2018 4th International Conference for Convergence in Technology (I2CT)
SDMIT Ujire, Mangalore, India. Oct 27-28, 2018
Analysis on Preservation Characteristics of Modular
Structure during HIV-1 Progression using Weighted
and Normalized Graphlet Frequency Distribution
Sourav Biswas
Machine Intelligence Unit Dept. of Computer Science
Indian Statistical Institute Aliah University
Kolkata, India Email: sumantababai86@gmail.com
Email: sourav8051@gmail.com
Abstract—In this paper, we have proposed a computational
framework to measure the preservation characteristics of modu-
lar structures between two biological networks. The preservation
characteristics of co-expressed gene modules are identified by
comparing the frequencies of few predefined small substructures
called graphlets in the coexpression networks of three HIV-
1 infection stages: acute, nonprogressor, and chronic. A novel
similarity measure has been proposed based on the frequencies
and significances of those graphlets occurring in the networks. A
widely used tool GtrieScanner is utilized to find the frequen-
cies and significance of those graphlets in networks. Results
confirm high similarity of topological properties between co-
expressed modules of acute and chronic stages than acute and
nonprogressor stages. Our method contributes to an important
understanding of preservation characteristics of the modular
organization in two different biological networks.
Keywords—HIV-1 infection stages, HIV-1 modules, Graphlets,
Normalized and weighted graphlet frequency distribution,
Preservation score
I. I NTRODUCTION
One vital task in HIV-1 research is to understand the pattern
changes of HIV-1 infection progression. The first stage of HIV-
1 infection is acute retroviral syndrome which induces the
production of virus in our body [1], [2]. In this stage, large
amount of viruses are being produced which diminishes CD4+
cell count rapidly. As an immediate response, the immune
system reduces the virus count to a moderate level, called
‘virus set point’, afterwards CD4+ count begins to increase.
The majority of HIV-1 infected individuals progresses to the
latent stage called ‘chronic’ stage, in which CD4+ cell count
begins to drop severely. This results progression of AIDS in
the human body which becomes vulnerable to opportunistic
infections. Few infected people remain clinically stable by
maintaining a high amount of CD4+ and CD8+ cell for
long time which is known as long term nonprogressor stage
[3], [4]. The transition from acute infection stage to other
latent stages can be dissected by examining the topological
pattern of the transcriptomal network for each infection stage.
System biological approach, based on microarray data, have
been widely used to elucidate the pattern of the transcriptome
across different stages of disease progression. Although there
978-1-5386-5232-9/18/$31.00 ©2018 IEEE
Sumanta Ray Sanghamitra Bandyopadhyay
Machine Intelligence Unit
Indian Statistical Institute
Kolkata, India
Email: sanghami@gmail.com
exists several studies that examine the preservation of modular
structure in different disease progression [5], [6], [7], to the
best of our knowledge, very few studies exist in the field
of HIV-1 infection stages. In [2], the three stages of HIV-
1 progression are analyzed based on coregulation pattern
of infected genes. In [8], the samples of three stages of
HIV-1 infection are distinguished based on differentially co-
expressed interacting protein pairs (DEPs). These works are
mainly focused on the detection of coexpressed modules and
differentially coexpressed protein pairs in three stages of HIV-
1 infection. In [9] a multi-objective modeling is proposed to
detect perturbation patterns of coexpressed modules across the
progression of HIV-1 infection. Later in [10] the author pro-
posed an eigengene based approach for analyzing microarray
gene expression data of HIV infected individuals and detect
preservation characteristics of coexpressed modules during
HIV infection progression.
In [11], the authors first proposed an idea to compare
two biological networks using frequency distribution of a set
of predefined graphlets. One of the major drawbacks of the
method is that it missed scalability. For two input networks of
different sizes, the frequency of a particular graphlet will be
higher for the network of larger size. So, it lacks a suitable
normalization technique which scales the metric uniformly for
networks of different sizes. Here, we have taken care of it by
using appropriate normalization technique. We have further
extended the idea by including ‘weight’ of each graphlet.
As all the predefined graphlets are not equally important, so
comparing the frequencies of all is not required. Rather it
is important to know which graphlets are more significant
over others in the network. To find significant graphlets of
a network we have utilized the concept of network motif.
Network motif is a small graphlet that occurs in a network
more frequently than similar random networks [12]. It is said
to be the building blocks of a network. The metric z-score
is utilized to determine whether the graphlet is statistically
significant or not. We have utilized the metric for determining
the significance of a graphlet in a network, and put it as a
weight. Finding frequencies and z-scores of all graphlets are
1
done using a motif finding tool called GtrieScanner[13]. matrices for the modules. We obtained a cut-off value of 0.8
for the acute and the chronic stage, while 0.6 cut-off was
choosen for the nonprogressor stage. These cut-off values are
Acute adopted by finding the knee of the graph in Fig. 1 which
0.8
describes the cut-off values and corresponding average density
0.6 of the network formed by the co-expression matrix. For the
0.4 remaining part of this article, we used ‘module’ to simply
represent the sub network constructed from the coexpression
0.2
module.
0
0.2 0.4 0.6 0.8 1
k-cutoff
Chronic
0.8

