Comparison of genetic sequences from donor and recipient viruses from linked
subtype A transmission pairs
Samantha McInally
Eric Hunter’s Laboratory
Emory University
Atlanta, GA
Global Distribution of HIV-1 subtypes
Adapted from McCutchan FE. J Med Virol 2006 and Arien, K.K. et al. 2007
Previous subtype A studies
• Female sex workers infected with nonsubtype A viruses were 8 times more likely to develop AIDS over the study period vs. those infected with subtype A (Kanki et al. 1999 Journal of Infectious Diseases)
• HIV-1 subtype A viruses replicate at a lower level compared to subtype D viruses (Baalwaet al. 2013 Virology)
• HIV-1 subtype A viruses were more virulent compared to subtype C viruses (Venner et al. 2016 EBioMedicine)
Differences between subtype A and C in IAVI Protocol C Acute Infection cohort
• 60% faster disease progression with subtype C vs. subtype A infections (Amornkul et al. 2013 AIDS)
• Higher post-counseling HIV-1 incidence in Zambia vs. Rwanda (Kamali et al. 2015 PLOS One)
• Higher proportion of non-progressors infected with subtype A vs subtype C (Price et al.
2017 PLOS Medicine submitted)
Comparison of subtype A and C viral loads
Prentice et al. 2014 Virology
Characteristics of Rwandan subtype A cohort and 10 transmission pairs
Rwandan subtype A cohort characteristics
Percentage of single and multi-variant infections in Rwandan cohort
25% Multi-variant transmission
75% Single-variant transmission
Percentage of subtypes and recombinants in Rwandan cohort
21 % AC recombinant 4 % CD recombinant
8% Subtype C 67% Subtype A
Ling Yue, Rui Xu, Giselle Umviligihozo, Erick Muok
Sample collection from transmission pairs
Epidemiologically linkedtransmission pair
Experimental parameters
• 10 subtype A infected transmission pairs
• Normalized for gender
• All single variant infections
• Exclude acutely infected donors
Donor Coded ID EDI
# of amplicons generated
(Y/N) Included in analysis
R880M 21 11 Y
R463M 21 11 Y
R53M 27 8 Y
R49F 26 12 Y
R65F 51 15 Y
R1135F 18 15 Y
R977M 17 17 Y
R269F 23 10 Y
R885M 32 7 Y
R3103F 13 11 Y
R3671M 27 16 N
Viral Kinetics of 10 linked recipients from the transmission pairs
0 3 6 9 12 15 18 21 24 27 300
2
4
6
8
TIme after Infection (months)
HIV
-1 V
iral L
oad
(L
og
10)
Viral Load 2 year kinetics
Subtype A
Subtype C
0 3 6 9 12 15 18 21 24 27 300
2
4
6
8
TIme after Infection (months)V
iral L
oad
(L
og
10)
Viral load 2 year individual kinetics
Subtype A
Subtype C
cDNA synthesis and limiting dilution genome-length PCR
Plasma sample near time of transmission
Plasma virus
Affinity purification of total RNA
Lyse cells, and spin to remove large particulates/cell debris
Apply lysate (containing nucleic acids and cellular contaminants)
to column with glass membrane
Wash with alcohol to remove contaminants; nucleic acids
stick to glass membrane while contaminants wash
through. Treat with DNase enzyme to remove
contaminating DNA.
Apply water to the column;
purified RNA washes off the
glass and is collected
RNA Extraction
Agarose Gel
DNA Sequencing9kb
Near-full length genome amplification
• Sequencing using Pacific Biosciences platform and MDseq (Dilernia et al. NAR 2015)
Bias for consensus-like selection in subtype C infections
Deymier, Ende et al 2015 PLOS Pathogens
Phylogenetic analysis of subtype A transmission pairs
Yue et al. PLOS Pathogens. 2015
Nucleotide sequence of TF is not closer to A1 consensus
R88
0
R26
9R53
R46
3R65
R49
R11
35
R97
7
R88
5
R31
03
Z331
Z3576
Z3618
Z3678
Z4248
Z4473
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
Pair
wis
e d
ista
nce t
o D
NA
co
nsen
su
s
(Patr
isti
c D
ista
nce)
Individual protein sequences are not closer to consensus
R88
0
R26
9R53
R46
3R65
R49
R11
35
R97
7
R88
5
R31
03
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Pair
wis
e D
ista
nce t
o A
A c
on
sen
su
s
(Patr
isti
c D
ista
nce)
Comparison of subtype A
gag sequences to A1 consensus
R88
0
R26
9R53
R46
3R65
R49
R11
35
R97
7
R88
5
R31
03
0.00
0.05
0.10
0.15
0.20
0.25
Pair
wis
e D
ista
nce t
o A
A c
on
sen
su
s
(Patr
isti
c D
ista
nce)
Comparison of subtype A
pol sequences to A1 consensus
R88
0
R26
9R53
R46
3R65
R49
R11
35
R97
7
R88
5
R31
03
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Pair
wis
e D
ista
nce t
o A
A c
on
sen
su
s
(Patr
isti
c D
ista
nce)
Comparison of subtype A
env sequences to A1 consensus
R88
0
R26
9R53
R46
3R65
R49
R11
35
R97
7
R88
5
R31
03
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Pair
wis
e D
ista
nce t
o A
A c
on
sen
su
s
(Patr
isti
c D
ista
nce)
Comparison of subtype A
nef sequences to A1 consensus
Summary• Amplified near full-length genomes for 10 subtype A
transmission pairs where the donors show significant evolution• Initial phylogenetic analyses suggests that subtype A TF viruses do not exhibit
a bias for consensus-like residues. However, this may reflect the small number of samples analyzed in the current study.
Future Directions• More detailed analysis of selection bias (Jonathan Carlson)
• Compare replicative capacity of subtype C and A IMCs
• Compare replicative capacity of subtype A TF and NT IMCs
AcknowledgementsEric Hunter, PhD
• Zach Ende, PhD
• Kelsie Brooks
• Sarah Connolly
• Elina El-Badry
• Ling Yue, MD
• Erick Muok, PhD
• Giselle Umviligihozo
• Dario Dilernia, PhD
• Daniela Monaco, PhD
• Evonne Woodson, PhD
• Danna Cunningham
• Daniel Wilkins
• Rui Xu
• Qianhong Qin
• Jon Allen
• Paul Farmer, PhD
• Effie Bartley
Rwanda-Zambia HIV
Research Group
(RZHRG)
• Study Participants
• Susan Allen, MD, MPH,
DTM&H
Funding
• R01 AI 51231
• R01 AI 64060
• IAVI