Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(4): 680-688 (2017)

Genome-wide comparative analysis of the codon usage

pattern in

Flaviviridae

family

Anjusha Mune

, Ajay Pandey

and Khushhali Menaria Pandey

Department of Biological Science and Engineering, MANIT Bhopal (M.P.) India

Department of Applied Mechanics, MANIT Bhopal (M.P.) India

ABSTRACT

Flaviviridae family is a group of viruses that cause several deadly diseases like Dengue Fever Virus, Zika Virus, Japa-

nese Encephalitis Virus, and Hepatitis C Virus etc. The codon usage analysis can serve as a tool to understand about

the molecular evolution and regulation of viral gene expression. The objective of this study is to  nd the key deter-

minants of codon usage in the family. In this study, the codon usage pattern for 114 genomes of Flaviviridae family

(with four genus Flavivirus, Hepacivirus, Pegivirus, Pestivirus) was analysed through codon usage indices (like NC,

RSCU, ENC, PCA) and multivariate statistical methods. Our results show that among the four genus Flavivirus and

Pestivirus show similarity in preferred base on the count of being AG rich. On the other hand, Pegivirus and Hepacivi-

rus show similarity in preferred base on the count of being GC rich. The overall codon usage bias in the entire family

is slightly biased. RSCU analysis showed that Flavivirus and Pestivirus prefer AG ending codons, whereas Pegivirus

and Hepacivirus show preference to GC ending codons. Many unclassi ed members show similarity with members

of genus Flavivirus in choices of codon. The ENC -GC3 plot show that mutation pressure is dominating evolutionary

driving force in making codon usage preferences. The study represents comprehensive analysis of codon usage pat-

tern and help to better understand the mechanism of codon usage bias.

KEY WORDS: EFFECTIVE NUMBER OF CODON, MUTATION PRESSURE, NUCLEOTIDE CONTENTS, PRINCIPAL COMPONENT ANALYSIS, RELATIVE

SYNONYMOUS CODON USAGE

680

ARTICLE INFORMATION:

*Corresponding Author: kmpbiomanit@gmail.com

Received 25

Oct, 2017

Accepted after revision 19

Dec, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

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NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.4/11

INTRODUCTION

Flaviviridae family is composed of fast evolving

RNA viruses. The members of the family are positive

single stranded RNA viruses that are causative agents

for number of neglected tropical diseases in humans and

animals. Flaviviridae family is mainly classi ed into four

genera: Flavivirus, Hepacivirus, Pegivirus and Pestivirus

(Lobo et al., 2009; Lu et al., 2017). The viral genomes

vary from 9 to 13 Kb and contain a single known ORF

Anjusha Mune etal.

that codes for a polyprotein, which is processed co- and

post-translationally by host and viral proteases into at

least 10 functional, individual polypeptides (Blitvich and

Firth, 2015; Brand etal., 2017).

Flavivirus genus comprises arthropod born viruses

(arboviruses) which are transmitted to host by vectors

(mosquitoes or ticks) via blood feeding. Birds and mam-

mals are usual primary hosts. Members of this genus are

further classi ed into groups on the basis of vector. They

are mosquito born, tick born, and non-known arthropod

vector (NKV) (Velazquez-Salinas etal., 2016). Flavivirus

includes viruses which cause several diseases includ-

ing: Dengue fever, Japanese encephalitis, Murray valley

encephalitis, West Nile fever, Zika fever. These viruses

are distributed worldwide but individual species are

restricted to particular epidemic areas (Moosavi et al.,

2011; Huang etal., 2014; Zhang etal., 2017).

Hepacivirus are the group of viruses mainly trans-

mitted by blood contact in mammals (horses, rodents,

bats, cows and primates); its best species being Hepati-

tis C virus. Genus Pestivirus infect mammal’s members

of family Bovidae (cattle, sheep, and goats) and suidae

family (various species of swine). Compared to the other

viruses in thefamily Pestivirus encode two unique gene

products, namely N

pro

and E

rns

. These unique proteins are

involved in repression of the host type I IFN response.