0.6

0.4

0.2

0
0.2 0.4 0.6 0.8 1
k-cutoff
Nonprogressor
0.4

0.3

0.2

0.1
Fig. 2. Fig. shows twenty nine graphlets which are used to compare two
0 networks
0.2 0.4 0.6 0.8 1
k-cutoff
B. Finding frequencies and z-scores of graphlets
GtrieScanner (http://www.dcc.fc.up.pt/gtries/)[13] is a net-
Fig. 1. correlation coefficient cutoff vs average network density plot work motif finding tool which is used to find the frequencies
and z-scores of all twenty nine graphlets for modules of
II. M ETHOD the three stages of HIV-1 infection. It finds frequencies of
graphlets in the original network and creates similar random
A. Data preprocessing networks for comparing the frequencies of those graphlets
We have downloaded the gene expression dataset (GSE6740 in original and those random networks. z-score is used to
series) from GEO database [14] which has expression values compare the significance of the frequency of a graphlet which
of CD4+ and CD8+ cells of the three HIV-1 infection stages is defined as: i i
of some untreated HIV-1 positive people. It consists of 22284 z-score= FG (SσR)−μ R (S )
(S i ) , where FG (S i ) is the frequency
genes and 10 samples at each progression stage, five samples of subgraph S in the original network. μR (S i ) and σR (S i )
i

are for CD4+ T cells and five samples are for CD8+ T
cells. For obtaining gene modules in three stages of HIV
progression we have utilizes PROCOMOSS [15] algorithm.
PROCOMOSS uses a multiobjective technique to cluster genes
based on the interaction and functional similarity between
gene pairs.For each stage of HIV infection, we have collected
interaction information of expressed genes and compiled func-
tional similarity matrix by using gene ontology data. These
two metrics are utilized by PROCOMOSS to cluster genes
into functional homogeneous modules. We have found 30
modules in the acute stage, 21 in the nonprogressor stage and
32 in the chronic stage. Now in each stage, a co-expression
matrix is formed using the microarray gene expression data
for each of the modules. A set of 30, 21 and 32 co-expression
matrices are built for the acute, nonprogressor and chronic
stage respectively. Using a cut-off value, these matrices are
turned into binary matrices and we called these as adjacency
represent average mean and standard deviation of the subgraph
S i in the similar random networks. Positive z-score of a
subgraph implies it is more abundant in the original network,
while a negative score signifies that it occurs fewer in original
than the normal. As both positive and negative z-score implies
significance of a graphlet, so we took the absolute value of it
and put this as a weight to a graphlet. Graphlets of a random
network tend to have z-scores near about zero.
C. Normalization of graphlet frequencies
As already mentioned, normalization was one of the key
motivations of this work. To deal with normalization we
introduced a concept called ‘reference network frequency’.
For a graphlet G and a network m, its ‘reference network
frequency’ or f req(Grefm ) is the maximum frequency of the
graphlet G in a network of the same order as m. It is the
number at which this graphlet G can occur in a network with

