The emergence of pathogenic viruses: an evolutionary approach Michael Worobey Department of Ecology and Evolutionary Biology, University of Arizona August

The emergence of pathogenic viruses: an evolutionary approach

Michael Worobey

Department of Ecology and Evolutionary Biology, University of Arizona

August 25, 2008

Questions:

(1)(1) Where did HIV come from? Where did HIV come from?

(2) (2) When, where, and how did HIV-1 first emerge from sub-When, where, and how did HIV-1 first emerge from sub-Saharan Africa?Saharan Africa?

(3) (3) When and how did the pandemic AIDS virus jump to When and how did the pandemic AIDS virus jump to humans and begin its spread?humans and begin its spread?

(4) What factors mediate the emergence of new flu lineages?(4) What factors mediate the emergence of new flu lineages?

1: The origin of HIV

Where did AIDS come from?

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

As a national correspondent for the San Francisco Chronicle, Shilts was the first newspaper reporter to cover the AIDS epidemic full time. In his book And the Band Played On—AIDS: The First Five Years (1980-1985), he took almost everyone to task on how the first years of the epidemic were handled

Randy Shilts



• First identified in US gay males in the early 1980s, severe immunosuppression

• Pneumocystis carinii pneumonia

• Other rare opportunistic infections, horrendous suffering and death

HIV/AIDS basicsHIV/AIDS basics

Early history

• New syndrome recognized by 1981

• Retroviral agent isolated in 1983

• Sexually transmitted, but also via needles, transfusions, birth

• Hit these risk groups hard in the US, but also high prevalence in Haiti, Central Africa

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

Françoise Barre-Sinoussi


• Viruses are made up of a set of genetic instructions wrapped inside a protective shell

• HIV is particularly succinct at around 3000 amino acid residues that hijack the cell’s own machinery

• Genome is in the form of RNA, so it also includes a reverse transcriptase (RNA to DNA enzyme)

• About 20 % of your genome is made up of similar “selfish DNA” (more than 10X the amount of your 30,000 protein genes)



Evolution in the fast lane:Evolution in the fast lane:

• About 10 billion virions are generated daily in an infected host (2.5 days per cycle)

• Each has a compact genome made up of about 10,000 nucleotides

• Approximately one mutation is generated for each new genome

• Every possible mutation occurs every day


Current status: DisasterCurrent status: DisasterHIV/AIDS basicsHIV/AIDS basics

The past…

Where did HIV/AIDS come from?

Origins of HIV/AIDSOrigins of HIV/AIDS

Where did HIV come from?• Divine retribution

• Doesn’t matter--it doesn’t cause AIDS

• Conspiracy theories - e.g. the CIA did it

• Voodoo rituals

• Ritualistic use of monkey blood

• Contamination of vaccines

• Zoonosis (a disease communicable from animals to humans under natural conditions)

How can we discriminate between these hypotheses?



“The poor homosexuals--they have declared war upon nature, and now nature is exacting an awful retribution” -Pat Buchanan

"With 80,000 dead of AIDS, our promiscuous homosexuals appear literally hell-bent on Satanism and suicide” -Pat Buchanan

"AIDS is not just God's punishment for homosexuals; it is God's punishment for the society that tolerates homosexuals.”-Jerry Falwell

“Grown men should not be having sex with prostitutes, unless they are married to them” -Jerry Falwell





“The poor homosexuals--they have declared war upon nature, and now nature is exacting an awful retribution” -Pat Buchanan

"With 80,000 dead of AIDS, our promiscuous homosexuals appear literally hell-bent on Satanism and suicide” -Pat Buchanan

"AIDS is not just God's punishment for homosexuals; it is God's punishment for the society that tolerates homosexuals.”-Jerry Falwell

“Grown men should not be having sex with prostitutes, unless they are married to them” -Jerry Falwell




Where did HIV come from?• Doesn’t matter--it doesn’t cause AIDS


Where did HIV come from?