The genus Pegivirus commonly causes persistent infec-

tion in a broad range of mammals (humans, non-human

primates, pigs, horses and a range of rodent and bat

species).Less information is available on transmission of

these viruses in different host species (Theze etal., 2015;

Zhou etal., 2012; Tautz etal., 2015).

Many new viruses have been documented but their

relationship with other virus, mode of transmission

and vector association is not clear, hence they are not

assigned under any genus and we consider them unclas-

si ed. The genetic code comprises 64 codons that can

be divided into 20 groups. Each group corresponds to

each of the standard amino acids and consists of one

to six codons (Butt etal., 2013; Chen, 2013; Gu etal.,

2003). Alternative codons within the same group cod-

ing for the same amino acid are often termed ‘synony-

mous’ codons. Most amino acids can be translated by

more than one codon. This redundancy is an important

factor that provides accuracy in production of protein.

These synonymous codons are not used randomly. There

are some codons that are used more often than other

codons. This phenomenon is referred to as codon usage

bias (Tao etal., 2009; Moratorio etal., 2013; Wang etal.,

2016; Van etal., 2016).

Studies on codon usage have determined several fac-

tors that could in uence codon usage pattern, including

mutational pressure, natural or translational selection,

secondary protein structure, replication and selective

transcription, hydrophobicity of the protein, and the

external environment. Among these, the major factors

responsible for codon usage variation among different

organism are considered to be compositional constraints

under mutation pressure and natural selection. Numbers

of previous studies on codon usage of different viruses

have highlighted mutation pressure as the major factor

in shaping codon usage patterns compared with natural

selection. But with increasing understanding of codon

usage it appears that although mutational pressure is

still a dominating force, it is certainly not the only one

when different viruses are considered (Sharp et al.,

1988; Cristina etal., 2015; Xiang etal.,2015, Butt etal.,

2016 ).

Analysis of codon usage patterns of Flaviviridae

would not only provide a base for better understanding

of biased usage of synonymous codons, the evolution

and pathogenesis of Flaviviridae, but also improve our

understanding of the regulation of viral genes expres-

sion and aid vaccine design, where the ef cient expres-

sion of viral protein may be required to generate immu-

nity. In order to gain insight into these matters, we have

analysed codon usage and base composition of the 114

species of Flaviviridae family. The patterns of preferred

codons for each individual amino acid in each species

were identi ed.

MATERIALS AND METHODS

Total 114 complete genome sequences of viruses of Fla-

viviridae family were downloaded from the National

Centre for Biotechnology (NCBI) database (http://www.

ncbi.nlm.nih.gov) in FASTA format. The accession

numbers and other detailed information of the selected

genomes were listed in [supplementary material Table 1].

Open reading frames (ORF) of all the genomic sequences

were identi ed by using NCBI ORF  nder (https://www.

ncbi.nlm.nih.gov/orf nder/).

In order to understand the frequencies of occurrence

of each nucleotide in ORFs, composition analysis was

conducted. The overall frequency of occurrence of the

nucleotides (A %, C %, U %, and G %) was calculated

along with the frequency of each nucleotide at the third

site of the synonymous codons (A

, C

, U

and G

). Also

the overall GC, AU and GC

content were calculated to

investigate the compositional properties. The codons

AUG and UGG are the only codons for Met and Trp,

respectively, and the termination codon UAA, UAG, and

UGA do not encode any amino acids. Therefore, these

 ve codons are excluded from the analysis.

The ENC is a very effective estimator to measure the

magnitude of codon usage bias in the coding sequences

of members of Flaviviridaefamily. The ENC value ranges

from 20 (when only one synonymous codon is chosen

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS GENOME-WIDE COMPARATIVE ANALYSIS OF THE CODON USAGE PATTERN IN

FLAVIVIRIDAE

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Anjusha Mune etal.

by the corresponding amino acid) to 61 (when all syn-

onymous codons are used equally) (Lu etal., 2005). In an

extremely biased gene where only one codon is used for

each amino acid, this value would be 20; if all codons

are used equally, it would be 61; and if the value of

ENC is greater than 40, the codon usage bias is regarded

as low (Wright F, 1990). We calculated ENC to meas-

ure the degree of departure from equal use of synony-

mous codons of ORF of members of Flaviviridaefamily.

