DISCLAIMER
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the Zika open site, according to the protocol for public health emergencies for international
concern as described in Christopher Dye et al. (http://dx.doi.org/10.2471/BLT.16.170860).
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RECOMMENDED CITATION
Shrinet J, Agrawal A, Bhatnagar RK, Sujatha Sunil S. Analysis of the genetic divergence in
Asian strains of ZIKA virus with reference to 2015-2016 outbreaks. [Submitted]. Bull World
Health Organ. E-pub: 22 Apr 2016. doi: http://dx.doi.org/10.2471/BLT.16.176065
Analysis of the genetic divergence in Asian strains of ZIKA
virus with reference to 2015-2016 outbreaks
Jatin Shrinet,a Aditi Agrawal,a Raj K Bhatnagara & Sujatha Sunila
a
International Centre for Genetic Engineering and Biotechnology, New Delhi-110067, India
Correspondence to: Sujatha Sunil (e-mail: [email protected])
(Submitted: 20 April 2016 – Published online: 22 April 2016)
Abstract
Objective: To compare Zika virus (ZIKV) genomes of the 2015-2016 outbreaks with the
older strains and evaluate evolution of ZIKV.
Method: We performed several genetic analyses to 50 ZIKV genomes currently
available in the public domain. Phylogenetic and mutation analysis, recombination
analysis, molecular evolution and selection analysis identified amino acid variations that
were unique to the 2015-2016 outbreak strains and the status of recombination and
evolution amongst these sequences.
Findings: We report distinct amino acid variations in the structural and nonstructural proteins of all 2015-2016 outbreak strains that are conserved amongst these
strains. Our results also reveal unique motifs in the UTRs of the new ZIKV strains. We
identified recombination events in the African strains but not in the recent isolates of
Asian lineage. Population level analysis revealed over dominant selection of alleles in
the genome.
Conclusion: 2015-2016 strains of ZIKV show distinct molecular signatures in
their genomes that are conserved across strains isolated from different parts of the
globe during the outbreak period. Our analysis at the population level emphasizes on a
possibility of balancing selection of the alleles.
Introduction
Arboviruses are an important group of viruses of medical relevance due to the wide range of
illnesses they cause. In the last two decades, infections caused by these viruses have been major
public health concerns resulting in pandemics and epidemics (1, 2). The latest addition to this list
is Zika virus (ZIKV) with the World Health Organization declaring Zika fever (ZF) as a Public
Health Emergency of International concern due to its possible association with neurological and
birth conditions(3).
Zika virus is a member of the genus Flavivirus, family Flaviviridae (4)that has other
medically important flaviviruses like dengue, yellow fever, West Nile, Japanese encephalitis
viruses. Originally maintained in a sylvatic cycle (5), the first virus was isolated from a Macaca
monkey in 1947 in the Zika forest region of Uganda (6). In these conditions humans are
considered to be incidental hosts; however, in the absence of non-human primates, humans
probably serve as the primary amplification hosts (7). The first human case was reported in 1954
in Nigeria (8),and sporadic cases have been reported from different regions around the globe
over the years (9-12). In addition to clinical cases, isolation of ZIKV from vectors has also been
reported (13-15).
The ZIKV genome consists of a 10794 bps long single stranded RNA of positive sense
encoding a single open reading frame (ORF). Flanked by two non-coding regions (5’ and 3’
untranslated regions), the ORF encodes a polyprotein: C-prM-E-NS1-NS2A-NS2B-NS3-NS4ANS4b-NS5, which is cleaved into three structural proteins, namely, capsid (C),
premembrane/membrane (prM) and envelope (E) and seven non-structural proteins (NS1, NS2A,
NS2B, NS3, NS4A, NS4B, NS5) (16, 17). Based on serologic and genetic properties, three
lineages, namely, East African, West African and Asian, have been identified (18).
In 2015, the Americas witnessed a huge outbreak of ZF with neurological implications
and symptoms of Guillian-Barre syndrome in affected individuals (19).Epidemiological studies
reveal the transmission to have originated on Yap in Micronesia in 2007 (18) that spread to other
Pacific islands (20) and to South and Central America (21). With the rapid spread of this virus to
several parts of the globe, it is imperative to understand the cause of spread. Until 2012, there
were eight genomes available; however, post 2012, 42 genomes have been reported in the public
domain (till 20th March 2016) of which 25 genomes reported post January 2016. Analyzing these
genomes at a molecular level may reveal the genetic divergence the newer viruses may exhibit
thereby providing insights to the evolution of the virus. The present report is a bioinformatics
characterization of the genomes of ZIKV isolated post 2015 and comparison with the older
strains of ZIKV.
Materials and Methods
Genome sequences and Phylogenetic analysis
A total of 50 genome sequences of ZIKV were retrieved from NCBI database. The sequences
were multiple aligned and manually edited to discard any aberration in the sequences. Twelve
different gene sequences of ZIKA virus namely, Capsid, pr, M, Envelope (E), NS1, NS2A,
NS2B, NS3, NS4A, 2K, NS4B and NS5 were extracted from the multiple aligned genome
sequences and were further used for analysis of variations in the proteins. The phylogenetic
analysis of trimmed genome sequences were performed using MEGA6 tool (22). Neighborjoining method, Minimum Evolution method with Gamma parameter 1 and 100 bootstrap
replications, Maximum likelihood method, UPGMA method and Maximum Parsimony method
models were used to construct the phylogenetic tree. Phylogeny test was performed using
bootstrap method and by taking 1000 number of bootstrap replications.
