Entry and Uncoating of Entry and Uncoating of VirusesViruses

Virology Unit 4, 2015 VersionVirology Unit 4, 2015 Version

Part BPart B

Influenza A EvolutionInfluenza A Evolution

Why influenza? Why now?Why influenza? Why now?

Important human diseaseImportant human disease

Historically significantHistorically significant

Virus EvolutionVirus Evolution

Lead-in from Virus EntryLead-in from Virus Entry

H17 H18

Widely distributed inwild waterfowl

Gene sequences stableCause mild disease

usually

Naming Convention for Influenza Strains:

A/Singapore/6/86/(H1N1)

Some HA propertiesSome HA propertiesVery well studiedVery well studied

18 HA subtypes recognized 18 HA subtypes recognized

And many variations within each subtypeAnd many variations within each subtype

All HAs have similar shape but amino acid All HAs have similar shape but amino acid

sequence may vary widelysequence may vary widely

Signature amino acidsSignature amino acids

HA is main target of neutralizing antibodies HA is main target of neutralizing antibodies

Most react to globular headMost react to globular head

Some react to stalkSome react to stalk

3 Functions3 Functions

AttachmentAttachment

FusionFusion

AntigenAntigen

General Ecology of Influenza AGeneral Ecology of Influenza A

Avian reservoirs, GI virusAvian reservoirs, GI virus

Occasional transfer to humans, Occasional transfer to humans,

mammals, livestockmammals, livestock

Mechanisms of Evolution-Influenza Mechanisms of Evolution-Influenza VirusesViruses

Antigenic shiftAntigenic shift - a reassortment mechanism - a reassortment mechanism

Antigenic driftAntigenic drift - amino acid substitution via - amino acid substitution via mutation mutation

Antigenic ShiftAntigenic Shift

Multiple infection of alternate host (e.g. swine)Multiple infection of alternate host (e.g. swine)Reassortment of genome segmentsReassortment of genome segmentsNew combination of genes produced by reshuffling, not breakage and New combination of genes produced by reshuffling, not breakage and

reunion or copy choicereunion or copy choiceAlternate host as “mixing bowl”Alternate host as “mixing bowl”Can produce new array of HA and NA-pandemic strains?Can produce new array of HA and NA-pandemic strains?H5N5 x H2N2 -> H5N2 and H2N5H5N5 x H2N2 -> H5N2 and H2N5

Antigenic Drift Antigenic Drift

Changes in coding Changes in coding sequence = mutationssequence = mutations

Some mutations result in Some mutations result in amino acid substitutionsamino acid substitutions

Changes in antigenic Changes in antigenic properties of HA (shown) properties of HA (shown) or NA (not shown) or NA (not shown) proteinsproteins

Example of Antigenic DriftExample of Antigenic Drift

Change in reactivity over timeChange in reactivity over timeImmunological pressure produces positive Immunological pressure produces positive

selection for drift strainsselection for drift strains

Drift Due to Replication Errors-Misincorporation of Nucleotides

Viral RNA polymerase forReplication

No 3’-5’ exonucleaseActivity (proofreading)

Average of one misincorporationerror per 1000-100,000NTPS

One error per 10 kb genome

This is not the same as RNA editing!!!!!!!!!!!!!

The Quasi-species ConceptDue to misincorporation without proofreading, progeny RNA genomes are diverse.

