COLLEGE OF MEDICINE

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Nov 8, 2013 (3 years and 9 months ago)

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PATRI CI A K PENTON

J ASON T
BLACKARD

UNI VERSI TY OF CI NCI NNATI

COLLEGE OF MEDI CI NE

DI VI SI ON OF DI GESTI VE DI SEASES


Background & Rationale


HIV generates sequence diversity via…


Poor proofreading capability of reverse transcriptase


Rapid viral turnover


Large number of infected cells


Recombination


Unique intra
-
host microenvironments







Lamers
, et. al. 2009

Background & Rationale


Previous documentation of HIV compartmentalization/quasispecies


Brain, CSF, and Testes


Used postmortem organs from patients who died of AIDS related causes


Patient HIV
Ams
198


Brain/CSF quasispecies were highly homogeneous


May be explained by the fact that HIV
-
1 lacks the capacity to replicate in
these compartments


There may be slower turnover of virus/infected cells in the brain


Liver quasispecies clustered closely together


All liver sequences were generated from only one positive PCR, creating
low proviral copy numbers


No distinct clustering was observed for any other organ tested



Van’t

Wout
, et. al. 1998


Van’t

Wout
, et. al. 1998

Background & Rationale


Chronic liver disease is a major cause of morbidity and mortality in
HIV
-
infected individuals.


A direct correlation between HIV RNA levels and liver fibrosis has been
reported


HIV patients exhibit an increase in liver enzyme levels


Suggests that HIV plays a role in liver disease progression


HIV/HCV
coinfection

is associated with enhanced HCV replication


Evidence for HIV interaction with other
hepatotropic

viruses


HIV can infect hepatocyte
-
derived cell lines and hepatocytes


Liver
-
specific HIV quasispecies have not been extensively explored





Blackard
, et. al. 2011

Methods


Mantel Test


Statistical test for correlation between two matrices


Common for ecology/
PopGen


Used to measure the genetic distance between pairs of compartments


The correlation and a
p
-
value are calculated


VESPA
-

Viral Epidemiology Signature Pattern Analysis


Detects specific AA or NT differences between two sets of sequences


Calculates frequency of each AA at each position in a set of aligned
sequences


Looks for a “signature” pattern


AAs that are conserved within one set but differing between the two sets


Threshold of 0.8 requires an AA to be present 80% of the time in the liver
sequences to be considered conserved



Methods


Bootstrap Neighbor
-
Joining Phylogenetic Trees


Data Clustering: assign a set of observations into subsets so they are
similar


The NJ approach applies data clustering techniques to sequences
analysis, where genetic distance is the observation


Bootstrapping is a test of phylogeny


If a bootstrap value for an interior branch is 70% or higher, then the
topology at that branch is considered accurate


Results

Table 1.
Mantel Test results from

the
V3 region f
or intrapatient paired
comparisons involving the liver.

Compartment

P
-
Value

Bone Marrow

0.0002

Brain

0.0001

CSF

0.0001

Kidney

0.0014

Lung

0.0001

Lymph Node

0.0002

PBMC

0.0050

Spleen

0.0017

Sequence data from
Van’t

Wout
, et. al. 1998 J
Virol

72(1):488.

Table

2.
Mantel Test results (p
-
values) for
HIV
-
1 gp120 viral variants from
intrapatient paired comparisons involving
the liver. Patients

are identified with a two
letter code (AM, AZ, DY).

Compartment

AM

AZ

DY

BslGngla

-

-

0.0001

Diaphragm

0.0062

-

-

FrntCrtx

-

0.0001

-

FtLobeGM

-

-

0.0001

FtLobeWM

-

-

0.0001

Kidney

0.0001

-

-

LmphNde

-

0.0001

0.0002

LmphNdeL

0.0001

-

-

LmphNdeR

0.7105

-

-

LvrTmr1

0.0001

-

-

LvrTmr2

0.0001

-

-

Meninges

-

0.0001

0.0001

Prostate

0.6841

-

-

Spleen

0.0071

0.0012

0.0001

StmchLma

0.0001

-

-

TmplCrtx

-

-

0.0001

Table

3.
Mantel Test results (p
-
values) for
HIV
-
1
nef

viral variants from intrapatient
paired comparisons involving the liver.
Patients

are identified with a two letter code
(AM, AZ, DY).