UTR analysis
5’ UTR and 3’ UTR sequences were extracted from the genome and aligned using MEGA6.
UTR sequences were not present for all the genomes and also some of the genomes have short
UTR sequences. The aligned sequences were then analyzed to study the conservation of residues.
The multiple aligned sequences were also subjected to RNAalifold web server to predict
consensus secondary structures of both the UTR sequences(23).
Recombination analysis
The multiple aligned ZIKV genome sequences were subjected to recombination analysis using
RDP tool (24). RDP analyze the sequences using 7 methods namely, RDP (R), GENECONV
(G), MaxChi (M), Chimaera (C), Bootscan (B), 3Seq (T) and SiScan (S). The events predicted
by more than 5 methods and without any unknown parent and p-value<0.05 were considered
recombination event.
Molecular evolution and selection analysis
Transition/Transversion bias, Substitution matrix, overall means distance variations were
calculated using MEGA 6. Tajima’s test of neutrality was also performed using MEGA6 tool.
Results and Discussion
Fifty ZIKV genome details that were used in the study are listed in Supplementary Table 1. Of
these sequences, 15 were belonged to year 2015; ten belonged to 2016 (as of March, 2016).
Amongst the remaining sequences, two sequences each were isolated in the years 2014, 2013,
2001, 1968 and 1974. One sequence each was reported from years 2012, 2010, 2007, 2000,
1997, 1984, 1976 and 1966. Information about the isolation date was not available for seven
sequences. The geographical distribution of these sequences showed that nine sequences were
from Brazil and all were isolated in year 2015. Two sequences of 2015 were isolated from
Guatemala and one sequence each of 2015 belongs to Suriname, Puerto Rico, Martinique and
Colombia. Several of these sequences have been previously used to study molecular evolution of
ZIKV in the earlier years (25, 26).
The phylogenetic tree of 50 ZIKV sequences was constructed using Neighbor-joining
methods (Figure 1). The tree was also constructed using other methods, namely, Minimum
Evolution method with Gamma parameter 1 and 100 bootstrap replications, Maximum likelihood
method, UPGMA method and Maximum Parsimony method with 1000 bootstrap replications
(Supplementary Figure 1). The sequences from 2015-2016 showed similarity to Asian lineage
and grouped in the same clade. These results showed that the Asian strain has caused the recent
outbreak in western part of the world as reported by others (27).
To study the molecular variations specific to Asian strains, Malaysian isolate
(HQ234499.1; 1966) (13) was used as reference for all further analyses. Sequence comparison of
structural and non-structural ZIKA virus proteins revealed several variations in the 2015-2016
genomes that are discussed in detail below.
Sequence analysis of the 2015-2016 isolates with Asian genotype
Structural region
Year 2015 and 2016 outbreak samples (n=25) were compared against the year 1966 sequence
from Malaysia. Nucleotide variations were too numerous to discuss here. With respect to amino
acid variations, structural proteins showed several variations in their sequences revealing high
mutational rate of the new ZIKV strains (28). Variations observed were classified into two
categories, those that were seen in all 2015-2016 samples and variations that were strain-specific.
Both these categories will be discussed in detail in the following sections. For better clarity of
analyzing common variations in the samples, consensus sequences were acquired for each region
year-wise and compared with the reference sequence (Table 1a). Capsid showed variations at
five aa positions, namely, N25S, L27F, R101K, I110V and I113V in all the sequences. Amino
acid variations in individual samples are listed in Table 1b. In Capsid, apart from the abovementioned variations, sequence KU729218.1 (from Brazil) showed variation at G105S. Five
samples, KU647676.1 (from Martinique), KU820897.1 (from Colombia), 820898.1 (from
China), KU922960.1, KU922923.1 (from Mexico) showed variation at position D107E.
Sequences KU866423.1, KU820899.2 (from China) showed variation at position S109N. Amino
acid E76D was seen in KU744693.1 (from China).
Sequence comparison of pr protein showed three aa variations, namely, V1A, S17N,
V31M in all the 2015 and 2016 sequences (Table 1a). Sequence KU312312.1 (from Suriname)
showed an additional change at M44T. No changes in M protein showed a single amino acid
variation, P72L in a sequence from Mexico (KU922923.1) (table 1b).
Envelope protein of 2015-2016 isolates of ZIKA virus when compared to the reference
Malaysian strain revealed changes at three positions, D393E, V473M and T487M in all
sequences (Table 1a). Amino acid variations T47S, S64T, M68I and V255A were observed in
KU729217.2 (from Brazil). Sequences KU729218.1 and KU497555.1 (from Brazil) showed
M349T and S260T changes respectively. Sequences KU501216.1 and KU501217.1 (from
Guatemala) showed V56I variation, isolate KU312312.1 (from Suriname) showed T479A,
sequences KU866423.1 and KU820899.2 (from China) displayed K419R variation in their
respective genomes. Of special mention is one isolate from China (KU744693.1) that displayed a
total of 12 aa variations including 3 conserved changes (Table 1b).