The values of ENC were obtained by EMBOSS CHIPS

program. These ENC values were further analysed as

suggested by Wright’s ENC – plot (Zhang etal., 2011).

The ENC values are plotted against GC

as a method

to understand the pattern of codon usage. The viruses,

whose codon choice is constrained only by a mutation

bias, will lie on or just below the curve of the predicted

values. The predicted values of ENC were calculated as

Wheresrepresents the given (G+C)

% value.

Relative synonymous codon usage (RSCU) Analysis:

The RSCU values of codons in each ORF for all the mem-

ber of Flaviviridaefamily were calculated by the given

formula to determine the characteristics of synonymous

codon usage. The synonymous codons with RSCU values

> 1.0 have positive codon usage bias and were de ned

as preferred codons, while those with RSCU values <

1.0 have negative codon usage bias and were de ned as

less- preferred codons. When the RSCU value is 1.0, it

means there is no codon usage bias for that amino acid

and the codons are chosen equally or randomly. More-

over, the synonymous codons with RSCU values >1.6

and < 0.6 were treated as over-represented and under-

represented codons, respectively (Wong etal., 2010; Ma

etal., 2013).

Where g

is the observed number of the ith codon for

the jth amino acid, which has n

types of synonymous

codons.

The correlation analysis was performed between each

general nucleotide composition (U%, A%, C%, and G

%) and each nucleotide composition in the third site of

codon (U

%, A

%, C

%, and G

%) and the value A%,

T%, C%, G% and A

%, T

%, C

%, G

% were compared

with GC%,, GC

% and ENC Using statistical software

SPSS 19 for windows. It is used to identify the relation-

ship between nucleotide composition and synonymous

codon usage pattern of viruses Flaviviridae family.

Principal component analysis (PCA): In this study

PCA was performed to analyse the major trend in codon

usage pattern among members of  aviviridae family.

Principal component analysis is one of the most fre-

quently used multivariate statistical techniques (Su MW

et al., 2009; Yadav and Swati D, 2012; Kanaya et al.,

2001; Wang et al., 2011). PCA is an orthogonal linear

transformation that is used to transform the original

data set into a new coordinate system. It involves a

mathematical transformation procedure that transforms

some correlated variable (RSCU) into a smaller number

of uncorrelated variables called principal components.

The greatest variance represented by the data lies on

the  rst coordinate, thus known as the  rst principal

component (PC), the second greatest variance is on the

second PC, and so on. One can use top 2 or 3 PCs to rep-

resent the data instead of the large number of original

variables (in this case, 59 Variables). In this study PCA

was done by constructing a 114  59 RSCU data matrix.

In the matrix each row denotes the codon usage pattern

of a speci c virus, demonstrated by its RSCU value. Each

member of Flaviviridae family was represented as a 59

dimensional vector and each dimension corresponds to

the RSCU value of one sense codon, which only included

several synonymous codons for a particular amino acid,

excluding Met (AUG), Trp (UGG) and three stop codon.

RESULTS AND DISCUSSION

COMPOSITIONAL PROPERTIES OF ORFS OF 114

FLAVIVIRIDAE GENOMES

The nucleotide contents (A, U, C, G and AU, GC %) and

each nucleotide contents in the third site of codon (A

, C

, G

and GC

%) in the orf of members of Flaviviri-

dae family do not show similarity and are found to be

quite different from each other. [Table 1 supplementary

material].The genome of genus Flavivirus is enriched for

purines (A and G) compared to Pyrimidines (U and C)

with high frequency of base G ranging from 21.52-34.01.

The purine richness is maintained throughout the genus

without getting affected by the vector choice of that

virus. But the content of G is higher in tick born viruses

as compared to others. The effect of purine richness can

be observed on the selection of codons as the most pref-

erentially used codons are A - ended or G – ended codons

with higher preference to A – ended codons except AUC

for Ile in Flavivirus. Members of genus Pestivirus are also

enriched for purine bases, with high A content ranging

from 31.54-36.94 unlike Flavivirus .Most preferentially

used codons are A - ended or G – ended codons with

higher preference to A – ended codons except AGU for

Ser in Pestivirus. The genus Pegivirus and Hepacivirus

are rich in GC content with High content of G base in

Pegivirus ranging from 27.67-32.41 and content of base

C is almost similar in both the genus. The GC and GC

682 GENOME-WIDE COMPARATIVE ANALYSIS OF THE CODON USAGE PATTERN IN

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Anjusha Mune etal.

