Influenza

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Flu Pandemics• Russian Flu (1889 – 1890)

– Approx. 1 million deaths – H2N2 subtype

• Spanish Flu (1918 – 1919) – 40 – 100 million deaths– H1N1 subtype

• Asian Flu (1957 – 1958) – 1 – 1.5 million deaths– H2N2 strain

• Hong Kong Flu (1968- 1969) – 0.75 to 1 million deaths– H3N2 subtype

• 2009 H1N1 – Between 151,700 and 575,4000

with 80% of deaths in those 65 and younger– H1N1 subtype

Flu• RNA Virus• 3 Influenza types

– A• Initial host - wild aquatic birds

but have now infected many different animals

• Divided into 2 categories – Low pathogenic and Highly Pathogenic

– B • Primary host - Humans

– C• Only hosts - Swine and Humans

• Types A and B cause yearly outbreaks• A mutates the fastest• B mutates at a rate 2-3 times lower than type A• However, since it does mutate, then no lasting immunity

is possible • C is the slowest to mutate

Influenza A and B • A is most virulent • A can be subdivided into serotypes

– Hemagglutinin • 17 subtypes

– Neuraminidase(Sialidase)

• 10 subtypes– Genome is generally divided

between 8 separate gene segments

• B can not be broken down into subtype

• Both have various strains• Current Vaccine help A and B

but not C– an A/California/7/2009 (H1N1);– an A(H3N2) virus A/Victoria/361/2011;– a B/Massachusetts/2/2012-like.

• Quadrivalent vaccine this year includes a B/Brisbane/60/2008-like (B/Victoria lineage) virus.

http://www.microscopy.fsu.edu/cells/viruses/images/influenzafigure1.jpg

Last year a New Flu – H3N2v which had Pig to human transmission

Influenza A Nomenclature

http://en.wikipedia.org/wiki/File:Influenza_nomenclature.svg

Human, Swine or Avian Flu?• Most of the time they all are Influenza Type A• The subtype can be the same

– for example - H1N1 or H3N2• But different strains have adapted for the particular host

– Seasonal flu is caused by Influenza A viruses that have changed to spread in humans (human influenza)• Currently circulating in humans are H3N2 and H1N1 viruses

– Avian flu is caused by Influenza A viruses that affect birds.– Swine flu is caused by Influenza A viruses that affect pigs

• Influenza A viruses that typically infect one species can cross over and cause illness in a different species– Until 1998 only one H1N1 circulated in pigs then a human H3N2

crossed over– Recently a H3N8 from horses moved to dogs

• Influenza A viruses “mutate” allowing yearly outbreaks and species jumps– Random mutation or reassortments

Antigenic Drift• Random mutations that

change the antigens in the virus

• These mutations allow the virus to evade the immune response

• Primarily concerned with mutation in hemagglutinin which is the cell surface protein responsible for entry into cells

Diagram from the National Institute of Allergy and Infectious Diseases

Antigenic Shift• Reassortment of two

or more different strains

• Typically from two different species

• Often enables a flu strain to jump from one animal species to another

• Could be from direct or intermediate transmission with no genetic changes

Diagram from the National Institute of Allergy and Infectious Diseases

• Avian Influenza A viruses typically are transmitted to humans in two ways– Through direct contact– Through an intermediate host

• Avian Flu replicates less efficiently in humans

• Human less efficiently in birds• Avian viruses preferentially bind to

N-acetylneuraminic acid- α2,3 galactose

• Human viruses preferentially bind to N-acetylneuraminic acid- α2,6 galactose

• Swine have BOTH!

• Overlaid images of α(2,6)- and α(2,3)-linked sialylpentasaccharides LSTc and LSTa, respectively, in the HA

receptor binding site

Hurdles to Cross• “Species Jump”

– adaptation that allows the strain to pass to another species

– Infection generally occurs via direct contact with animals who are ill.

• Human to Human– development of changed viruses

with the ability to cause infection and spread in the human population

• Examples– 1918 flu pandemic which is

believed to have been initially caused by an antigenic drift from an avian flu to a human flu

– antigenic shifts from the reassortment of an avian flu and a human flu (e.g Asian and Hong Kong pandemics)

• There may be little or no immunity in the human population to these new viruses.