Non-structural region
The non-structural protein sequences comparison of 25 isolates of 2016 (10) and 2015 (15) with
the reference sequence from Malaysia (HQ234499.1) indicates that the non-structural proteins of
ZIKA virus is more conserved than the structural proteins. Non-structural proteins namely, NS1,
NS2A, NS2B and NS3 showed very few conserved changes as compared to NS4B and NS5
which showed 7 and 15 aa variations respectively.
NS1 showed two changes namely A188V and V264M that were present in all 25
sequences (Table 2a). Sequences KU729217.1 and KU321639.1 (from Brazil) has additional aa
variations at G190E and Y122H respectively (Table 2b). Some sequences revealed two types of
aa variations at position R324. While sequences KU647676.1 (from Martinique), KU922923.1
and KU922960.1 (from Mexico) and KU820897.1 (from Colombia) showed R324W,
KU866423.1 and KU820899.2 (from China) had R324Q instead of R324W indicating the
evolving nature of the site. KU853013.1 (Italy) has an additional variation M349V. Sequences
KU501216.1 and KU501217.1 (from Guatemala) showed an additional mutation at position
G100A. One isolate from China (KU744693.1) showed a total of seven variations including two
conserved variations (Table 2b).
NS2A has only one conserved change A143V that was present in all the sequences.
Analyses of individual protein sequences showed some additional variations - L113F in
KU49755.1 (Brazil), I80T in KU647676.1 (Martinique), KU922923.1 and KU922960.1 (from
Mexico) Variation I139V was present in three sequences of China (KU820898.1, KU740184.2
and KU761564.1). NS2B protein was found to be conserved when 2015-2016 sequences were
compared against reference sequence with one exception of KU729217.2 (Brazil) with variation
M32I.
NS3 sequence has two conserved variation i.e., N400H and M472L seen in all sequences
(Table 2a). Apart from these positions, isolates KU729218.1 and KU321639.1 (from Brazil)
showed M334T and H355Y amino acid residue variations. Likewise both the sequences
KU501216.1 and KU501217.1 (from Guatemala) have a variation at position M572L. Isolates
KU922960.1 and KU922923.1 (from Mexico) showed A106E variation (Table 2b).
NS4B protein has changes at seven positions namely, G14S, M26I, L49F, M98I, I180V,
V184I and L186S in all sequences analyzed. Apart from these changes, only KU321639.1
(Brazil) at one position I176M and KU744693.1 (China) at four additional sites, A44P, T48S,
D150E and I176M showed variations. NS5 protein showed 15 numbers of evolved sites and this
could be due to its large size of 902 amino acid residues. Details of all the aa variations in the
non structural proteins are listed in Table 2a and 2b.
5’ and 3’ UTRs
Untranslated regions (5’ and 3’) are known to play important roles in flavivirus replication and
virulence (29). The untranslated regions (UTR) sequences of ZIKA virus from recent outbreak
were aligned and checked for variations. Malaysian strain did not have 5’ and 3’ UTR sequence
available for analysis and also UTR information were absent for two sequences each from 2015
and 2016 isolates respectively. The analysis revealed that both UTR sequences (5’ UTR and 3’
UTR) were mostly conserved. The sequences were also subjected to UTRscan web server to
predict any conserved UTR motif. UTRscan analysis showed presence of two motifs in the 3
‘UTR sequence, namely, uORF (upstream open reading frame) and MBE (Musashi binding
element) and no motifs were detected in 5’ UTR. Analysis of these motifs revealed that there are
nomenclature differences between prediction softwares and literature and uORF nomenclature
was homologous to dORF (downstream open reading frame) that was observed in the case of 3’
UTR (30). This motif has been reported in flaviviruses for the first time in this study even though
it has been shown to be present in mammalian UTRs and is found to be conserved thereby
highlighting their importance (30). The relevance of dORF in ZIKV warrants in-depth functional
studies. MBE, earlier referred to as polyadenylation response element are also known to play part
in temporal regulation in Xenopus (31, 32). Studies have shown the importance of this conserved
domain in promoting RNA genome cyclization (29, 33). In addition, secondary structures of both
the UTR sequences were predicted. RNAalifold was used for this purpose and the structures are
detailed in Figure 2. The results revealed that was conservation of the structures as previously
shown in a recent study (34).