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Table 1. List of Over-represented and Under-represented codons of four genus and unclassi ed members of

Flaviviridae family.

Genus Over-represented codons Under- represented codons

Flavivirus

(mosquito born)

UUG and CUG (Leu), GUG (Val), GGA (Gly), UCA

(Ser), CCA(Pro), ACA(Thr), AGA and AGG (Arg),

UUA (Leu), GUA (Val), CCG (Pro), GCG (Ala), GGU

(Gly)

Flavivirus

(tick born)

CUG (Leu), AUC ( Ile), GUG (Val), CCA (Pro), AGA

and AGG (Arg), GGA (Gly), AGU and AGC (Ser)

UUA and CUA (Leu), GUA (Val), UCG (Ser), CCG

(Pro), GCG (Ala), CGU and CGA (Arg)

Flavivirus

(NKV)

UUG (Leu), UCA (Ser), CCA (Pro), AGA & AGG

(Arg), GGA (Gly), ACA (Thr), GUG (Val)

GUA (Val), CGC & CGU (Arg)

Pestivirus CUA and CUG (Leu), AUA (Ile), GUG and GUA

(Val), UCA, AGC and AGU (Ser), CCA for Pro, GCA

for Ala, AGA and AGG for Arg , GGG for Gly,

ACA (Thr)

UCG (Ser), GCG (Ala), CGU, CGC, CGA and CGG

(Arg), UCG (Ser) and CCG (Pro).

Pegivirus UUG and CUG (Leu), AUC (Ile), GUG (Val), UCU

and UCC (Ser), ACU and ACC (Thr), CGC and CGG

(Arg)

GUA (Val), UUA and CUA (Leu), AUA (Ile), and AGA

(Arg) and GGA (Gly).

Hepacivirus AUC (Ile), CUC (Leu), GGC (Gly), AGG (Arg), UCC

(Ser)

UUA and CUA (Leu), GUA (Val), CGA (Arg)

Unclassi ed UUG (Leu), GUG (Val), CCA (Pro)

GGA (Gly)

GUA (Val), UUA (Leu)

compositions also highlight the richness of these nucleo-

tides in Pegivirus and Hepacivirus.

As a result of this they prefer using G - ended or

C - ended codons with higher preference to C – ended

codons. The unclassi ed group of virus show similarity

with genus Flavivirus, enriched for purines, hence most

preferentially used codons are A- ended or G with higher

preference to A – ended codons. Two out of the four

genus, Flavivirus and Pestivirus, are AG rich and show

similarity in preferred base. On the other hand, Pegivirus

and Hepacivirus are GC rich and show similarity in pre-

ferred base. We observed that the four possible nucleo-

tides are not used at equal frequencies. Flavivirus and

Pestivirus genus show low C content whereas Pegivirus

and Hepacivirus genus show low A content. The base U

is observed to be stable in the entire Flaviviridae family.

EFFECTIVE NUMBER OF CODON USAGE (ENC)

Different species have different tendencies to prefer spe-

ci c codons, symbolized by Effective number of codons

values. To investigate the overall codon usage pattern

of Flaviviridae family, the ENC values for each orf is

calculated and compared among the four genus [Supple-

mentary material Table 1]. The values were analysed and

compared within a genus and between different genus.

Overall, the observed ENC values range between 44.99

(Norway rat Pestivirus) to 58.97 (Aedes Flavivirus) with

the average being 53.74 across the Flaviviridae family.