Viral Infection

RNA dependent RNA Polymerasehas no proof reading activity, soError rate of 105 which results in antigenic drift

1. Binding2. Uncoating3. Replication/

transcription4. Translation5. Protein processing6. Reassembly7. Budding

Influenza Genes and ProteinsGenes are on 8 separate segments of negative-sense single-stranded RNA that encode 11

known proteins

General category Protein(s) encoded Protein functions Surface proteins Hemagglutinin (HA) Viral attachment and fusion

proteinNeuraminidase (NA) Virus releaseM2 ion channel Facilitates viral RNP uncoating

Internal proteins Matrix (M1) protein Viral structural protein regulates RNP nuclear import

Nucleoprotein (NP) Required for viral RNA synthesis

Polymerase Components PA Polymerase subunitPB1 Polymerase catalytic subunit

endonuclease activityPB2 Polymerase subunit binds caps

of host cell mRNAs

Non-structural proteins NS1 Evasion of interferon responsesPB1-F2 Proapoptotic factor, possible

immune evasion functionNuclear export protein Mediates viral RNA nuclear export

(NEP or NS2)

Antiviral Research Volume 79, Issue 3, September 2008, Pages 166-178

Hemagglutinin• Lectin (Glycoprotein) that

mediates binding of virus to cell

• Viruses bind through hemagglutinin onto sialic acid sugars on the surface of epithelial cells triggering uptake of the virus via endocytosis

pH structure change

Low pH in endosome causes conformational changes that facilitate fusionof viral and host membranes. Postulated to be similar to SNARE complexesFusion between endosome and virus forms a pore through which the viral genetic material enter the cytosol

Hemagglutinin

The presence of a multibasic cleavage site is not the only determinant of viral pathogencity . The 1918 Flu had a single site while H5N1 has a multibasic site

• HA0 must undergo proteolytic cleavage into HA1 and HA2 to allow the conformational change required for membrane fusion.

• LPAI have a single basic singe basic cleavage site

• HPAI have a multibasic cleavage site• Variations in host protease determines

location and effectiveness of infection • LPAI is cleaved by a host

trypsin like protease limited to the respiratory tract

• HPAI is a furin-like protease which present in virtually all cells thus allowingHPAI to spread easily throughout the body often causing fatal systemic infections

M2 protein • Proton selective ion channel• located in the viral envelope • Transports protons from the endosome into

the virus particle• Lower pH allows dissociation of the viral

matrix protein M1 from the ribonucleoprotein RNP

• Crucial step in “uncoating” of the RNP and exposing the content to the cytoplasm of the host cell

• The RNP complex is transported to nucleus where replication and transcription occurs

• The gene for M2 is susceptible to mutations, so strains are developing resistance

M2 Antiviral drugs• Symmetrel and Flumadine

– function by blocking the channel– effective against influenza A – Many strains are resistant too

Neuraminidase

• Glycoprotein• Glycosidase• Cleaves the sialic acid

residue on the host cell allowing the mature virus to detach

• Flu treatment– neuraminidase inhibitors– effective against both

influenza A and B

Neuraminidase Inhibitors

Tamiflu RelenzaStructure based design

Avian “Bird” Flu• Only a few subtypes have

been highly pathogenic in humans. – Spanish, Asia and Hong Kong

• Most recent are H5N1 and H7N9 in Asia

• Most infections were contracted after direct contact with infected bird

• Limited evidence of human to human transmission

• Expected to be the next deadly global pandemic

Highly Pathogenic H5N1 which generally results in severe pneumonia has

60% mortality world wide

1918 flu pandemic• H1N1 subtype• Initially caused by an antigenic drift • High death rate was due to cytokine storm

– too many immune cells are activated in a single place

• In lungs fluids and immune cells accumulate and eventually block off the airways

• More susceptible were those with healthy immune systems

Mortality Age Distribution for

1918 epidemic and normal epidemics

• (CDC is author, Taken from "1918 Influenza: the Mother of All Pandemics" Jeffery K. Taubenberger and David M. Morens, http://www.cdc.gov/ncidod/EID/vol12no01/05-0979.htm )