• Divine retribution




• Zoonosis (a disease communicable from animals to man under natural conditions)


• THE PLAUSIBLE HYPOTHESES ALL HAVE IN COMMON THE INCRIMINATION OF SIMIAN IMMUNODEFICIENCY VIRUSES (SIVcpz) FROM CHIMPANZEES

• THE KEY DISCOVERY WAS THE FINDING THAT AFRICAN PRIMATES ARE INFECTED WITH SIMILAR VIRUSES…


Early history

• New syndrome recognized by 1981

• Retroviral agent isolated in 1983

• Sexually transmitted, but also via needles, transfusions, birth

• Hit these risk groups hard in the US, but also high prevalence in Haiti, Central Africa

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

Françoise Barre-Sinoussi





• Voodoo rituals



• Zoonosis (a disease communicable from animals to humans under natural conditions)

How can we discriminate between these hypotheses?


Pan troglodytesPan troglodytes

Cercocebus Cercocebus atysatys

Cercopithecus Cercopithecus lhoestilhoesti

CercopithecusCercopithecusalbogularisalbogularis

Colobus guerezaColobus guereza

Chlorocebus aethiopsChlorocebus aethiops

Key discovery: SIVs are found naturally in African primates

SIVcol

SIVsun

SIVlhoest

SIVmnd

SIVsm

HIV-2/A

HIV-2/B

SIVsyk

SIVagmVER

SIVagmGRI

SIVagmTAN

HIV-1/M

HIV-1/N

SIVcpz

HIV-1/O

SIVcpz

0.1

x

x

x

x

x

• Divine retribution




• Zoonosis (a disease communicable from animals to man under natural conditions)


• THE PLAUSIBLE HYPOTHESES ALL HAVE IN COMMON THE INCRIMINATION OF SIMIAN IMMUNODEFICIENCY VIRUSES (SIVcpz) FROM CHIMPANZEES

• THE KEY DISCOVERY WAS THE FINDING THAT AFRICAN PRIMATES ARE INFECTED WITH SIMILAR VIRUSES…



A direct test: non-invasive sampling of SIVcpz from the supposed “source” (and a big blank space on the map of SIVcpz distribution)



Worobey et al. 2004

Phylogenetic position Expected for source population

Phylogenetic position of Kisangani SIV


•>1500 samples collected from across DRC

•212 SIVcpz Ab positive

•22 SIVcpz RNA positive

Worobey, Hahn, Li, Ndjango, unpublished results

Extensive non-invasive sampling continues:

2: The Worldwide Emergence of HIV-1

B RF

A U455

C ETH2220

H VI991

K EQTB11

SIVcpz/Ant

SIVcpz/Gab1

SIVcpz/Cam5

B LAID 94UG114

D ELIA 92UG037

G SE6165G 92NG083J SE9280

J SE9173

C 92BR025

H 90CF056

F1 VI850F1 93BR020

F2 MP255F2 MP257

O MVP1580

N YBF30

N YBF106

SIVcpz/US

O ANT70

K MP535

Subtypes A-K

30% amino acid divergence in Env between subtypes

Utility:

Tracing the global pandemic Documenting recombination

subtypes

HIV-1 group M Diversity

Molecular Epidemiology of HIV-1 group M

Revisiting an old hypothesis…

Questions:

Did HIV-1 move from Haiti to US, or US to Haiti?Did HIV-1 move from Haiti to US, or US to Haiti?

When and how did these events take place?When and how did these events take place?

Approach:

•Archival Haitian-linked samples, Pitchenik Archival Haitian-linked samples, Pitchenik et al, AIM, et al, AIM, 19831983

•Some of the earliest known AIDS patients in the US(Haitian immigrants to the US 1970s/80s)

•Full-length Full-length envenv alignment of published B and D alignment of published B and D subtype sequences (117 B plus 5 D)subtype sequences (117 B plus 5 D)

•Bayesian MCMC approach, MrBayes and BEASTBayesian MCMC approach, MrBayes and BEAST

Phylogenetic patterns under different scenarios:

Haiti first US first

Simultaneous Unknowable

Results: the Emergence of HIV-1 in the Americas

Posterior probability of “pandemic clade” = 1.0

Posterior probability of Haitian origin = 0.999 Posterior probability of Trinidad &

Tobago clade = 1.0

n = 13

n = 96

Gilbert et al. 2007, PNAS

Estimating rates of viral evolution

dt

Rate =

Average: 5 x 10-3/site/year

divergence rate: 1% /site/year

Mutation rate : 3.4 x 10-5 /site/replication

Replications : about 300/year

300 x 3.4 x 10-5 = 10.2 x 10-3

Nonsynonymous, dN = 2-5 x 10-3/site/year

Synonymous, dS = 10 x 10-3/site/year

Using the “molecular clock”Calibrate the clockCorrect for multiple hitsAccount for methodological bias

The Emergence of HIV-1/AIDS in the Americas: when?