We also observed the values across 4 Genus. The codon

bias of Flavivirus genus was on average 53.54 and

ranged from 58.97 (Aedes Flavivirus) to 47.30 (Tamana

bat virus) with standard deviation of 2.14. The overall

codon bias of Hepacivirus genus was on average 55.07

and ranged from 57.02 (Hepatitis GB virus B) to 51.27

(Norway rat Hepacivirus) with standard deviation of

1.85. The overall codon bias of Pegivirus genus had an

average value of 54.17 and ranged from 57.37 (Human

Pegivirus 2) to 50.59 (Rodent Pegivirus) with standard

deviation of 2.48.The overall codon bias of Pestivirus

had an average value of 50.71 and ranged from 54.08

(Atypical porcine Pestivirus 1) to 44.99 (Norway rat

Pestivirus) with standard deviation of 2.19. Finally, the

codon bias of unclassi ed members was represented

by an average value of 55.89 and ranged from 58.15

(Anopheles  avivirus variant 1) to 52.30 (Bamaga virus)

with standard deviation of 1.75.

Among the entire genus Flavivirus showed highest

variation in ENC value and members of unclassi ed

group have shown the least variation. The codon varia-

tion of Flavivirus genus is higher than the variation of

other three genus, implying that the evolution speed of

these viruses is higher than the speed of the remain-

ing viruses of the family. Conceptual value is com-

prised between 21 (if only single codon is used for each

amino acid) and 61 (if all codons are used with equal

frequency). In general, the overall codon bias of the four

genus and unclassi ed members of Flaviviridae viruses

is considerably weak. This is in agreement with previ-

ous reports about some other RNA viruses, for example

BVDV (ENC=51.42), H5N1 (ENC=50.91) and SARS-covs

(ENC=48.99), NDV (ENC=56.15) (Wang etal., 2011; Zhou

Anjusha Mune etal.

684 GENOME-WIDE COMPARATIVE ANALYSIS OF THE CODON USAGE PATTERN IN

FLAVIVIRIDAE

FAMILY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

et al., 2005; Gu et al., 2003; Wang et al., 2011). The

possible explanation of weak codon bias in RNA virus

is that a weak bias is helpful for ef cient replication of

virus in host cells. (Zhong etal., 2007)

MUTATION PRESSURE AFFECTS THE CODON

USAGE PATTERN

Mutational pressure and natural selection are considered

the two major factors that shape codon usage patterns

(Jenkins and Holmes, 2003). A general mutational pres-

sure, which affects the whole genome, would certainly

account for the majority of the codon usage among cer-

tain RNA viruses (Tatarinova et al., 2010). To identify

whether the evolution and variation pattern of codon

usage had been driven alone by mutation pressure or

also contributed by natural selection, we compared the

correlation between overall nucleotide composition (A,

U, C, G) and nucleotide composition at the third position

of codon (A

, U

, C

, G

) and correlation between overall

nucleotide composition (A, U, C, G, A

, U

, C

, G

) and

GC, GC

and ENC for individual genus using Pearson s

correlation [supplementary material ( Tables 2-3).

In genus Flavivirus GC and GC

show signi cant pos-

itive correlation with G(r=0.87, P<0.01) (r=0.80, P<0.01),

C (r=0.75, P<0.01) (r=0.80, P<0.01) and G

(r=0.86,

P<0.01) (r=0.85, P<0.01), C

(r=0.75, P<0.01) (r=0.82,

P<0.01).and negative correlation with A (r=-0.84,

P<0.01) (r= -0.85, P<0.01), U (r= -0.70, P<0.01) (r= -0.65,

P<0.01), and A

(r= -0.76, P<0.01), (r= -0.81, P<0.01),

(r= -0.69, P<0.01) (r= -0.68, P<0.01). ENC shows posi-

tive signi cant correlation with C (r= 0.73, P<0.01) and

(r= 0.70, P<0.01), and negative correlation with A (r=

-0.69, P<0.01) and A

(r= -0.65, P<0.01)

and non-sig-

ni cant correlation with U, G, and U

A shows positive

correlation with A

, negative correlation with C

and G

and non-signi cant correlation with U

U shows signi -

cantly negative correlation with C

and G

3 ,

positive cor-

relation with U

, and non-signi cant correlation with

G and C show signi cantly negative correlation with

and U

and signi cantly positive correlation with C

and G

. When we study correlation vector wise, tick born

and NKV viruses show signi cant correlation in com-

parison with mosquito borne viruses of the genus. In

genus Pestivirus an interesting and complex correlation

was observed.