Analysis of Recombination events
Recombination analyses were performed using RDP4 tool on all the 50 ZIKV genomes. Total of
11 events were predicted by more than five algorithms (p-value<0.05). Out of these, six events
were shown to be having one of the unknown parents, so they were not considered for further
analysis. The remaining five events consist of eight sequences namely, KF383115.1,
KF383116.1, KF383117.1, KF383118.1, KF383120.1, KF383121.1, HQ234501.1 and
HQ234498.1 (Table 3a). Further analysis showed that these sequences belong to African strains
(East African and West African). Recombination analyses were also performed for the individual
genes to predict the presence of any recombination event (Table 3b). The analysis done using all
algorithms with the above mentioned criteria showed one recombination event each for Envelope
and NS1 genes and two in NS3. In the case of Envelope, isolate KF383118.1 was a recombinant
with site 1-459 and 1041-1512 from major parent (KF383117.1) and site 460-1040 from minor
parent (LC002520.1). For NS1, recombinant was KF383117.1 (site 2-641) and minor and major
parent were KF383119.1 and KF383116.1 respectively. NS3 showed two events, out of which
one event showed KF383117.1 as recombinant (site 610-1070) with HQ23450.1 as major parent
and KF383119.1 as minor parent. In second NS3 event, KF383116.1 was recombinant (site 7321035) with HQ234501.1 as major parent and KF383119.1 as minor parent. The p-value for all
the events was found to be significant p-value<=0.05) for all the events. This result clearly
indicates that recombination events are only present in African isolates and absent in Asian
lineage at present. While studies have highlighted that flaviviruses have infrequent
recombination events in the field (35), a study have provided evidence of the presence of such
events in ZIKV (25).
Molecular Evolution - Selection test
The estimated Transition/Transversion bias value is 6.00. Substitution pattern and rates were
estimated under the Kimura-2 parameter model (+G+I). Selection analysis of genome sequences
was performed using Tajima’s neutrality test involving 50 nucleotide sequences. Tajima’s test
showed nucleotide diversity of 0.064089 and D value of 0.124450. Positive value of Tajima’s D
test suggests over dominant selection of these alleles in the population resulting on negative
selection (36, 37). Several studies have emphasized on the infection and transmission modes to
influence accumulation of negatively selected sites (25, 38).
Conclusion
In conclusion, our study is a comprehensive analysis of ZIKV genomes available till date. With
ZIKV infection spreading across the globe at an alarming rate, it is important to understand the
underlying molecular mechanisms that could aid the spread. Our analysis reveals balancing
selection of the identified amino acid variations thereby favoring fitness to the strains.
Acknowledgements
We thank ICGEB for the support. This work was supported by ICGEB core funds.
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Figure Legends
Figure 1. The phylogenetic tree constructed using Neighbor-Joining method is represented in the figure.
Bootstrap values are written next to the branches. For computing evolutionary distance, maximum
Likelihood method was used. Asian strains and African strains formed two distinct clusters and the tree
is rooted using Spondweni Virus as outgroup.
Figure 2. The figure represents the consensus secondary structure of UTR generated using RNAalifold tool. The bases written in black font are
conserve and the bases written in grey are absent or not sequenced in some of the isolates. a) Consensus secondary structure of 5’ UTR. b) Consensus
secondary structure of 3’ UTR.
Table 1a. The table represents the mutation identified in the consensus sequences of the structural protein of the isolates of year 20152016. The number of sequences used in the consensus for each region is also shown. The mutations were identified by comparing the
sequences with Malaysian isolate.
Protein
Polypeptide
position
Protein
position
Capsid
25
27
76
101
105
107
109
110
113
123
139
153
166
287
323
337
346
354
358
442
503
520
545
550
612
613
620
623
639
683
709
739
763
769
777
25
27
76
101
105
107
109
110
113
1
17
31
44
72
33
47
56
64
68
152
213
230
255
260
322
323
330
333
349
393
419
449
473
479
487
pr
M
Envelope
Malasiya
1966
(n=1)
N
L
E
R
G
D
S
I
I
V
S
V
M
P
V
T
V
S
M
I
V
D
V
S
L
H
V
A
M
D
K
F
V
T
T
Frenchpolynasia
2013 (n=1)
.
.
.
.
.
.
.
.
.
A
N
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
E
.
.
M
.
M
Puertrico
2015
(n=1)
S
F
.
K
.
.
.
V
V
A
N
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
E
.
.
M
.
M
Brazil
2015
(n=9)
S
F
.
K
G/S
.
.
V
V
A
N
M
.
.
.
T/S
.
T/S
I/M
.
.
.
V/A
S/T
.
.
.
.
M/T
E
.
.
M
.
M
Martinque Colambia Guatemala Suriname
2015
2015
2015
2015
(n=1)
(n=1)
(n=2)
(n=1)
S
S
S
S
F
F
F
F
.
.
.
.
K
K
K
K
.
.
.
.
E
E
.
.
.
.
.
.
V
V
V
V
V
V
V
V
A
A
A
A
N
N
N
N
M
M
M
M
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
I
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
E
E
E
E
.
.
.
.
.
.
.
.
M
M
M
M
.
.
.
A
M
M
M
M
Mexico
2016
(n=2)
S
F
.
K
.
E
.
V
V
A
N
M
.
P/L
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
E
.
.
M
.
M
China
2016
(n=6)
S
F
E/D
K
.
D/E
S/N
V
V
A
N
M
.
.
V/A
.
.
.
.
I/L
V/A
D/A
.
.
L/V
H/D
V/G
A/G
.
E
K/R
F/I
M
.
M
Italy
2016
(n=2)
S
F
.
K
.
.
.
V
V
A
N
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
E
.
.
M
.
M
Table 1b. The table represents the mutation identified in the structural protein sequences of the isolates of year
2015-2016. The mutations were identified by comparing the sequences with Malaysian isolate.