2
order similar to m. A reference network mref of a network
m can be formed by creating a fully connected network of
same node size as m. Normalization is done by dividing the
frequency of a graphlet in a network by its frequency in the
reference network. Mathematically, the normalized frequency
of a graphlet G in a network m can be defined as:
f req( Gm )
Fnorm (Gm ) = (1)
f req( Grefm )
Another important aspect is how to prioritize the graphlets
for a given network. For this, we have chosen z-score, which
determines the importance of the graphlets. Now to put
together the concept of normalized frequency and significance
of a graphlet with respect to a network, ‘normalized weighted
graphlet frequency’ can be formulated as below:
Fnw (Gm ) = Fnorm (Gm ) × |Zm (G)| (2)
which depicts the normalized and weighted frequency of a
graphlet G in a network m and |Zm (G)| is the absolute z-score
value of graphlet G in the network m.
D. Similarity between two modules using weighted graphlet
frequency distribution
Studying local structure of a network is an important
step to understand topological characteristics appeared in the
network. Comparing these, we can facilitate the understanding
of structural similarity among the networks.
To compare preservations among the three stages of HIV-
1 infection, first we need to find pairwise similarity between
all modules of the three stages. Here, we used the measure
for finding similarity between two graphs proposed in [11]
which was based on graphlet frequency distribution. The only
difference lies in the concept of normalizing and adding
some weights to the graphlets before comparing them. Given
two networks m and p, the structural similarity according to
normalized weighted graphlet frequency distribution can be
formulated as:
 within it. Hence they possess some kind of similarity among
k
1 Fnw (Gm
i ) Fnw (Gpi )
Sim(m, p) =  (3)
k i=1 Fnw (Gm
i ) Fnw (Gpi )
Fnw (Gm
i ) is the normalized and weighted frequency of
graphlet Gi in network m. K is the number of graphlets whose
normalized and weighted frequencies are being compared
here. For computational convenience, we have considered total
29 graphlets structures (Fig.2) having maximum 5-nodes. If
computation power permits one can go beyond size five.
The measure is commutative. It captures similarity between
two networks in all twenty nine weighted graphlet frequency
distribution. If the value is near about 1, it signifies the more
topological similarity exists between the two input networks.
It lies between zero to one.
3
E. Finding preservation score between two sets of modules
A similarity between two modules is given by the formula
described in the above section. To find preservation score
between two sets of modules we can use this formula re-
peatedly. The preservation score between two sets of modules
M = m1 , m2 , . . . mk P = p1 , p2 , . . . pn is defined as follows:
k
M axj∈n Sim(mi , pj )
i=1
P reserv(M, P ) = (4)
k
where the similarity between two modules mi and pj is
defined as in above section. If the value approaches one, it
means more similarity in the two groups of input networks
exist. Note that, it is not necessary for the two groups to
contain the same number of modules.
F. Preservation in random networks
To validate our method we have generated some random
networks by following the famous Erdős Rényi model [16].
In this random graph model, all graphs with a fixed set of
vertices and edges are equally likely. There are two variants
of the ErdsRnyi random graph model. In the G(n, M ) model,
a graph G is selected at random from an ensemble of all
possible graphs which have n nodes and M edges. For
example, in the G(3, 2) model, each of the three possible
graphs on three vertices and two edges are included with
probability 1/3. Another model G(n, p) consists of a graph
which is constructed by connecting nodes randomly. Edges
have probability p independent from every other edge.
We have used an R package called “igraph”
(http://igraph.org/r/)[17] to generate a set of random graphs
using G(n, p) variant of ErdősRényi model. Total five sets of
modules are created where each set consists of ten random
networks with different n, p parameter. Random node size n
(from 20 to 70) and random p (from 0.3 to 0.6) are taken for
G(n, p) model. In table I, preservation scores between all sets
of random networks are shown. All diagonal entries are found
to be ones due to self-similarity. For rest of the entries, the
score is roughly around 0.5. This is due the fact that, all the
networks are generated using same random graph generation
model and each set contain the same number(ten) of networks
themselves which justifies this low similarity values.
III. R ESULT
A. Comparing individual modules in HIV-1
For comparing two modules, the first step was to find the
normalized frequency of all 29 graphlets in those modules.
As already mentioned earlier we have used gtrieScanner
tool [13] to find the frequencies of the graphlets. To create
a visualization, we have sorted all modules in each stage
according to their mean graphlet frequency and took the top
five modules from each stage. We have plotted normalized
frequencies of top five graphlets of these modules in Fig. 3.
Ten graphlets are observed in the acute stage, out of which
three graphlets are of size 4 (g11, g12, g9) and the rest are
 TABLE I
P RESERVATION S CORES AMONG FIVE SETS OF RANDOM NETWORKS

random random random random random

set 1 set 2 set 3 set 4 set 5 0.6

random graphlet
1.0000 0.4470 0.4440 0.4600 0.4370 g1
set 1 g11

normalized_frequency
random g12

0.4140 1.0000 0.4180 0.4460 0.4210 0.4 g2

set 2

Acute
g3
g4
random
0.4410 0.4250 1.0000 0.4400 0.4350 g5

set 3 g6
g8
random 0.2 g9
0.4520 0.4550 0.4260 1.0000 0.4190
set 4
random
0.4570 0.4250 0.4490 0.4000 1.0000
set 5
0.0