To sum up, the GC, GC

and ENC have highly posi-

tive signi cant correlation with C (r=0.84, P<0.01)

(r=0.86, P<0.01) (ENC=0.87), C

(r=0.90, P<0.01) (r=0.94,

P<0.01) (ENC=0.89) and G (r=0.94, P<0.01) (r=0.91,

P<0.01) (ENC=0.74),G

(r=0.98, P<0.01) (r=0.97, P<0.01)

(ENC=0.82)

And signi cantly negative correlation with

A (r= -0.99, P<0.01) (r= -0.99, P<0.01) (ENC= -0.92),

(r= -0.97, P<0.01) (r= -0.99, P<0.01) (ENC= -0.90)

and U (r= -0.89, P<0.01) (r= -0.84, P<0.01), U

(r= -0.82,

P<0.01) (r= -0.80, P<0.01) (ENC= -0.66, P<0.05). A

and

show signi cantly positive correlation with A and

U, and signi cantly negative correlation with C and

G, whereas C

and G

show signi cantly negative cor-

relation with A and U and signi cantly positive cor-

relation with C and G. In genus Hepacivirus A

shows

positive correlation with A and has non-correlation with

U, C, G and GC and GC

. Similarly, A shows non-cor-

relation with U

, C

, G

and GC, GC

and ENC. GC and

show signi cantly negative correlation with U (r=

-0.94, P<0.01) (r= -0.97, P<0.01) U

(r= -0.92, P<0.01)

(r= -0.96, P<0.01) and highly positive correlation with

G (r=0.95, P<0.01) (r=0.94, P<0.01), G

(r=0.96, P<0.01)

(r=0.97, P<0.01) and C (r=0.98, P<0.01) (r=0.95, P<0.01),

(r=0.96, P<0.01) (r=0.99, P<0.01). ENC of Hepacivirus

show non-correlation with A, U, C, G, U

In genus Pegivirus the GC and GC

show signi cantly

positive correlation with G (r=0.83, P<0.01) (r=0.83,

P<0.01), C (r=0.84, P<0.01) (r=0.71, P<0.05), C

(r=0.82,

P<0.01) (r=0.84, P<0.01) and signi cantly negative cor-

relation with U (r= -0.77, P<0.05) (r= -0.91, P<0.01)

and A

(r= -0.89, P<0.01) (r= -0.73, P<0.05). ENC have

highly signi cant correlation with A, U, C, G, A

, C

and

non-correlation with U

and G

A shows signi cant cor-

relation with A

but does not show signi cant correla-

tion with U

, C

3 ,

. U shows signi cant correlation with

, C

and G

, and non-signi cant correlation with A

show signi cant correlation with A

and C

and non-

signi cant correlation with U

and G

. G shows signi -

cant correlation with G

and non-signi cant correlation

with A

and C

The members of unclassi ed group

do not show signi cant correlation with other nucleo-

tides, they show signi cant positive correlation with the

same type of nucleotide like A show positive correla-

tion with A

. The GC and GC

show positive correlation

with C (r=0.73, P<0.01) (r=0.62, P<0.05) and G (r=0.81,

P<0.01) (r=0.67, P<0.01) and negative correlation with

A (r=-0.75, P<0.01) (r=-0.69, P<0.01) and U (r=-0.78,

P<0.01) (r=-0.59, P<0.05). ENC does not show signi -

cant correlation with any nucleotide. This analysis col-

lectively indicates that mutational pressure is most likely

responsible for the patterns of nucleotide composition

and, therefore, codon usage patterns in all four genus of

Flaviviridae family.