Protein
KU501215.1_Puertrico_2015
Capsid
N25S, L27F, R101K,
I110V, I113V
N25S, L27F, R101K,
KU729217.2_Brazil_2015
I110V, I113V
N25S, L27F, R101K,
KU707826.1_Brazil_2015
I110V, I113V
N25S, L27F, R101K,
KU729218.1_Brazil_2015
G105S, I110V, I113V
N25S, L27F, R101K,
KU321639.1_Brazil_2015
I110V, I113V
N25S, L27F, R101K,
KU497555.1_Brazil_2015
I110V, I113V
N25S, L27F, R101K,
KU365780.1_Brazil_2015
I110V, I113V
N25S, L27F, R101K,
KU365779.1_Brazil_2015
I110V, I113V
N25S, L27F, R101K,
KU365778.1_Brazil_2015
I110V, I113V
N25S, L27F, R101K,
KU365777.1_Brazil_2015
I110V, I113V
KU647676.1_Martinque_2015 N25S, L27F, R101K,
D107E, I110V, I113V
KU820897.1_Colambia_2015 N25S, L27F, D107E,
R101K, I110V, I113V
KU501217.1_Guatemala_2015 N25S, L27F, R101K,
I110V, I113V
KU501216.1_Guatemala_2015 N25S, L27F, R101K,
I110V, I113V
KU312312.1_Suriname_2015 N25S, L27F, R101K,
I110V, I113V
N25S, L27F, R101K,
KU922960.1_Mexico_2016
D107E, I110V, I113V
N25S, L27F, R101K,
KU922923.1_Mexico_2016
D107E, I110V, I113V
N25S, L27F, R101K,
KU866423.1_China_2016
S109N, I110V, I113V
N25S, L27F, R101K,
KU820898.1_China_2016
D107E, I110V, I113V
N25S, L27F, R101K,
KU740184.2_China_2016
I110V, I113V
N25S, L27F, S109N,
KU820899.2_China_2016
R101K, I110V, I113V
N25S, L27F, R101K,
KU761564.1_China_2016
I110V, I113V
N25S, L27F, E76D,
KU744693.1_China_2016
R101K, I110V, I113V
N25S, L27F, R101K,
KU853013.1_Italy_2016
I110V, I113V
N25S, L27F, R101K,
KU853012.1_Italy_2016
I110V, I113V
pr
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M, M44T
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
V1A, S17N,
V31M
M
-
E
D393E, V473M, T487M
-
T47S, S64T, M68I, V255A, D393E, V473M,
T487M
D393E, V473M, T487M
-
M349T, D393E, V473M, T487M
-
D393E, V473M, T487M
-
S260T, D393E, V473M, T487M
-
D393E, V473M, T487M
-
D393E, V473M, T487M
-
D393E, V473M, T487M
-
D393E, V473M, T487M
-
D393E, V473M, T487M
-
D393E, V473M, T487M
-
V56I, D393E, V473M, T487M
-
V56I, D393E, V473M, T487M
-
D393E, V473M, T479A, T487M
-
D393E, V473M, T487M
P72L D393E, V473M, T487M
-
D393E, K419R, V473M, T487M
-
D393E, V473M, T487M
-
D393E, V473M, T487M
-
D393E, K419R, V473M, T487M
-
D393E, V473M, T487M
-
V33A, I152L, V213A, D230A, L322V, H323D,
V330G, A333G, D393E, F449I, V473M, T487M
D393E, V473M, T487M
-
D393E, V473M, T487M
Table 2a. The table represents the mutation identified in the consensus sequences of non-structural protein of the isolates of year
2015-2016. The number of sequences used in the consensus for each region is also shown. The mutations were identified by
comparing the sequences with Malaysian isolate.
Protein Polypeptide Protein Malasiya Frenchposition
position
1966
polynasia
(n=1)
2013
(n=1)
NS1
NS2A
NS2B
Puertrico
2015
(n=1)
Brazil
2015
(n=9)
Martinque Colambi Guatemala Surinam
2015
a
2015
e
(n=1)
2015
(n=2)
2015
(n=1)
(n=1)
Mexico
2016
(n=2)
China
2016
(n=6)
Italy
2016
(n=2)
795
1
D
.
.
.
.
.
.
.
.
D/G
.
894
100
G
.
.
.
.
.
A
.
.
.
.
916
122
Y
.
.
Y/H
.
.
.
.
.
.
.
970
176
S
.
.
.
.
.
.
.
.
S/W
.
982
188
A
V
V
V
V
V
V
V
V
V
V
984
190
G
.
.
G/E
.
.
.
.
.
.
.
1005
211
R
.
.
.
.
.
.
.
.
R/W
.
1050
256
T
.
.
.
.
.
.
.
.
T/A
.
1058
264
V
M
M
M
M
M
M
M
M
M
M
1107
313
C
.
.
.
.
.
.
.
.
C/S
.
1118
324
R
.
.
.
W
W
.
.
W
R/Q
.
1143
349
M
.
.
M/V
.
.
.
.
.
.
V
1226
80
I
.
.
.
T
.
.
.
T
.
.
1259
113
L
.
.
L/F
.
.
.
.
.
.
.
1285
139
I
.
.
.
.
.
.
.
.
I/V
.