Module1 Module2 Module3 Module4 Module5

module

of size 3. Similarly, the nonprogressor and the chronic stages

0.6
have seven and eight graphlets only. This result suggests
only some structures/graphlets are playing important roles
and most of them are of size 3. Please note that others graphlet
g1

normalized_frequency
graphlets are also present in all modules but these graphlets 0.4 g2
g3

Chronic
are occurring in high frequency. g4
g5
g6
g8

Next step is to calculate weights of the graphlets in each 0.2 g9

module. Pairwise similarity scores among all modules in the

three stages are computed using the equation (3). In Fig. 4,
top five pairwise similarity scores among the three stages are 0.0

Module1 Module2 Module3 Module4 Module5

shown using a point plot. In the first subplot of the Fig. module

4, acute and chronic modules share high sim scores. For 0.03

acute and nonprogressor stages, individual similarity scores

are substantially less compared to the other cases. From the
last subplot of Fig.4, it is found nonprogressor and chronic graphlet
0.02

modules do have both high (about 0.5) and very low (around
normalized_frequency

g1
g2

Nonprogressor
0.05) sim scores. Fig. 5 shows three boxplots of all pairwise g4
g5

similarity scores between modules of acute-chronic, acute- g6

g8

nonprogressor and nonprogressor-chronic stages, respectively. 0.01 g9

By analyzing this distribution we can get an intuitive idea that,

acute-chronic stages are more similar than the other two cases.
B. Preservation scores among three stages of HIV-1 pro- 0.00

Module1 Module2 Module3 Module4 Module5

gression module

We have found preservation score 0.3361 for acute and

nonprogressor modules whereas for acute and chronic modules
it is 0.5582. It signifies the more topological similarities
between acute and chronic stages than acute and nonprogressor
stages. We have also calculated preservation score between
nonprogressor and chronic stages, which accounts to 0.3678.
Both acute and chronic stages are fatal as HIV-1 infection
progresses rapidly in these two stages. It is only during
nonprogressor stage, where the infection is low and people can
survive by maintaining a sufficient number of CD4+ cells.
C ONCLUSIONS
In this paper, we have proposed a novel framework to
compare two different sets of biological networks based on
graphlet frequency distribution. We have incorporated weight
to each graphlet and normalized it before comparing its
frequency. We have carried out a comprehensive analysis
of three sets of coexpression networks formed by the gene
Fig. 3. Showing normalized frequencies of top five graphlets of five modules
in each of the stages.(Note the different y-axis scaling for nonprogressor stage)
modules of the HIV-1 infection stages. By comparing the
individual modules in the three stages, it appears that acute and
chronic have more similar pattern than acute-nonprogressor or
nonprogressor-chronic. One of the proposed measure, which
combines the information of pairwise similarities between the
two sets of networks confirms this. This similarity reflects the
actual medical conditions of people suffering from HIV-1 in
real life. Hence it can be concluded that, topological properties
of biological networks surely reflect their functionality. In this
aspect, our method can be further utilized in any two networks

4
 0.6
chronic_modules
0.6
7 23
17 25 nonprogressor_modules
6 16 21
8 31 1
3 4 2
0.5 5 14 0.4 3
Sim_score

Sim_score
27 12 17
9 24 19
10 26 5
13 30 8
2 15 4
0.4
11 32 6
0.2
29 20 9

21 18 18

28 1
22
0.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
acute_modules acute_modules

0.5

chronic_modules
31

0.4 12
30
14
Sim_score

24
26
0.3
18
15
19
4
0.2 25

0.1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
nonprogressor_modules

Fig. 4. Displaying pairwise similarity scores between top five modules in each of the acute-chronic, acute-nonprogressor and nonprogressor-chronic stages

