1. Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960
Paper by Michael Worobey
Presentation by Mary O’Connor

2. Goal of Paper
Define the time of most recent common ancestor (TMRCA) for the HIV-1 M group by conducting a “comparative evolutionary genetic study of the pre-AIDS epidemic HIV-1 group M viruses”
Illuminate the timescale of the HIV-1 virus’s evolution
Instrumental pre-AIDS sequences used:
DRC60 (dating from 1960, Kinshasa)
ZR59 (dating from 1959, Kinshasa)

3. Definition of Terms Pandemic Relaxed Molecular Clock (unconstrained)
BMCMC
95% Highest Probability Distribution (HPD)
Uncorrected Pairwise Distance
Blinded Assay
Reference Sequence
BEAST
Coalescent Tree Prior

4. Background: General Information
HIV = Human Immunodeficiency Virus
Member of the Lentivirus family (slowly replicating retroviruses associated with long-term illnesses)
Symptoms: Severe flu-like symptoms, contraction of various illnesses
Transmitted as single stranded RNA through bodily fluids

5. Background: General Information
Eventually causes AIDS and failure of immune system
34 million people infected with HIV by 2011
1.8 million people died of AIDS in 2010
68% of HIV cases and 66% of deaths occur in sub-Saharan Africa
Nearly 30 million people have died of AIDS since it was first recognized in 1981.

6. Background: Origins
AIDS first seen in US in 1981 when people started dying of rare diseases
Earliest documented case of HIV was in DRC in 1959
HIV-1 originated in chimpanzees in west Africa as SIVcpz
Transferred to humans in the early 20th century, likely through predation
SIV may mutate into HIV if it is rapidly transmitted from person to person. This gives it time to mutate before any one immune system can suppress it.

7. Background: Phylogeny

8. Background: Geography of HIV M Group Subtypes
This graphic describes the distribution of HIV-1 M group subtypes
throughout the world.

9. Discussion of Methods
Screened 27 Bouin’s-fixed paraffin-embedded tissue blocks from Kinshasa with PCR and reverse transcription and found one containing HIV-1 RNA (DRC60). Note: Researchers started with pre-sequences ZR59.
Viral sequence was extracted with special procedure specific for degraded sequences.
Fragments were amplified through PCR with primers that are known to correspond to sections of the gag, pol, and env gene regions.

10. Discussion of Methods
PCR products for ZR59, DRC60, and three modern control sequences were cloned and sequenced.
Blinded Assay conducted: A second lab successful identified and sequenced the same DRC60 gene regions with nearly the same results (1.4% uncorrected pairwise distance).

11. Discussion of Methods
Sequences were phylogenetically analyzed with an unconstrained BMCMC method (relaxed molecular clock).

12. Discussion of Methods
Validated time of origin of DRC60 and ZR59 by treating the origin date as unknown and calculating the rate of evolution based on the tree that had already been generated.
Uncorrected Pairwise
distance between
DRC60 and ZR59 in
env region calculated
to be 11.7% (Greater
than 99.2% of
within-subtype
comparisons)

13. Discussion of Methods
Estimated TMRCE of HIV-1 M group with BEAST using a relaxed molecular clock.

14. Estimation of TMRCA
Calibrated substitution rates at many data points to account for relaxed molecular clock
Inclusion of older sequences (DRC60 and ZR59) improved estimations for the TMRCA for M group

15. Best-fit model assumed a constant population size of the virus
Estimation of TMRCA
Best-fit model assumed a constant population size of the virus

16. Summary and Analysis of Results
Significant genetic difference between ZR59 and DRC60 proves that the “diversification of HIV-1 in west-central Africa occurred in the early twentieth century, long before the recognition of the AIDS pandemic”
HIV-1 group M experienced a long period of slow growth in first half of twentieth century.
Close timing of the growth of big cities such as Kinshasa with the TMRCA implies that cities helped the initial establishment and spread of HIV-1

17. Limitations of Study
Potentially damaged/ modified sequences from the Bouin’s fixative solution.
Lack of information before 1976.

18. Future Work
Are there differences in transmission, virulence, and treatment?
How quickly is HIV evolving and how do we prepare for the emergence of new subgroups or even new clades?
Should we be studying the SIV strains that got transmitted to humans? Why did certain ones carry over to humans?
Archival banks of Bouin’s-fixed paraffin embedded tissue specimens

19. References