1289
143
A
V
V
V
V
V
V
V
V
V
V
1404
32
M
.
.
M/I
.
.
.
.
.
.
.
NS3
NS4B
NS5
1608
106
A
.
.
.
.
.
.
.
E
.
.
1836
334
M
.
.
M/T
.
.
.
.
.
.
.
1856
354
D
.
.
.
.
.
.
.
.
D/E
.
1857
355
H
.
.
H/Y
.
.
.
.
.
H/Y
.
1867
365
S
.
.
.
.
.
.
.
.
S/R
.
1902
400
N
H
H
H
H
H
H
H
H
H
H
1938
436
D
.
.
.
.
.
.
.
.
D/G
.
1974
472
M
L
L
L
L
L
L
L
L
L
L
2027
525
R
.
.
.
.
.
.
.
.
R/K
.
2074
572
M
.
.
.
.
.
L
.
.
.
.
2283
14
G
S
S
S
S
S
S
S
S
S
S
2295
26
M
I
I
I
I
I
I
I
I
I/M
I
2313
44
A
.
.
.
.
.
.
.
.
A/P
.
2317
48
T
.
.
.
.
.
.
.
.
T/S
.
2318
49
L
F
F
F
F
F
F
F
F
F
F
2367
98
M
I
I
I
I
I
I
I
I
I
I
2419
150
D
.
.
.
.
.
.
.
.
D/E
.
2445
176
I
.
.
I/M
.
.
.
.
.
I/M
.
2449
180
I
V
V
V
V
V
V
V
V
V
V
2453
184
V
I
I
I
I
I
I
I
I
I
I
2455
186
L
S
S
S
S
S
S
S
S
S
S
2611
91
A
.
V
.
.
.
.
.
.
.
.
2634
114
T
M
V
V
V
V
V
V
V
M/V
V
2644
124
V
.
.
.
.
.
.
.
.
V/I
.
2659
139
S
P
P
P
P
P
P
P
P
P
P
2694
174
K
.
.
.
.
.
R
.
.
.
.
2749
229
I
T
T
T
T
T
T
T
T
T/I
T
2778
258
N
.
.
N/D
.
.
.
.
.
.
.
2787
267
A
V
V
V
V
V
V
V
V
V/A
V
2795
275
L
M
M
M
M
M
M
M
M
M
M
2800
280
N
.
.
N/D
.
.
.
.
.
.
.
2802
282
V
I
I
I
I
I
I
I
I
I
I
2807
287
S
.
.
.
.
.
.
.
.
S/A
.
2809
289
H
.
.
.
.
.
.
.
Q
H/K
.
2831
311
E
.
.
E/V
.
.
.
.
.
E/D
.
2833
313
P
.
.
.
.
.
.
.
.
P/A
.
2842
322
I
.
.
.
.
.
.
.
.
.
V
2896
376
N
S
S
S
S
S
S
S
S
S
S
2974
454
N
.
.
.
.
.
.
.
.
N/I
.
2975
455
M
.
.
.
.
.
.
.
.
M/T
.
3030
510
G
.
.
.
.
.
.
.
V
.
.
3045
525
R
.
.
.
.
.
C
.
.
.
.
3046
526
T
I
I
I
I
I
I
I
I
I
I
3050
530
K
R
R
R
R
R
R
R
R
R
R
3107
587
R
K
K
K
K
K
K
K
K
K
K
3144
624
N
.
.
.
.
.
.
.
.
S/N
.
3162
642
P
S
S
S
S
S
S
S
S
S
S
3167
647
S
N
N
N
N
N
N
N
N
N
N
3190
670
K
.
.
.
.
.
.
.
.
R/K
.
3223
703
S
D
D
D
D
D
D
D
D
D
D
3239
719
Y
H
H
H
H
H
H
H
H
H
H
3334
814
V
.
.
.
.
.
.
.
.
V/A
.
3353
833
T
.
.
.
A
A
.
.
A
.
.
3387
867
D
N
N
N
N
N
N
N
N
N
N
3392
872
V
.
.
.
.
.
.
.
.
V/M
.
3398
878
D
.
.
.
.
.
.
.
.
.
E
3403
883
M
.
.
.
.
.
.
.
.
M/V
.
Table 2b. The table represents the mutation identified in the non-structural protein sequences of the individual isolates of year 20152016. The mutations were identified by comparing the sequences with Malaysian isolate.
