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

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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

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Fever (up to 104 • Sweating • Dry Cough • Sore throat • Headache • Malaise • Fatigue

°

• Nasal congestion F) • Chills (sometimes shaking) • Muscle aches and pains

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.

Type

Naming influenza viruses

16

Hemagglutinin

9

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

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Genus: Influenza C

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Seven segments

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Genus: (unnamed, Thogoto-like viruses)

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Seven segments

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Genus: (unnamed, Infectious Salmon Anemia virus)

•

Seven segments

Influenza A virus

Family:

Orthomyxoviridae

•Segmented negative sense single strand RNA genome

M2 HA NA



2-6Gal



2-3Gal M1 NEP PB1, PB2, PA NP www.cdc.gov

NS1, PB1-F2 Infected cells

HA top view SA α2,3-gal HA side view SA α2,6-gal

Type A influenza cannot be eradicated



2-3Gal 16 HA subtypes 9 NA subtypes



2-6Gal



2-3Gal



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

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

1977 H1N1 “Russian Flu” 2009 H1N1 “American Flu”

cH1N1 Pandemic Influenza 2009 - Natural history of swine influenza North American Swine Influenza (~last 12 years) Eurasian Swine Influenza trH3N2 trH3N2 trH1N2 H3N2 cH1N1 trH1N1 H1N1 PB2 = North Am. Avian PB1 = Human PA = North Am. Avian HA = Human 2009 swine-like H1N1 NP = Classical swine NA = Human M = Classical swine NS = Classical swine 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 NA

Master donor strain genes

PB2 NP PB1 PA M NS

Transfection of plasmids into cells Receptor binding and Entry

Neuraminidase Inhibitors “Tamiflu” “Relenza”

Packaging and Budding ER - Golgi AAAA Host mRNAs AAAA AAAA AAAA AAAA Nucleus Uncoating

Amantadine “Symmetrel” “Flumadine” X +

Endocytosis

Influenza virus Reverse Genetics

viral protein mRNA cap translation AAAA RNA polymerase II (pol II) pII CMV tI pA BGH ATG--------- viral cDNA -------TAA pIh RNA polymerase I (pol I) (-) vRNA 3‘ ppp 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 -

R R R R

G L H7N3 BC H7N3 Chile H7N7 Human

Influenza A viruses from domestic poultry can cause disease in humans.

1997 – – – – – – 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.

1999 – – – 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.

2003 – H7N7 outbreak in domestic poultry in the Netherlands 89 H7N7 human infections, 1 fatal.

HA subtypes in different animal species

Subtype H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 Human Swine Horse

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

Bird

NA subtypes in different animal species

Subtype N1 N2 N3 N4 N5 N6 N7 N8 N9 Human Swine Horse

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

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Northern Hemisphere: October-November

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Southern Hemisphere: April-May Antiviral treatment

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Therapeutic

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Prophylactic

Viral immunity - Vaccines

• • • Infection: solid immunity to homologous virus – – – – 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

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Prevalent A subtype (two) and B viruses

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Virus grown in the allantoic cavity of 10-days old chicken embryos (reassortants derived from A/PR/8/34 and B/Lee/1/40)

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Viruses are inactivated with formalin and standardized for HA content

Efficacy of inactivated vaccines

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Efficacy

– –

Varies with age and immunocompetence Depends on match between projected vs actual strains

• •

Children/Teens

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Stimulates high HA-inhibition antibody titers

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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 10 6.5-7.5

TCID 50 /ml)

Flumist

®

: Efficacy and limitations

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Live viruses with limited replication in the upper respiratory tract

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Prevents (>90%) disease symptoms

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Limited use: Only approved for people 5 to 49 years old in good health condition

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Children between 5 and 8 years old: two doses with an interval of 60 days (if not previously vaccinated with Flumist ® )

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Expensive ($70?)

Antivirals

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M2 ion channel inhibitors

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Amantadine

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Rimantidine Neuraminidase inhibitors

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Tamiflu™ (Roche)

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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 76 105 C B A 55 153 G 146 B 129 1 105 C F E D 113 D 130 E F 170 H 1 G 159 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

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PB2 primer-dependent initiation

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Cleavage of host mRNA to use as primers PA primer-independent initiation

P complex: synthesis of RNA molecules

3’ UCGCUUUCGUCC U vRNA(-) 5’ GGAACAAAGAUGAppp 5’ m7GpppX m Y…AGCGAAAGCAGG G A (10-13 nts.) mRNA(+) A (n) (15-22 nts.) 3’ 5’ pppAGCGAAAGCAGG A cRNA(+) 3’ CCUUGUUUCUACU

Matrix (M1): most abundant viral protein

M1 M2 252aa 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 NEP 237aa 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

1

RNA binding

73

Effector domain

142

NES

186 223 231

30kDa PABII CPSF

NS1 and NEP (NS2):

NS1 NEP 237aa 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 – 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”