Asian and Hong Kong Flu

• H2N2• A mutation in wild ducks combed with a

pre-existing human strain• H3N2 evolved via antigenic shift and

caused a smaller outbreak

Protein Changes in function that could affect virulence• Increased virulence of the 1918 H1N1 and H5N1 Bird flu has been linked to:

the HA and NA proteins and the polymerase proteins (PB1,PB2, and PA) and the Non-structural proteins

• HA– Altered cleavage site allowing change in tropism; – altered receptor specificity allowing increased transmissibility and

altered tropism in the airway• NA

– Ability to promote HA cleavage• M2

– Mutations that confer resistance to amantadine and rimantadine• PB1

– Enhanced virus RNA synthesis• PB2

– Enhanced virus RNA synthesis in mammalian cells, enhanced polymerase function at high temperature

• NS1– Enhanced ability to suppress innate immunity

• PB1-F2– Enhanced pro-apoptotic activity

• None suggest so far for– M1, NP, PA or NS2

1918 Influenza • Avian HA consensus sequence

– Six residues are conserved in the HA1 domains of most avian H1 HAs

– implicating these residues in the ability to bind to the avian 2,3Gal receptor

• Comparison of the avian HA consensus sequence with the 1918 Virus shows that only one or two of these conserved residues are different – 190 and 225

• For H1 1918– Residue 190 alone determined

shift• Know already that H5N1 found in

humans have mutations at several positions which allow better recognition of the human receptor

H5N1

Might only require a singe BASE change of switch to human receptor

Cell, Volume 153, Issue 7, 1475-1485, 06 June 2013

H7N9• The hemagglutinin of

H7N9 virus does not efficiently bind human receptors

• •A single residue change in receptor binding site increases binding to human receptors

• •Mutations on hemagglutinin may reduce the effectiveness of current H7 vaccines

Cell, Volume 153, Issue 7, 1486-1493, 06 June 2013

Minimum Changes Needed• 1st better binding to mammalian receptor• Hemagglutinin

– Only 2-4 amino acid substitutions are required to switch binding from α2,3 to α2,6

• 2nd Better uptake and release of the virus• Neuraminidase

– In the 1918 virus, Neuraminidase plays an unusual role in binding and activating the host protease that cleaves HA rendering the virus infectious

• Cleavage of the Hemagglutinin precedes the conformation change which leads to cell fusion

• Cleavage is generally dependent on the type and distribution of the host trypsin like protease

– H5N1 Neuraminidase is rather slow in cleaving the sialic acid to release the new virus particle

– A few mutations will take the H5N1 closer to the 1918 neuraminidase

Minimum Changes Needed• Polymerase proteins (PB1,PB2, and PA)

– The 1918 virus contains only 10 aa changes in the sequences of the polymerase proteins

– H5N1 already has 7 of the 10– Important because avian viruses replicate around 41C but

effective replication in mammals is 37C– Both 1918 and H5N1 have a single mutation, N66S, which has

been linked to increased virulence

2009 H1 N1:Genetics of New strain• What is special about this

H1N1 outbreak? • Reassortment of:

– One human strain– Two pig strains– One bird strain

• Hemagglutinin gene is similar to current swine flu viruses present in U.S. pigs

• Neuraminidase and matrix protein genes resembled versions present in European swine flu isolates

• This is different from the previous seasonal H1N1 strain

• High rate of Human to Human transmission

• But mild symptoms so far N Engl J Med 361:115-119

Infectivity and Virulence

 • H5N1 and H7N9 currently has low Human to Human

transmission but are highly virulent • The 2009 H1N1 quickly became a pandemic (highly

pathogenic) but was not very virulent• What will be the next pandemic• Will we see a reassortment (antigenic shift) of 2009

H1N1 and H5N1 or H7N(? • Will H5N1 have a minor antigenic drift like 1918 H1N1

allowing it to be transmitted human to human?• Are we prepared?

Pathogenic – cause disease Virulent – extend of pathology

Infectivity – the ability of a organism to invade and replicate