Sequences/Proteins
KU501215.1_Puertrico_2015
NS1
A188V,
V264M
NS2A
A143V
NS2B
NS3
N400H,
M472L
NS4B
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU729217.2_Brazil_2015
A188V,
G190E,
V264M,
M349V
A188V,
V264M
A143V
M32I
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
A143V
-
N400H,
M472L
KU729218.1_Brazil_2015
A188V,
V264M
A143V
-
KU321639.1_Brazil_2015
Y122H,
A188V,
V264M
A188V,
V264M
A143V
-
L113F,
A143V
-
M334T,
N400H,
M472L
H355Y,
N400H,
M472L
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, L49F, M98I,
I176M, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU365780.1_Brazil_2015
A188V,
V264M
A143V
-
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU365779.1_Brazil_2015
A188V,
V264M
A143V
-
N400H,
M472L
KU365778.1_Brazil_2015
A188V,
V264M
A143V
-
N400H,
M472L
KU365777.1_Brazil_2015
A188V,
V264M
A143V
-
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU647676.1_Martinque_2015
A188V,
V264M,
R324W
I80T,
A143V
-
N400H,
M472L
KU707826.1_Brazil_2015
KU497555.1_Brazil_2015
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
NS5
A91V, T114V, S139P, I229T, A267V,
L275M, V282I, N376S, T526I, K530R,
R587K, P642S, S647N, S703D, Y719H,
D867N
T114V, S139P, I229T, A267V, L275M,
N280D, V282I, N376S, T526I, K530R,
R587K, P642S, S647N, S703D, Y719H,
D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, E311V, N376S, T526I, K530R,
R587K, P642S, S647N, S703D, Y719H,
D867N
T114V, S139P, I229T, N258D, A267V,
L275M, V282I, N376S, T526I, K530R,
R587K, P642S, S647N, S703D, Y719H,
D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, D867N
T114V, S139P, I229T, N258D, A267V,
L275M, V282I, N376S, T526I, K530R,
R587K, P642S, S647N, S703D, Y719H,
D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, T833A,
D867N
A188V,
V264M,
R324W
A143V
-
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU501217.1_Guatemala_2015 G100A,
A188V,
V264M
A143V
-
N400H,
M472L,
M572L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU501216.1_Guatemala_2015 G100A,
A188V,
V264M
A188V,
KU312312.1_Suriname_2015
V264M
A143V
-
A143V
-
N400H,
M472L,
M572L
N400H,
M472L
KU820897.1_Colambia_2015
KU922960.1_Mexico_2016
A188V,
V264M,
R324W
I80T,
A143V
-
A106E,
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU922923.1_Mexico_2016
A188V,
V264M,
R324W
I80T,
A143V
-
A106E,
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU866423.1_China_2016
A188V,
V264M,
R324Q
A143V
-
N400H,
M472L,
R525K
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU820898.1_China_2016
A188V,
V264M
I139V,
A143V
-
N400H,
M472L
KU740184.2_China_2016
A188V,
V264M
I139V,
A143V
-
N400H,
M472L
KU820899.2_China_2016
A188V,
V264M,
R324Q
A143V
-
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
KU761564.1_China_2016
A188V,
V264M
I139V,
A143V
-
N400H,
M472L
KU744693.1_China_2016
D1G, S176W,
A188V,
R211W,
T256A,
A143V
-
D354E,
H355Y,
S365R,
N400H,
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
G14S, A44P, T48S,
L49F, M98I,
D150E, I176M,
I180V, V184I,
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, T833A,
D867N
T114V, S139P, K174R, I229T, A267V,
L275M, V282I, N376S, R525C, T526I,
K530R, R587K, P642S, S647N, S703D,
Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, H289Q, N376S, G510V, T526I,
K530R, R587K, P642S, S647N, S703D,
Y719H, T833A, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, H289Q, N376S, G510V, T526I,
K530R, R587K, P642S, S647N, S703D,
Y719H, T833A, D867N
T114M, V124I, S139P, I229T, A267V,
L275M, V282I, N376S, T526I, K530R,
R587K, N624S, P642S, S647N, K670R,
S703D, Y719H, D867N, V872M, M883V
T114V, S139P, L275M, V282I, N376S,
T526I, K530R, R587K, P642S, S647N,
S703D, Y719H, D867N
T114V, S139P, L275M, V282I, N376S,
T526I, K530R, R587K, P642S, S647N,
S703D, Y719H, D867N
T114M, S139P, I229T, A267V, L275M,
V282I, N376S, T526I, K530R, R587K,
N624S, P642S, S647N, K670R, S703D,
Y719H, D867N
T114V, S139P, L275M, V282I, N376S,
T526I, K530R, R587K, P642S, S647N,
S703D, Y719H, D867N
T114V, S139P, I229T, A267V, L275M,
V282I, S287A, H289K, E311D, P313A,
N376S, N454I, T526I, K530R, R587K,
P642S, S647N, S703D, Y719H, V814A,
V264M, C313S
KU853013.1_Italy_2016
A188V,
V264M,
M349V
A143V
-
KU853012.1_Italy_2016
A188V,
V264M
A143V
-
D436G,
M472L
N400H,
M472L
L186S
D867N
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
N400H,
M472L
G14S, M26I, L49F,
M98I, I180V,
V184I, L186S
T114V, S139P, I229T, A267V, L275M,
V282I, I322V, N376S, T526I, K530R,
R587K, P642S, S647N, S703D, Y719H,
D867N, D878E
T114V, S139P, I229T, A267V, L275M,
V282I, I322V, N376S, T526I, K530R,
R587K, P642S, S647N, S703D, Y719H,
D867N, D878E
Table 3a. Recombination analysis of whole genome sequences of ZIKA virus. ‘+’ sign represents the prediction of event by
respective method and ‘-‘symbol represent no result predicted by respective method.