Progeny are not necessarily identical either in genotype or phenotype

Within a cell, an individual or a population

The virus constitutes a “quasispecies” or mutant swarm

Different sequences have different fitness-but they can complement

There tends to be one “master” sequence with mutants surrounding

The unit of selection is the quasi-species-not any individual sequence

A fitness landscape (aka adaptive landscape) shows all possible sequences in xy dimensions

Fitness or reproductive success is the z axis

A tool for visualizing adaptation of a mutant swarm

Master sequences near a peak

Swarm can quickly shift to a nearby peak if conditions change

But very hard to “climb” out of avalley because negative mutations are hard to remove(Muller’s Ratchet)

Fitness Landscapes

RNA Viruses Live on the Edge

Pressure to exhibit variationBut too much variation leads to extinction of the quasi-speciesFor each genome there is a limit to the number of errors it can tolerate

(size dependent)This is the error thresholdPassing the threshold leads to error catastrophe for a quasi-species

Shift-relatively fast, may involve any RNA segment, may generate pandemic strains

Drift-relatively slow, usually refers to HA/NA, usually generates seasonal strains

Shift Strains Can Give Rise to Shift Strains Can Give Rise to PandemicsPandemics

Wild bird reservoirs for genome segmentsWild bird reservoirs for genome segmentsNew forms of HA and NA in combinationNew forms of HA and NA in combinationWorld population is immunologically naïveWorld population is immunologically naïveNo “herd immunity”No “herd immunity”Periodic replacement of pandemic virus withPeriodic replacement of pandemic virus with

another another 1918: H1N11918: H1N11957: H2N21957: H2N21968: H3N21968: H3N22009: H1N12009: H1N1

HA & NA Composition of Isolates Causing Pandemics

Obviously a pre-2009 cartoon

New PandemicNew PandemicVirus in 2009 Virus in 2009

Highly contagious H1N1 Highly contagious H1N1 Mexico, California-Mexico, California-

Spring 2009Spring 2009

Replaces all other strains by OctoberReplaces all other strains by October Probably moved from pigs to humans (but NOT Probably moved from pigs to humans (but NOT

called swine flu)called swine flu) Circulating in pigs for many years?Circulating in pigs for many years? Contains bird, pig, human RNA segmentsContains bird, pig, human RNA segments European, Asian, North American European, Asian, North American

Genesis of A/H1N1pdm09-a “triple reassortant”

1918 Pandemic-worst ever1918 Pandemic-worst ever

The 1918 “Spanish Flu” World-Wide Pandemic

- Killed 30 to 50 million people worldwide. - >640,000 deaths in the USA alone. - Infected 28% of all Americans 2.5% died.- Half of the people who died were between 20 & 40; formed a majority of work force.- Flu altered course of World War I.

Devastated the German army.Accounted for 50% of US deaths

Some Unanswered Questions Some Unanswered Questions About the 1918 PandemicAbout the 1918 Pandemic

Where did it come from?Where did it come from?

What happened to it?What happened to it?

Why was it so deadly?Why was it so deadly?

Very stubborn questions with or without Very stubborn questions with or without modern technologymodern technology

Resurrection of 1918 Flu From Sequenced Fragments Can

Provide AnswersAnn Reid

Johan Hultin

Jeffrey Taubenberger

Brevig Mission,Alaska

Lung damage in mice infected with 1918 flu. Science 310:77-80 (2005)

Experiments with 1918 Virus or SegmentsResurrected 1918 virus highly pathogenic in mice, ferrets, primates (nonhuman)Reassortants including 1918 HA or NA segments pathogenic in mice-produce hyper immune response*“cytokine storm”

Other genome segments importantNS-1 an interferon agonistPB-2 replicationLungs infected with 1918

strain had 39,000 times as many virus particles four days after infection as mice infected with TX strain.

All of the mice died within six days after infection compared with none infected with the TX strain

*”Gain of function”

Source of 1918 FluSource of 1918 Flu

Did it enter the human Did it enter the human population prior to 1918?population prior to 1918?

Probably not a direct jump Probably not a direct jump from birdsfrom birds

An avian-like H1N1 but not An avian-like H1N1 but not the bird flu the bird flu

First reported infection of First reported infection of swineswine

Where did the 1918 Pandemic Where did the 1918 Pandemic Strain Go?Strain Go?

