14

H13

H12H11

H9H10

H8

H3

H5H6H7

H15

SubtypeSubtype HumanHuman SwineSwine HorseHorse BirdBird

HA subtypes in different animal speciesHA subtypes in different animal species

HA subtypes in Humans: H1, H2, H3, H5, H7, H9HA subtypes in Humans: H1, H2, H3, H5, H7, H9

N1N2

N4

N9N8

N3

N5N6N7

SubtypeSubtype HumanHuman SwineSwine HorseHorse BirdBird

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

– Southern Hemisphere: April-MaySouthern Hemisphere: April-May

• Antiviral treatmentAntiviral treatment

– TherapeuticTherapeutic

– ProphylacticProphylactic

Viral immunity - VaccinesViral immunity - Vaccines

• 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

Cold adapted influenza viruses: Cold adapted influenza viruses:

caca A/Ann Arbor/6/60 (H2N2)A/Ann Arbor/6/60 (H2N2)

caca B/Ann Arbor/1/66 B/Ann Arbor/1/66

Viruses grown at 25ºC in chicken embryosViruses grown at 25ºC in chicken embryos

Intranasal administration (0.5 ml allatoic fluid Intranasal administration (0.5 ml allatoic fluid

diluted to contain 10diluted to contain 106.5-7.56.5-7.5 TCID TCID5050/ml)/ml)

Live Attenuated Influenza VaccineLive Attenuated Influenza Vaccine(LAIV, Flumist(LAIV, Flumist®®))

•Live viruses with limited replication in the upper respiratory Live viruses with limited replication in the upper respiratory

tract tract

•Prevents (>90%) disease symptomsPrevents (>90%) disease symptoms

•Limited use: Only approved for people 5 to 49 years old in Limited use: Only approved for people 5 to 49 years old in

good health conditiongood health condition

•Children between 5 and 8 years old: two doses with an Children between 5 and 8 years old: two doses with an

interval of 60 days (if not previously vaccinated with Flumistinterval of 60 days (if not previously vaccinated with Flumist®®))

•Expensive ($70?)Expensive ($70?)

FlumistFlumist®®: Efficacy and limitations: Efficacy and limitations

AntiviralsAntivirals

• M2 ion channel inhibitorsM2 ion channel inhibitors– AmantadineAmantadine– RimantidineRimantidine

• Neuraminidase inhibitorsNeuraminidase inhibitors– Tamiflu™ (Roche)Tamiflu™ (Roche)– RelenzaRelenza®® (Glaxo-SmithKline) (Glaxo-SmithKline)

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

• Tamiflu (oseltamivir phosphate)Tamiflu (oseltamivir phosphate)

– Treatment, people ≥ 1Treatment, people ≥ 1

– 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

130

B

AA

CC

DD

105

153

159170

38

55

76

FFEE GG

HH

153

105

129146GG

CC

BB

DD

113

11

11EEFF

AA

Replication complex-P complexReplication complex-P complex

• Replication complex or Ribonucleoprotein complex (vRNP)Replication complex or Ribonucleoprotein complex (vRNP)

– Four viral proteinsFour viral proteins

• PB1, segment 2, 2341 nts, 757 aaPB1, segment 2, 2341 nts, 757 aa

• PB2, segment 1, 2341 nts, 759 aaPB2, segment 1, 2341 nts, 759 aa

• PA, segment 3, 2233 nts, 716 aaPA, segment 3, 2233 nts, 716 aa

• NP, segment 5, 1565 nts, 498 aaNP, segment 5, 1565 nts, 498 aa

– vRNA segmentsvRNA segments

– 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

Replication complex - P-complexReplication complex - P-complex

• 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

– Swine influenza outbreaksSwine influenza outbreaks

– 1977 Russian flu (man-made?)1977 Russian flu (man-made?)

• How much technology is needed to create a harmful virus?How much technology is needed to create a harmful virus?

Vaccine for pandemic preparednessVaccine for pandemic preparedness

• Is the circulating virus amenable for vaccine Is the circulating virus amenable for vaccine

development? development?

– How well does it grow in eggs?How well does it grow in eggs?

– Does it kill the embryo?Does it kill the embryo?

• Surrogate virus available?Surrogate virus available?

• Can we prepare a vaccine by reverse genetics?Can we prepare a vaccine by reverse genetics?

– Asian H5N1 viruses put reverse genetics to the testAsian 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?

Additional material: Fields Virology 3rd and 4th Ed.,“Orthomyxoviruses”

If an influenza pandemic strain were to emerge tomorrow, what would you do to ameliorate the spread of the disease?