Recombinant
Major
Minor parent
parent
RDP
GENECONV
BootScan
MaxiChi (P-
Chimaera (P-
SiScan
3Seq
(P-Val)
(P-Val)
(P-Val)
Val)
Val)
(P-Val)
(P-Val)
KF383117.1
KF383116.1
KF383115.1
+ (6.975E-32)
+ (2.081E-32)
- (NA)
+ (8.794E-7)
+ (9.072E-12)
+ (7.309E-11)
+ (1.917E-9)
KF383118.1
KF383121.1
KF383117.1
+ (4.050E-31)
+ (1.319E-26)
- (NA)
+ (4.147E-8)
+ (7.136E-8)
+ (7.859E-8)
+ (4.460E-4)
KF383117.1
HQ234501.1
KF383121.1
+ (7.007E-19)
+ (1.688E-18)
- (NA)
+ (1.461E-5)
+ (1.020E-5)
+ (7.178E-7)
+ (2.394E-8)
KF383117.1
KF383116.1
KF383118.1
+ (5.698E-22)
+ 5.905E-20)
- (NA)
+ (7.158E-7)
+ (5.999E-7)
+ (2.570E-7)
+ (2.754E-3)
KF383118.1
HQ234498.1
KF383120.1
+ (3.315E-19)
+ (2.896E-9)
- (NA)
+ (3.221E-7)
- (NA)
+ (5.905E-3)
+ (1.334E-2)
Table 3b. The recombination analysis results of individual genes of ZIKA virus. ‘+’ sign represents the prediction of event by
respective method and ‘-‘symbol represent no result predicted by respective method.
Genes
Recombinant
Major
Minor
parent
parent
RDP (P-Val) GENECONV
(P-Val)
BootScan
MaxiChi
Chimaera
SiScan (P-
3Seq
(P-Val)
(P-Val)
(P-Val)
Val)
(P-Val)
Envelope
KF383118.1
LC002520.1 KF383117.1 + (1.065E-10) + (6.088E-10) + (7.162E-10) + (6.415E-10) + (4.014E-10) + (7.242E-10) + (5.422E-12)
NS1
KF383117.1
KF383116.1 KF383119.1 + (1.553E-07) + (6.592E-06) + (1.587E-05) + (1.418E-03) + (4.026E-07) + (2.529E-07) + (1.290E-12)
NS3
KF383117.1 HQ234501.1 KF383119.1 + (2.504E-13) + (6.474E-11) + (5.521E-09) + (7.745E-10) + (6.190E-10) + (2.222E-11) + (5.188E-18)
NS3
KF383116.1 HQ234501.1 KF383119.1 + (3.031E-08) + (2.575E-09) + (5.414E-11) + (9.034E-04) + (4.071E-05) + (5.747E-07) + (2.674E-09)
Supplementary Material
Supplementary Table 1: Sequences used in the study.
S. No
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
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Accession Number
KU501217.1
KU501216.1
KU501215.1
KU647676.1
KU729217.2
KU729218.1
KU853013.1
KU853012.1
KU321639.1
KU497555.1
KU312312.1
KU707826.1
KF268950.1
KF268949.1
KF268948.1
KF993678.1
KJ776791.1
KF383121.1
KF383119.1
KF383118.1
KF383115.1
KF383120.1
KF383117.1
KF383116.1
JN860885.1
HQ234499.1
HQ234498.1
HQ234501.1
HQ234500.1
DQ859059.1
EU545988.1
AY632535.2
KU922960.1
KU922923.1
KU866423.1
KU820898.1
KU740184.2
KU820899.2
KU820897.1
KU761564.1
KU681082.3
KU681081.3
KU744693.1
KU509998.1
KU365780.1
KU365779.1
KU365778.1
KU365777.1
KU720415.1
LC002520.1
Country
Guatemala
Guatemala
Puertrico
Martinque
Brazil
Brazil
Italy
Italy
Brazil
Brazil
Suriname
Brazil
Central African Republic
Central African Republic
Central African Republic
Canada
French Polynasia
East African
Senegal
Senegal
Central African Republic
Senegal
Senegal
Senegal
Cambodia
Malasiya
Uganda
Senegal
Nigeria
Uganda
Micronesia
Uganda
Mexico
Mexico
China
China
China
China
Colambia
China
Phillipines
Thialand
China
Haiti
Brazil
Brazil
Brazil
Brazil
Uganda
Uganda
Year
2015
2015
2015
2015
2015
2015
2016
2016
2015
2015
2015
2015
1976
2013
2013
2001
2001
1968
2000
1997
2010
1966
1947
1984
1968
2007
2016
2016
2016
2016
2016
2016
2015
2016
2012
2014
2016
2014
2015
2015
2015
2015
1947
-
References (PMID/DOI)
10.3201/eid2205.160065
10.3201/eid2205.160065
10.3201/eid2205.160065
10.1016/j.nmni.2016.02.013
27013429
27013429
26987769
26987769
26941134
26897108
26775124
26401719
25514122
25514122
25514122
25294619
24903869
24421913
24421913
24421913
24421913
24421913
24421913
24421913
22389730
22389730
22389730
22389730
22389730
19741066
18680646
16223950
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Supplementary Figure 1: Phylogenetic trees of ZIKA virus predicted using other methods. a) Maximum
Likelihood tree. b) Minimum-Evolution tree. C) Maximum Parsimony tree. d) UPGMA tree.