Pandemic slows and stops in USA by Pandemic slows and stops in USA by November, 1918November, 1918

Too few susceptibles remaining-herd Too few susceptibles remaining-herd immunityimmunity

Selection against highly virulent forms of Selection against highly virulent forms of influenzainfluenza

Many genome segments derived from 1918 Many genome segments derived from 1918 pandemic strain are still in circulationpandemic strain are still in circulation

““Swine Flu” of 1976Swine Flu” of 1976Death of 2 Army recruits in Death of 2 Army recruits in

January spark fears of a new January spark fears of a new pandemic like 1918pandemic like 1918

USG sponsors national USG sponsors national immunization programimmunization program

Vaccine problems, absence of Vaccine problems, absence of pandemic call a halt to programpandemic call a halt to program

Bird Flu Not the Same as Swine Flu Bird Flu Not the Same as Swine Flu or Spanish Fluor Spanish Flu

Economic and potentiual public Economic and potentiual public health problemshealth problems

1983-1985 PA outbreak 1983-1985 PA outbreak considered the mother of all considered the mother of all poultry outbreakspoultry outbreaks

Some Serious Flu Outbreaks in PoultrySome Serious Flu Outbreaks in Poultry

1959 ScotlandchickenH5N11959 ScotlandchickenH5N11963 EnglandturkeyH7N31963 EnglandturkeyH7N31966 Ontario (Canada)turkeyH5N91966 Ontario (Canada)turkeyH5N91976 Victoria (Australia)chickenH7N71976 Victoria (Australia)chickenH7N71979 GermanychickenH7N71979 GermanychickenH7N71979 EnglandturkeyH7N71979 EnglandturkeyH7N71983–1985 Pennsylvania (USA)*chicken, 1983–1985 Pennsylvania (USA)*chicken,

turkeyH5N2turkeyH5N21983 IrelandturkeyH5N81983 IrelandturkeyH5N81985 Victoria (Australia)chickenH7N71985 Victoria (Australia)chickenH7N71991 EnglandturkeyH5N11991 EnglandturkeyH5N11992 Victoria (Australia)chickenH7N31992 Victoria (Australia)chickenH7N31994 Queensland (Australia)chickenH7N31994 Queensland (Australia)chickenH7N31994–1995 Mexico*chickenH5N21994–1995 Mexico*chickenH5N21994 Pakistan*chickenH7N31994 Pakistan*chickenH7N31997 New South Wales (Australia)chickenH7N41997 New South Wales (Australia)chickenH7N41997 Hong Kong (China)*chickenH5N11997 Hong Kong (China)*chickenH5N11997 ItalychickenH5N21997 ItalychickenH5N21999–2000 Italy*turkeyH7N11999–2000 Italy*turkeyH7N12002Hong Kong (China)chickenH5N12002Hong Kong (China)chickenH5N12002 ChilechickenH7N32002 ChilechickenH7N32003 Netherlands*chickenH7N72003 Netherlands*chickenH7N7

Pennsylvania Outbreak Pennsylvania Outbreak Pennsylvania has tasted such devastation Pennsylvania has tasted such devastation

before. In 1983-84, a highly infectious before. In 1983-84, a highly infectious H5N2 strain of avian flu erupted H5N2 strain of avian flu erupted among domestic poultry in the state. "It among domestic poultry in the state. "It started as a low pathogenic strain started as a low pathogenic strain (LPAI) and didn't cause much alarm at (LPAI) and didn't cause much alarm at first," recalls Michael Hulet. "But it first," recalls Michael Hulet. "But it continued to circulate in the bird continued to circulate in the bird populations for about six months and populations for about six months and then it mutated and became a highly then it mutated and became a highly pathogenic (HPAI) strain." pathogenic (HPAI) strain."

Adds Paul Patterson, the virus was Adds Paul Patterson, the virus was eventually eradicated through eventually eradicated through quarantine and the extermination of quarantine and the extermination of over 17 million birds at a huge cost to over 17 million birds at a huge cost to the industry, up to $65 million lost." the industry, up to $65 million lost." (Unlike the H5N1 strain circling the (Unlike the H5N1 strain circling the globe today, H5N2 didn't result in globe today, H5N2 didn't result in human illness or death.)human illness or death.)