VARIATION OF RELATIVE SYNONYMOUS

CODON USAGES IN FLAVIVIRIDAE FAMILY

In order to investigate the extent of codon usage bias in

 aviviridae family, all RSCU values of different codons

in genus Flavivirus (69), Hepacivirus (14), Pegivirus (8),

Pestivirus (11) and unclassi ed members (12) were cal-

culated. The heat map [supplementary material Fig. 1]

Anjusha Mune etal.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS GENOME-WIDE COMPARATIVE ANALYSIS OF THE CODON USAGE PATTERN IN

FLAVIVIRIDAE

FAMILY 685

show the RSCU results of all codons in the 114 viruses of

Flaviviridae family. Green represent lower RSCU value,

black represent moderate RSCU, and red represents

greater RSCU values. The common over - represented

and common under - represented codons are listed for

each genus of Flaviviridae family [Table 1]. As we know

genus Flavivirus is classi ed into three groups on the

basis of vector. The over - represented and under - rep-

resented codons are identi ed vector wise for this genus.

Viruses in this genus show similarity in choice of

codon with their subtype or genotype like the four sero-

type of Dengue show similar choice of codons. Preferred

codons in four serotype are UUG and CUG for Leu, AUA

for Ile, GUG for Val, UCA for Ser, CCA for Pro, ACA for

Thr, GCC and GCA for Ala, AGA and AGG for Arg, GGA

for Gly. Less preferred codons are GUA for val, UCG for

Ser, CCG for Pro, ACG for Thr, GCG for Ala, CGU and

CGC, CGA and CGG for Arg, GGU and GGC for Gly.

Similarly, westnile 1 and westnile 2 virus show similar

choice of codon usage UUG, CUC and CUG for Leu, AUC

for Ile, GUG for Val, UCA for Ser, CCA for Pro, ACC and

ACA for Thr, GCU and GCC for Ala, AGU and AGC for

Ser, AGA and AGG for Arg, GGA for Gly. The less pre-

ferred codons are UUA for Leu, GUA for Val,UCG for Ser,

CCG for Pro,GCG for Ala, CGU and CGA for Arg, GGU

for Gly. In genus Hepacivirus Equine and Bovine show

similar choices for preference of codon in comparison to

the other members of the group. The unclassi ed mem-

bers of Flaviviridae family show similarity with genus

Flavivirus like, Lammi virus shows similarity with mos-

quito born Flaviviruses especially with West Nile virus

in choice of preferred and less preferred codon. Nhu-

mirim virus shows similarity with nkv group of Flavi-

viruses especially with Paraiso virus show similarity in

preferred codons CCC, CCA & CCG for Pro, GCG for Ala,

CGA & CGG for Arg, AGU for Ser and GGA & GGG for

Gly. IIomantsi virus and Donggang virus show similar-

ity in preferred codon CCC & CCG for Pro, GCG for Ala,

CGA & CGG for Arg, AGU for Ser and GGA & GGG

for Gly. IIomantsi ,Lammiand Nienokoue viruses show

higher degree of similarity in choice of preferred codon

with mosquito born Flavivirus. GUG for Gly is the only

common codon in the entire Flaviviridae.

In general, the amount of the over - represented

codon is more than the amount of under - represented

codon in the four genus of family and this feature is

consistent with all the 114 ORF`s, suggesting that the

evolution process of viral genome of all four genus is

similar to some degree and the codon usage bias is weak

which is supporting the results we observed from ENC

values. The nucleotide composition also plays an impor-

tant role in choosing preferred codons, therefore Flavi-

virus and Pestivirus shows preference to A and G ending

codons, as they are rich in purines. And Hepacivirus and

Pegivirus show preference to G and C ending codons as

they are rich in GC content.

In addition, the RSCU values of the eight codon con-

taining CpG (CCG, GCG, UCG, ACG, CGC, CGG, CGU,

and CGA) in four genus were analysed. All of these eight

codons were not preferential codons and were found

suppressed in genus Flavivirus and Pestivirus. In genus

Pegivirus and Hepacivirus six codons are under - repre-

sented except CGC and CGG. The explanation for CpG

scarcity in these viruses is attributed to their property to

escape the host immune response. A high CpG content

leads to increased unmethylated CpGs which has immu-

nostimultory property and therefore are easily recog-

nized by the host’s innate immune system as a pathogen

signature. This is injurious to the small DNA (or RNA)

viruses. Thus high mutational rates are observed in CpGs

FIGURE 1. The ENC vs GC3 plots of four genus and unclassi ed members (Pestivirus,

Pegivirus, Hepacivirus, and Flavivirus) of Flaviviridae family.