Posterior probability of Haitian origin = 0.998

1969 [1966-72]

Conclusions:

It’s not a sampling artifact: the B epidemic is older in Haiti, >40 It’s not a sampling artifact: the B epidemic is older in Haiti, >40 yearsyears

Timing fits well with large movement of people between Haiti and Timing fits well with large movement of people between Haiti and DR Congo after independence in early 1960sDR Congo after independence in early 1960s

One such individual may have been the first to bring HIV out of One such individual may have been the first to bring HIV out of sub-Saharan Africasub-Saharan Africa

Conclusions•Strong support for a single-patient introduction of Strong support for a single-patient introduction of “pandemic clade” from Haiti“pandemic clade” from Haiti

•In or around 1969In or around 1969(long cryptic period in US)(long cryptic period in US)

•Ecological, not evolutionary factors determined “success”Ecological, not evolutionary factors determined “success”

•Do non-pandemic clade viruses have distinct Do non-pandemic clade viruses have distinct immunological properties? (Mascola immunological properties? (Mascola et alet al, , JIDJID, 1994), 1994)

•Why so few successful epidemic introductions after the Why so few successful epidemic introductions after the pandemic clade in or around 1969?pandemic clade in or around 1969?

3: When and how did HIV jump into humans and begin its initial

spread?

Molecular archeology of HIV: motivation

•archival sequences can provide direct tests of archival sequences can provide direct tests of evolutionary hypothesesevolutionary hypotheses

•1918 Spanish Flu virus has been resurrected and used to 1918 Spanish Flu virus has been resurrected and used to investigate emergence, pathogenesis and other questions.investigate emergence, pathogenesis and other questions.

•For HIV relevant frozen samples are For HIV relevant frozen samples are rarerare and already and already screened (one from 1959, then 1976)screened (one from 1959, then 1976)

Nature, 1998

Science, 2000

• Ambient temperature Ambient temperature specimens like blood specimens like blood smears and paraffin-smears and paraffin-embedded tissue are embedded tissue are not so rarenot so rare

•Between 5 and 10 microtome sections, 5-10 m in thickness

•or an approximately equivalent amount of tissue shaved from each block with a disposable scalpel blade

•Digestion/extraction optimzed for RNA recovery

•Primers designed for short fragments with primers in M-group or subtype A conserved regions

Table S1. Primers and sequence-confirmed amplification results.

Primer Primer Sequence 5'-3' Frag-

ment # DRC60

BE81 BE85 CAN97

HIVG1 (F)a ACCCACCTATCCCAGTAGGAGAAAT 1 (+)c (+) (+) (+)

HIVG2 (R) GGTCCTTGTCTTA TGTCCAGAATGC

Pol3290Fb

GCCAGAAAAAGACAGCTGGACTGTCAA 2 (+)c (+) (+) (+)

Pol3415R CCTTAGGGGARCCAAAGCTCT

env6371F CACCACTCTATTTTGTGCATCAG 3 (+) (+) (+) (-)

env6442R GCATGTGTAGCCCAGACATTAT

env6445F GTGTACCCACAGACCCCAA C 4 (+) (+) (+) (-)

env6542R CTCATGCATTTGTTCTACCATGT

env6828F ACACAGGCTTGTCCAAAGGT 5 (+)c (-) (-) (-)

env6890R ACCAGCTGGGGCACAATAAT

env7468F CACTCCCATGCAGAATAAAACA 6 (+)c (+) (+) (-)

env7535R AGGGGCATACATTGCTTGTC

env7717Fd

CCACCAAGGCAAAGAGAAGA 7 (-) (+) (-) (+)

env7796R TCCCAAGAACCCAAGGAAC

env7899F ATAGAGGCGCAACAGCATCT 8 (-) (-) (-) (-)

env7977R TTTCCACAGCCAGGACTCTT

env8047F TGCCCTGGAACTCTAGTTGG 9 (+)c (+) (+) (-)

