Transcript No Slide Title

Origins, Evolution, and Dissemination of Subtype C HIV-1 in Zimbabwe:
Bayesian and Phylogenetic Analysis
Sudeb Dalai, Seble Kassaye, Tulio de Oliveira, Gordon Harkins, Jennifer Lint, Elizabeth Johnston, and David Katzenstein.
Stanford University School of Medicine, Stanford, CA, USA; and South African National Bioinformatics Institute,
University of the Western Cape, Cape Town, South Africa.
Contact Information: [email protected]
I. Background and Objectives
HIV-1 subtype C now accounts for approximately 50% of the estimated 33 million people living with HIV/AIDS and half of the 1-2
million new infections annually. The predominance of a single clade of HIV-1 in the most severely affected sub-Saharan countries
has been ascribed to a founder effect, while the rapid heterosexual transmission and dissemination of subtype C has been attributed
to sexual-social factors, the frequency of concurrent partnerships, increased virus load, and shedding of virus alongside sexually
transmitted infections. Comparisons with subtype B virus isolates have demonstrated subtype C viruses have enhanced tropism for
macrophages and dendritic cells, increased viral replication rates through transcriptional regulation, and most recently, a higher rate
of mutation and drug resistance among women receiving single-dose nevirapine.
1991 N=39
1998 N=56
2001 N=27
2006 N=90
A high rate of infection is documented in young, antenatal women, where infection rates between ages 16-24 are 2-5% per year. HIV
testing of pregnant women for pMTCT programs provides a consistent population from which recently transmitted viruses can be
identified and the evolution of new subtypes, recombinants, and temporal changes in subtype C, can be characterized.
Routine sequencing of pol genes of circulating virus, as a surveillance tool for drug resistance, has increasingly been used for
evolutionary and phylogenetic mapping, and to explore the origins, molecular epidemiology, and genetic diversity of different HIV-1
subtypes. We analyzed subtype C pol sequence data over time from successive cohorts of women screening for HIV infection in
antenatal clinics in Zimbabwe. Evaluation of genetic diversity and phylogenetic relationships in these cohorts enabled formulation
of a Bayesian molecular clock, which provided information on the origins, timing, and epidemic growth patterns of the subtype C
epidemic in southern Africa.
Figure 1. Maximum-likelihood tree of 178 ZW subtype C sequences, demonstrating
temporal clustering and increasing divergence over the 15-yr epidemic period.
II. Methods
•We sequenced HIV-1 pol from samples obtained from 4
sequential cohorts of pregnant, HIV-positive women in Harare,
Zimbabwe presenting to antenatal clinics from 1991-2006.
•Maximum-likelihood phylogenetic trees were constructed with
PhyML v2.4.4.
•Ancestral sequences were reconstructed with HyPhy v1.0.
•Sequence divergence from ancestral sequences was used to
derive a nucleotide evolutionary rate, which we used to calibrate
a Bayesian Markov chain Monte Carlo analysis (BEAST v1.4)
under several different models of population growth. BEAST
software implementation was used to reconstruct most recent
common ancestor (MRCA) sequences, time the introduction of
infection in this region, and estimate a time-resolved phylogeny
and viral dynamics of the epidemic.
Figure 2. Time-resolved phylogenies constructed with Bayesian MCMC. Across various population growth models, rapid epidemic expansion is seen during 1979-1981 with
multiple clusters of introduction. Percent lineage calculations demonstrate most of current genetic diversity was introduced during 1980-1985.
•A number of other concurrent southern African subtype C
sequences were added to the dataset, and the Slatkin-Maddison
statistical test for gene flow (implemented in MacClade v4.0)
was used to test the hypothesis of compartmentalization vs.
migration of subtype C HIV-1 among southern African countries.
III. Results
•Pol sequences obtained from 212 women (39 in 1991; 56 in
1998; 27 in 2000; 90 in 2006) demonstrated a significant increase
in sequence diversity over the 16-year period, assessed by genetic
distance (p<0.0001, one-way ANOVA).
•Sequences demonstrated clustering by sampling year, with the
recent 2006 sequences most divergent from the ancestral node
(Figure 1).
Figure 3. Bayesian MCMC estimates of MRCA are consistent with a founder virus introduced in ZW ~1973-75, across various population models.
Figure 4. Skyline plot analysis indicates multi-phase epidemic patterns (lag, explosive, linear) for ZW subtype C, with rapid expansion in the early 1980’s.
•BEAST analysis calibrated a molecular clock evolutionary rate at
2-3x10-3 nucleotide subsitutions / site / year, across several
different models of population growth.
IV. Summary and
Conclusions
•The Zimbabwean HIV epidemic likely
originated from multiple introductions of
subtype C virus in the late 1970’s and early
1980’s. Historically, this corresponds to a
change in political boundaries (Zimbabwean
Independence) and rapid population influx
from neighboring countries.
•The estimated date of the MRCA was 1973 across all population
growth models (Figure 3), with clear evidence for multiple
introductions of subtype C HIV-1 from neighboring countries
during 1979-1981 (Figures 2 & 4).
•Lineage calculations at various timepoints (as a percentage of
current lineages) indicated that most lineage diversity was
introduced during 1980-1985 (Figure 2).
A
B
•Zimbabwean subtype C sequences clustered most closely with
sequences from neighboring African nations, and were more
divergent from subtype C sequences isolated from other regions
of Africa or the world.
Before 1998 n=563
After 1998 n=483
•Bayesian Skyline analysis implemented in BEAST (Figure 4)
demonstrated three epidemic growth phases: an initial, slow phase
seeded HIV-1 in the 1970’s, followed by exponential growth in
the 1980’s and a linear expanding epidemic to the present.
•Slatkin-Maddison tests of compartmentalization indicated
migratory patterns consistent with flow of HIV-1 between
Zimbabwe and South Africa, Zambia, and Botswana (before
1998) and additional migratory patterns between Zimbabwe and
Malawi/Mozambique (after 1998) (Figures 5A-D).
Acknowledgements
Sudeb Dalai is supported by the Howard Hughes Medical Institute Research Fellowship
and the Paul and Daisy Soros Fellowship for New Americans.
Before 1998
C
After 1998
D
•The timing, phylogenetic clustering, and
genetic diversity of Zimbabwean subtype C
sequences is consistent with an origin in
southern Africa, followed by rapid expansion
as modeled by Bayesian MCMC sampling of
trees.
•Characterizing the origins of subtype C HIV1 in southern Africa, its molecular evolution
in a changing landscape of host, virus, and
ARV pressures, and epidemic patterns in atrisk populations, are critical in guiding
development of the next generation of drugs,
vaccines, and prevention strategies. Further
studies should elucidate the complex selection
factors driving viral evolution and diversity.
Figure 5. Phylogeographic analysis (Slatkin-Maddison method), showing the frequency of gene flow (migrations) to/from various southern African nations. The size of
each circle is proportional to the percentage of observed migrations. A. Before 1998, ZW migrations mainly involve South Africa, Zambia, and Botswana. B. After 1998,
ZW migration expanded to include Mozambique and Malawi. C & D. Geographic representations of observed migrations. Size of arrow represents percent migration.