Anjusha Mune etal.

686 GENOME-WIDE COMPARATIVE ANALYSIS OF THE CODON USAGE PATTERN IN

FLAVIVIRIDAE

FAMILY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 2. Principal components (PCs) and variances explained in the analysis of the 59 relative syn-

onymous codon usage (RSCU) indices. The ﬁrst 9 PC vectors are listed on with accumulated variance

(%). The plot on the shows that the ﬁrst  ve PCs have explained more than 70% of the variance of the

original data.

FIGURE 3. Principal component analysis (PCA) plot

for analysis of the relative synonymous codon usage

(RSCU) indices of 114 RNA viruses. The PCA scores of

the 114 viruses were plotted in a three-dimensional

coordinate system using the ﬁrst three principal com-

ponent vectors as axes.

since its de cit will enable virus to infect the host. (Dorn

and Kippenberger, 2008; Krieg, 2003)

CORRELATION ANALYSIS BETWEEN ENC AND

VALUE

A plot of ENC versus GC

is widely used to study codon

usage variation among different organisms. It is the

most important part of investigation of codon usage

pattern. The ENC values of each member of Flaviviridae

family were plotted against its corresponding GC

values

and all values lie below the expected curve as shown in

[Fig. 1]. Therefore it can be hypothesized that the codon

usage bias, in all these 114 viruses is principally in u-

enced by the mutational pressure.

IDENTIFICATION OF SIMILARITIES AND DIFFERENCES IN

CODON USAGE PREFERENCES BY PCA

The identi cation of similarities and differences in

codon usage preferences is an involved process that can

be handled by using the Principal Component Analy-

sis (PCA) approach. The PCA is a classical data analysis

method that identi es patterns and focuses on similari-

ties and differences in a multivariate data set. The explo-

ration of codon usage pattern differences among these

RNA viruses involves processing of the 114 × 59 RSCU

matrix by PCA. This enables calculating the principal

components (PCs) which in turn are employed to high-

light the similarities and differences in codon usages.

[Fig.2] shows the trend of the  rst 9 PCs. PCs with Eigen

value greater than or equal to 1 are usually considered

as being of statistical signi cance (the Kaiser criterion)

as indicated in [Supplementary material Table 4] .The

 rst PC is associated with 36.73% of the variance among

the 59 RSCU indices. The  rst two PCs taken together

account for 53.61% of the variance whereas the  rst

three PCs combined together account for 63.30% of the

variance in codon usage.

The variances of a total of 114 PCs generated from

PCA are listed in [Supplementary Material Table 4].

Fig. 3 is the three-dimensional PCA plot using the  rst

three PCs of these 114 viruses as axes [the correspond-

ing PCA coordinates are listed in Supplementary Mate-

rial Table 5]. The PCA score diagram shows that the all

viruses can be broadly classi ed into four categories.

This classi cation is essentially based on different hosts,

Anjusha Mune etal.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS GENOME-WIDE COMPARATIVE ANALYSIS OF THE CODON USAGE PATTERN IN

FLAVIVIRIDAE

FAMILY 687

vectors and ecological niche. Genus Flavivirus display

negative values on the second and third PC axes. Mem-

bers of genus Pegivirus displayed positive values on the

three PC axes. The genus Hepacivirus displays more pos-

itive values on  rst and third PC axes. The genus Pes-

tivirus displays negative values on  rst and second PC

axes whereas all positive values appear on the third axis.

The unclassi ed members manifest a heterogeneous dis-

tribution of values and, consequently, do not represent

a  fth category but get merged into the four categories.

CONCLUSION

Our analysis reveals that the overall codon usage bias in

Flaviviridae family is slightly biased and mutation pres-

sure is the main factor that affects codon usage variation

in viruses. Other factors like Compositional constraint and

natural selection also signi cantly in uence codon usage

variation. Results show RNA viruses with same vector

choice share similar codon usage preferences. However,

more detailed analysis is needed to understand the rela-

tionship of codon choices between viruses and hosts.

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