env8112R CCATCCAGG TCATGTTCTCC

env8423F CGAAGAAGAAGGTGGAGAGC 10 (+)c (+) (-) (-)

env8498R GTCCCAGGCAAGTGCTAAGA

env7717Fd,e

CCACCAAGGCAAAGAGAAGA 11 (+) N/Df N/D N/D

env7805R TCCTGCTGCTCCTAAGAACC

env7771Fd,e GAGCTGTCTTCCTTGGGTTCT 12 (+) N/D N/D N/D

env7846R GCCTGTACCGTCAGCGTTA

env7835F d,e GACGGTACAGGCCAGACAAT 13 (+) N/D N/D N/D

env7937R CCAGACCGTGAGTTTCAACA

env7890F d,e CTGAGGGCTATAGAGGCTCAAC 14 (+)c N/D N/D N/D

env7960R CTTGCCTGGAGCTGTTTAATG aPreviou sly pub lished (S13 ).

HIV DNA/RNA can be recovered from “ancient” paraffin-embedded samples:

• The inclusion of the 1959 and 1960 sequences appears to improve estimation of the TMRCA of the M group.

• Because DRC60 and ZR59 limit the influence of the coalescent tree prior on the posterior TMRCA distributions, the different demographic models give consistent results, with tighter date ranges and 95% HPDs that extend no later than 1933.

• Suggest that HIV-1 has been circulating ca. 100 years.

• The Bayesian skyline plot tracks effective population size through time

• Suggests that HIV-1 group M experienced a long period of relatively slow growth in the first half of the 20th century followed by a rapid expansion thereafter.

• Similar pattern as observed by Yusim et al. (2001), Phil. Trans.

A/A1

A2

B

DF1

F2

C

J

Kinshasa, 1960

Kinshasa, 1959

+

+

+

•Strict ancient DNA protocols

•Topological position (A-like, monophyletic with other DRC sequences in a basal clade)

•Branch length

•Reproducibility

•Independent replication (blinded)

•No fragments longer than 126 nucleotides amplifiable

•RNA survived in the sample, but of quality expected for Bouin’s-fixed specimen (B2M quantitative RT-PCR)

•Tissue of origin makes sense

•Control sequences well behaved

•The estimated age of the M group ancestor and the estimated time for dispersion of the HIV-1 pandemic strains coincide with the societal and social changes throughout west-Central Africa under colonial rule.

•Historical changes in migration and population size that attended the founding and rapid growth of colonial administrative and trading centers like Kinshasa likely enabled the region to become the epicenter of the HIV/AIDS pandemic.

.

•There was no site in these countries with a population exceeding 10,000 until after 1910.

•Kinshasa, Brazzaville, Bangui, and Yaoundé were founded 1881-99

•Kinshasa had 5000 inhabitants in 1908, and 49,000 in 1940.

•It then grew to 420,000 by 1961.

Implications 1

•First putative case of HIV disease.First putative case of HIV disease.

•Multiple, unlinked infected individuals in Kinshasa by 1960sMultiple, unlinked infected individuals in Kinshasa by 1960s

•Extensive genetic diversity in 1959/1960 Kinshasa Extensive genetic diversity in 1959/1960 Kinshasa (early (early hub); hub); nascent subtypesnascent subtypes

•Opens the door to HIV “paleovirology” and comparative Opens the door to HIV “paleovirology” and comparative evolutionary genomicsevolutionary genomics

Implications 2

-Direct study of pathogenic potential of HIV and possible Direct study of pathogenic potential of HIV and possible changes in virulence over timechanges in virulence over time

-Need for recovery of complete genomesNeed for recovery of complete genomes

-Vaccine-relevant genetic change or stasis (real old Vaccine-relevant genetic change or stasis (real old sequences in addition to computed estimates)sequences in addition to computed estimates)

•The use of a consensus or reconstructed ancestral sequence effectively cuts diversity in half

•If you could create one of these that was immunogenic, it should have broader range and last longer than field isolate

•Currently, computer-reconstructed consensus strain is in trials in monkeys

•We’re working to generate real ancestral sequences…

vaccinesvaccines

Documents

The emergence of pathogenic viruses: an evolutionary approach Michael Worobey Department of Ecology and Evolutionary Biology, University of Arizona August