Kawaoka, Yoshihiro, Clayton W. Naeve, and Robert G. Webster. "Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin?." Virology 139.2 (1984): 303-316.The A/Chick/Penn/83 (H5N2) influenza virus that appeared in chickens in Pennsylvania in April 1983 and subsequently became virulent in October 1983, was examined for plaque-forming ability and cleavability of the hemagglutinin (HA) molecule. The avirulent virus produced plaques and cleaved the HA only in the presence of trypsin. In contrast, the virulent virus produced plaques and cleaved the HA precursor into HA1 and HA2 in the presence or absence of trypsin. The apparent molecular weight of the HA1 from the avirulent virus was higher than that from the virulent virus, but when the viruses were grown in the presence of tunicamycin, the molecular weights of HA were indistinguishable. Two of nine monoclonal antibodies to the HA of the avirulent virus indicate that there is at least one epitope on the HA that is different between the virulent and avirulent viruses. The amino acid sequences of the HAs from the two viruses were compared by sequencing their respective HA gene. The nucleotide sequence coding for the processed HA polypeptide contained 1641 nucleotides specifying a protein of 547 amino acids. The amino acid sequences of the virulent and avirulent viruses were indistinguishable through the connecting peptide region, indicating that the difference in cleavability of the H5 HA is not directly attributed to the amino acid sequence of the connecting peptide. Four of seven nucleotide changes resulted in amino acid changes at residues 13, 69, and 123 of HAI and at residue 501 of the HA2 polypeptide. Since there were no deletions or insertions in the amino acid sequence of the virulent or avirulent viruses, the possibility exists that the difference in molecular weight is due to loss of a carbohydrate side chain in the virulent strain. The amino acid change in the virulent strain at residue 13 is the only mutation that could affect a glycosylation site and this is in the vicinity of the connecting peptide. It is postulated that the loss of this carbohydrate may permit access of an enzyme that recognizes the basic amino acid sequences and results in cleavage activation of the HA in the virulent virus.

H5N1 Outbreak in Humans-Hong Kong

First Appearance of a “Bird Flu” in Humans

Direct transmission of all-bird genome segments to humans

New Lethal H5N1 Variant Circulating Poultry and Wild Birds

Hong Kong Market Avian Influenza

Highly lethal flu strain (H5N1) appeared in Hong Kong market in 1997

Caused an epidemic among chickens causing high proportion of deaths

Virus spread from chickens & killed 6 of 18 infected people

Slaughtered >one million chickens & changed caging & housing practices.

Other “bird flu” outbreaksOther “bird flu” outbreaks

H5N1-continues to circulate worldwide in H5N1-continues to circulate worldwide in birds: shift and driftbirds: shift and drift

In humans 364 deaths out of 615 cases In humans 364 deaths out of 615 cases (2013)(2013)

2003: H7N7, Europe, I death2003: H7N7, Europe, I death 2004: H7N3, Canada2004: H7N3, Canada 2004: H10N7, Egypt2004: H10N7, Egypt Human cases usually attributable to Human cases usually attributable to

contact with infected birdscontact with infected birds

Antivirals and VaccinesAntivirals and Vaccines

Neuraminidase InhibitorsNeuraminidase InhibitorsRelenza (zanamivir)Relenza (zanamivir)

Tamiflu (oseltamivir)Tamiflu (oseltamivir)

Ion Channel BlockersIon Channel BlockersRimantadineRimantadine

AmantadineAmantadine

Vaccines: Vaccines: Can be inactivated/subunit (injected) or Can be inactivated/subunit (injected) or

attenuated (inhaled= LAIV or FluMist) attenuated (inhaled= LAIV or FluMist)

Universal flu vaccine based on HA “stalk”?Universal flu vaccine based on HA “stalk”?