Compartment

AM

AZ

DY

BslGngla

-

-

0.0002

Diaphragm

0.0001

-

-

FrntCrtx

-

0.0001

-

FtLobeGM

-

-

0.0002

FtLobeWM

-

-

0.0001

Kidney

0.0091

-

-

LmphNde

-

0.0001

0.5057

LmphNdeL

0.0108

-

-

LmphNdeR

0.1416

-

-

LvrTmr1

0.0027

-

-

LvrTmr2

0.0009

-

-

Meninges

-

0.0001

0.001

Prostate

0.0011

-

-

Spleen

0.0714

0.0042

0.0046

StmchLma

0.0800

-

-

TmplCrtx

-

-

0.0001

Sequence data from
Lamers
, et. al. 2009
PLoS

ONE 4(3):e5065.

Table
4.
Intrapatient
mutations
in HIV
-
1 V3 region of various
tissues

and organs
compared
to the liver.
CSF= Cerebral Spinal Fluid, PBMC= Primary Blood
Mononuclear Cells.

Sequence data from
Van’t

Wout
, et. al. 1998 J
Virol

72(1):488.

Liver signature

.T...............K...T..E...............N.........I.....

Bone Marrow signature

.I...............R...A..D...............D.........T.....

Threshold signature

.T...............K...T..E...............N.........I.....

Liver signature

.............I.......T..E...N.........G...N...K..VI...E.

Brain signature

.............M.......A..D...D.........R...T...Q..AG...A.

Threshold signature

.............I.......T..E...N.........G...N...K..VI...E.

Liver signature

.............I.......T..E...N.........G...N...K..VI...E.

CSF signature

.............M.......A..D...D.........R...T...Q..AG...A.

Threshold signature

.............I.......T..E...N.........G...N...K..VI...E.

Liver signature

.T...............K...T..................N.........I.....

Kidney signature

.V...............R...A..................D.........T.....

Threshold signature

.T...............K...T..................N.........I.....

Liver signature

.................K...T..E...N.........G.......K...I.....

Lung signature

.................R...A..D...D.........R.......Y...E.....

Threshold signature

.................K...T..E...N.........G.......K...I.....

Liver signature

.T...............K...T................G.N..N..K...I.....

Lymph Node signature

.V...............R...A................R.D..S..E...T.....

Threshold signature

.T...............K...T................G.N..N..K...I.....

Liver signature

.T...............K...T................G.N........VI.....

PBMC signature

.I...............R...A................R.D........AT.....

Threshold signature

.T...............K...T................G.N........VI.....

Liver signature

.T...............K....................G.N.........I.....

Spleen signature

.V...............R....................R.D.........T.....

Threshold signature

.T...............K....................G.N.........I.....

Threonine


Isoleucine


0.002
0.001
0.001
0.001
Liver & Diaphragm (AM)

Liver & Kidney (AM)

Liver & Left

Lymph Node (AM)

Liver & Right

Lymph Node (AM)

802

1000

761

997

996

1000

768

873

998

809

1000

984

952

700

Figure 1.
Intrapatient neighbor
-
joining phylogenetic trees with
nef

viral variants from the liver
(open circles) and a different tissue or organ (closed circles). Patients are identified parenthetically
with a two letter code (AM, AZ, DY). Shown in the lower left corner is a bar depicting the percent
genetic distance for each tree. Only relevant bootstrap values greater than 700 out of 1000 are
shown.
Sequence data from
Lamers
, et. al. 2009
PLoS

ONE 4(3):e5065.

0.01
Lymph Node

Spleen

Liver

Frontal Cortex

Meninges

819

Figure 2.
Intrapatient phylogenetic tree showing
gp120 variants from patient AZ. Relevant bootstrap
values >700 are shown.
Sequence data from
Lamers
,
et. al.
2009
PLoS

ONE
4(3):e5065.

Conclusions


Open Discussion