The emergence of pathogenic viruses: an evolutionary approach

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The emergence of pathogenic viruses an
evolutionary approach

• Michael Worobey
• Department of Ecology and Evolutionary Biology,
  University of Arizona
• August 25, 2008

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Questions

• (1) Where did HIV come from?
• (2) When, where, and how did HIV-1 first emerge
  from sub-Saharan Africa?
• (3) When and how did the pandemic AIDS virus jump
  to humans and begin its spread?
• (4) What factors mediate the emergence of new flu
  lineages?

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1 The origin of HIV
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Where did AIDS come from?
HIV/AIDS basics

• First identified in US gay males in the early
  1980s, severe immunosuppression
• Pneumocystis carinii pneumonia
• Other rare opportunistic infections, horrendous
  suffering and death

Randy Shilts
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 OnAIDS The First
Five Years (1980-1985), he took almost everyone
to task on how the first years of the epidemic
were handled
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HIV/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

Françoise Barre-Sinoussi
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HIV/AIDS basics

• 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 cells 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)

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HIV/AIDS basics
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Evolution in the fast lane
HIV/AIDS basics

• 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

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Current status Disaster
HIV/AIDS basics
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Origins of HIV/AIDS

• The past
• Where did HIV/AIDS come from?

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Where did HIV come from?
Origins of HIV/AIDS

• Divine retribution
• Doesnt matter--it doesnt 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?
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Where did HIV come from?
Origins of HIV/AIDS

• Divine retribution
• 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

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Where did HIV come from?
Origins of HIV/AIDS

• Divine retribution
• 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

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Where did HIV come from?
Origins of HIV/AIDS

• Doesnt matter--it doesnt cause AIDS

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Where did HIV come from?
Origins of HIV/AIDS

• Doesnt matter--it doesnt cause AIDS

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Origins of HIV/AIDS

• Divine retribution
• Doesnt matter--it doesnt cause AIDS
• Conspiracy theories - e.g. the CIA did it
• Ritualistic use of monkey blood
• Zoonosis (a disease communicable from animals to
  man under natural conditions)
• Contamination of vaccines
• 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

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HIV/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

Françoise Barre-Sinoussi
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Where did HIV come from?
Origins of HIV/AIDS

• Divine retribution
• Doesnt matter--it doesnt 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?
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• Key discovery SIVs are found naturally in
  African primates

Cercocebus atys
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Origins of HIV/AIDS

• Divine retribution
• Doesnt matter--it doesnt cause AIDS
• Conspiracy theories - e.g. the CIA did it
• Ritualistic use of monkey blood
• Zoonosis (a disease communicable from animals to
  man under natural conditions)
• Contamination of vaccines
• 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

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Origins of HIV/AIDS
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Origins of HIV/AIDS
A direct test non-invasive sampling of SIVcpz
from the supposed source (and a big blank space
on the map of SIVcpz distribution)
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Origins of HIV/AIDS
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Origins of HIV/AIDS
Phylogenetic position Expected for source
population
Phylogenetic position of Kisangani SIV
Worobey et al. 2004
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Extensive non-invasive sampling continues

• gt1500 samples collected from across DRC
• 212 SIVcpz Ab positive
• 22 SIVcpz RNA positive
• Worobey, Hahn, Li, Ndjango, unpublished results

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2 The Worldwide Emergence of HIV-1
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HIV-1 group M Diversity
B RF
B LAI
Subtypes A-K 30 amino acid
divergence in Env between subtypes
Utility Tracing the global
pandemic Documenting
recombination
D 94UG114
D ELI
subtypes
A 92UG037
A U455
G SE6165
G 92NG083
J SE9280
J SE9173
C ETH2220
C 92BR025
H VI991
H 90CF056
K MP535
K EQTB11
F1 VI850
F1 93BR020
F2 MP255
F2 MP257
SIVcpz/US
SIVcpz/Cam5
N YBF30
N YBF106
SIVcpz/Gab1
O ANT70
O MVP1580
SIVcpz/Ant
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Molecular Epidemiology of HIV-1 group M
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Revisiting an old hypothesis
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Questions
Did HIV-1 move from Haiti to US, or US to
Haiti? When and how did these events take
place?
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Approach

• Archival Haitian-linked samples, Pitchenik et
  al, AIM, 1983
• Some of the earliest known AIDS patients in the
  US
• (Haitian immigrants to the US 1970s/80s)
• Full-length env alignment of published B and D
  subtype sequences (117 B plus 5 D)
• Bayesian MCMC approach, MrBayes and BEAST

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Phylogenetic patterns under different scenarios
US first
Haiti first
Simultaneous
Unknowable
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Results the Emergence of HIV-1 in the Americas
Posterior probability of Haitian origin 0.999
n 13
Posterior probability of Trinidad Tobago clade
1.0
n 96
Posterior probability of pandemic clade 1.0
Gilbert et al. 2007, PNAS
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Estimating rates of viral evolution
Average 5 x 10-3/site/year divergence rate 1
/site/year

Using the molecular clock Calibrate the
clock Correct for multiple hits Account for
methodological bias
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The Emergence of HIV-1/AIDS in the Americas when?
Posterior probability of Haitian origin 0.998
1969 1966-72
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Conclusions
Its not a sampling artifact the B epidemic is
older in Haiti, gt40 years Timing fits well with
large movement of people between Haiti and DR
Congo after independence in early 1960s One such
individual may have been the first to bring HIV
out of sub-Saharan Africa
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Conclusions

• Strong support for a single-patient introduction
  of pandemic clade from Haiti
• In or around 1969
• (long cryptic period in US)
• Ecological, not evolutionary factors determined
  success
• Do non-pandemic clade viruses have distinct
  immunological properties? (Mascola et al, JID,
  1994)
• Why so few successful epidemic introductions
  after the pandemic clade in or around 1969?

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3 When and how did HIV jump into humans and
begin its initial spread?
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Molecular archeology of HIV motivation

• archival sequences can provide direct tests of
  evolutionary hypotheses
• 1918 Spanish Flu virus has been resurrected and
  used to investigate emergence, pathogenesis and
  other questions.
• For HIV relevant frozen samples are rare and
  already screened (one from 1959, then 1976)

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Nature, 1998
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Science, 2000
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• Ambient temperature specimens like blood smears
  and paraffin-embedded tissue are not so rare

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• 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

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HIV DNA/RNA can be recovered from ancient
paraffin-embedded samples
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• 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.

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• 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.

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A2
A/A1
Kinshasa, 1960
B


D
Kinshasa, 1959
F1
F2
C
J
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• 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 Bouins-fixed specimen (B2M
  quantitative RT-PCR)
• Tissue of origin makes sense
• Control sequences well behaved

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• 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.

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.

• 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.

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Implications 1

• First putative case of HIV disease.
• Multiple, unlinked infected individuals in
  Kinshasa by 1960s
• Extensive genetic diversity in 1959/1960 Kinshasa
  (early hub) nascent subtypes
• Opens the door to HIV paleovirology and
  comparative evolutionary genomics

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Implications 2

• Direct study of pathogenic potential of HIV and
  possible changes in virulence over time
• Need for recovery of complete genomes
• Vaccine-relevant genetic change or stasis (real
  old sequences in addition to computed estimates)

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vaccines

• 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
• Were working to generate real ancestral
  sequences