or two sets of networks. [6] Miller, J., Horvath, S., Geschwind, D.: Divergence of human and mouse
brain transcriptome highlights alzheimer disease pathways. Proc Natl
ACKNOWLEDGEMENTS Acad Sci U S A 107, 12698–12703 (2010)
[7] Oldham, M., Horvath, S., Geschwind, H.: Conservation and evolution
This publication is an outcome of the R&D work undertaken of gene coexpression networks in human and chimpanzee brains. Proc
Natl Acad Sci U S A 103, 17973–17978 (2006)
project under the Visvesvaraya PhD Scheme of Ministry of
[8] Yoon, D., Kim, H., Suh-Kim, H., Park, R., Lee, K.: Differentially co-
Electronics & Information Technology, Government of India, expressed interacting protein pairs discriminate samples under distinct
being implemented by Digital India Corporation stages of hiv-1 infection. BMC Systems Biology 5(Suppl 2)S1(DOI:
10.1186/1752-0509-5-S2-S1) (2011)
R EFERENCES [9] Ray, S., Biswas, S., Mukhopadhyay, A., Bandyopadhyay, S.: Detecting
perturbation in co-expression modules associated with different stages of
[1] Bandyopadhyay, S., Ray, S., Mukhopadhyay, A., Maulik, U.: A review of hiv-1 progression: A multi-objective evolutionary approach. In: Proceed-
in silico approaches for analysis and prediction of hiv-1-human protein- ings of the 2014 Fourth International Conference of Emerging Applica-
protein interactions. Briefings in Bioinformatics 16(5), 830–851 (2015) tions of Information Technology. EAIT ’14, pp. 15–20. IEEE Computer
[2] Ray, S., Bandyopadhyay, S.: Discovering condition specific topological Society, Washington, DC, USA (2014). doi:10.1109/EAIT.2014.34.
pattern changes in coexpression network: an application to hiv1 pro- http://dx.doi.org/10.1109/EAIT.2014.34
gression. IEEE/ACM Trans Comput Biol Bioinform. 16(6), 1086–1099 [10] Ray, S., Hossain, M., Khatun, L.: Discovering preservation pattern from
(2016) co-expression modules in progression of hiv-1 disease: An eigengene
[3] Zeller, J., McCain, N., Swanson, B.: Immunological and virological based approach. In: Proceedings of the IEEE, 2016 International Con-
markers of hiv-disease progression. J Assoc Nurses AIDS Care 7(15-27) ference on Advances in Computing, Communications and Informatics
(1996) (ICACCI), pp. 10–110920167732146. IEEE Computer Society, ???
[4] Buchbinder, S., Katz, M., Hessol, N., O’Malley, P., Holmberg, S.: Long- (2016)
term hiv-1 infection without immunologic progression. AIDS 8, 1123– [11] Bhattacharjee, D., Hossain, S.M.M., Sultana, R., Ray, S.: Topological
1128 (1994) inquisition into the ppi networks associated with human diseases through
[5] Cai, C., Langfelder, P., Fuller, T., Oldham, M., et al.,, R.L.: Is human graphlet frequency distribution. In: International Conference on Pattern
blood a good surrogate for brain tissue in transcriptional studies? BMC Recognition and Machine Intelligence, pp. 431–437 (2017). Springer
Genomics 11(589), 1471–2164 (2010) [12] Milo, R., Shen-Orr, S., Itzkovitz, S., Kashtan, N., Chklovskii, D.,

5
 0.6

Sim_scores with nonprogressor modules

0.6
Sim_scores with chronic modules

0.4
0.4

0.2
0.2

0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
acute_modules acute_modules

0.5
Sim_scores with chronic modules

0.4

0.3

0.2

0.1

0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
nonprogressor_modules

Fig. 5. Three box-plots showing pairwise similarity scores between all modules in each of the acute-chronic, acute-nonprogressor and nonprogressor-chronic
stages

Alon, U.: Network motifs: simple building blocks of complex networks.

Science 298(5594), 824–827 (2002)
[13] Ribeiro, P., Silva, F.: G-tries: an efficient data structure for discovering
network motifs. In: Proceedings of the 2010 ACM Symposium on
Applied Computing, pp. 1559–1566 (2010). ACM
[14] Hyrcza, M.D., Kovacs, C., Loutfy, M., Halpenny, R., Heisler, L., Yang,
S., Wilkins, O., Ostrowski, M., Der, S.D.: Distinct transcriptional profiles
in ex vivo cd4+ and cd8+ t cells are established early in human
immunodeficiency virus type 1 infection and are characterized by a
chronic interferon response as well as extensive transcriptional changes
in cd8+ t cells. Journal of virology 81(7), 3477–3486 (2007)
[15] Mukhopadhyay, A., Ray, S., De, M.: Detecting protein complexes in
a ppi network: a gene ontology based multi-objective evolutionary
approach. Molecular BioSystems 8(11), 3036–3048 (2012)
[16] Erdos, P.: On random graphs. Publicationes mathematicae 6, 290–297
(1959)
[17] Csardi, G., Nepusz, T.: The igraph software package for complex
network research. InterJournal Complex Systems, 1695 (2006)