Vol. 18(30), pp. 928-934, October, 2019
DOI: 10.5897/AJB2018.16738
Article Number: BC40DDF62154
ISSN: 1684-5315
Copyright ©2019
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJB
African Journal of Biotechnology
Full Length Research Paper
Characterization of three full Iris yellow spot virus
genes of a garlic-infecting isolate from Zimbabwe using
next-generation sequencing technology
Charles Karavina1,2*, Jacques Davy Ibaba1 and Augustine Gubba1
1
Department of Plant Pathology, School of Agriculture, Earth and Environmental Sciences, University of KwaZulu-Natal,
Private Bag X01, Pietermaritzburg, South Africa.
2
Department of Crop Science, Bindura University of Science Education, Private Bag 1020, Bindura, Zimbabwe.
Received 28 December, 2018; Accepted 29 January, 2019
Iris yellow spot virus (IYSV) is an important pathogen of Allium species worldwide. It has a tripartite
genome consisting of the large (L), medium (M) and small (S) RNA segments. Despite its worldwide
distribution, very few complete gene and genome sequences are available in public databases. The aim
of this study was to obtain full gene sequences of a garlic-infecting IYSV isolate by next-generation
sequencing (NGS) for understanding its evolution. Total RNA was extracted from an IYSV-positive
garlic leaf and sequenced on the Illumina HiSeq platform using paired-end chemistry 125 × 125 bp
reads. The quality of raw reads was assessed using FastQC software before trimming with
Trimmomatic version 0.36. The resultant paired-end sequences were used for both de novo and
reference-based genome assembly. The resultant consensus gene sequences were analyzed using
SIAS (for sequence identity and composition), ExPASy (for protein molecular weight) and ORF Finder
(for open reading frame identification). Three full gene sequences, that is, nucleocapsid (N),
nonstructural protein (NSs) and movement protein (NSm) were recovered. The N gene did not display
any distinct clustering patterns based on geographical locations and was most identical to an onioninfecting isolate from Serbia (Accession KT272878). The NSs and NSm genes clustered closely with
homologous sequences of IYSV isolates that were retrieved from GenBank and EMBL. This study lays
the foundation for complete genome studies of IYSV in Zimbabwe.
Key words: Allium species, emerging pathogen, reverse transcription polymerase chain reaction (RT-PCR),
serology, tospovirus.
INTRODUCTION
Iris yellow spot virus, IYSV, is an important emerging
pathogen of Allium species worldwide responsible for
causing significant yield and quality losses in alliaceous
and ornamental crops (Bag et al., 2015). It was first
isolated and characterized in iris (Iris holandica) in The
Netherlands (Cortês et al., 1998) and has now been
confirmed to be present in over 40 countries worldwide
(CABI, 2018). In Zimbabwe, IYSV was first detected
infecting onions (Allium cepa) in 2014 (Karavina et al.,
2016) and was subsequently reported infecting garlic
(Allium sativum), leeks (Allium ampeloprasum) and
shallots (A. cepa var. aggregatum) (Karavina and Gubba,
Karavina et al.
2017).
IYSV belongs to the genus virus in the family
Tospoviridae. Like other tospoviruses, IYSV has quasispherical enveloped particles that are 80 to 120 nm in
diameter (Pappu et al., 2009). It has a tripartite singlestranded RNA genome consisting of the large (L),
medium (M) and small (S) segments. The L RNA
encodes the RNA-dependent RNA polymerase (RdRp) in
the viral complementary sense (Bag et al., 2010). The M
RNA is ambisense and has two open reading frames
(ORFs). The viral sense strand of the M RNA encodes
the non-structural movement (NSm) protein, while the
anti-viral sense strand encodes the Gn/Gc glycoproteins.
The NSm protein is responsible for virus particle
movement during systemic infection, while the two
glycoproteins serve as virus attachment proteins (Bandla
et al., 1998). The S RNA is also ambisense and encodes
the non-structural (NSs) protein in the viral sense strand
and the nucleocapsid (N) protein in the antiviral sense
strand. The NSs protein is involved in suppressing RNA
silencing, while the N protein encapsidates the RNA
segments (Bag et al., 2015). In addition to these genes,
both M and S RNAs contain intergenic regions (IGRs)
capable of forming stable hairpin structures (King et al.,
2012). IYSV is currently known to be transmitted by two
thrips species, Thrips tabaci and Frankliniella fusca in a
persistent and propagative mode (Srinivasan et al.,
2012).
IYSV identification and characterization is carried out
by employing biological, serological, morphological and
molecular techniques (Bag et al., 2015). Biological
characterization, also known as host indexing, involves
the inoculation of indicator plant species that produce
typical symptoms. However, it normally takes several
days for symptoms to develop on inoculated hosts (Bag
et al., 2012). Serological detection can lead to ambiguous
results, especially amongst closely related viruses like
IYSV and Tomato yellow ring virus (TYRV). Also,
antibodies to IYSV must be raised if serological detection
is to be successful. Morphological identification involves
the use of electron microscopy, a technique that is
technically challenging and not affordable in most
research and academic institutions in developing
countries. Being RNA viruses, reverse transcriptionpolymerase chain reaction (RT-PCR) can also be
employed for IYSV identification (Walsh et al., 2001).
However, primers specific for IYSV detection must be
available/designed.
Next-Generation Sequencing (NGS) provides an
unbiased,
quick,
costand
labour-effective
characterization procedure for plant viruses (Kreuze et
al., 2009). Two assembly methods, namely, de novo
929
assembly and reference-based mapping are used to
recover the virus genome from the sequenced data. With
NGS, full viral genomes can be recovered at once
(Adams et al., 2009).
In public databases like NCBI and EMBL, except for the
N gene, there are no more than five complete IYSV gene
sequences available. This has negatively impacted
evolutionary studies of this pathogen. In this study, NGS
was used to characterize three full genes of a garlicinfecting IYSV isolate from Zimbabwe.
MATERIALS AND METHODS
Sources of materials used
IYSV was isolated from garlic plants collected at a horticultural farm
in Chegutu, Zimbabwe, in 2015. Garlic plants that displayed
necrotic, irregularly-shaped and grey-to-bleached lesions typical of
IYSV infection were targeted during sample collection. Young
leaves from symptomatic plants were collected in RNAlater®
solution (Thermo Fisher Scientific, USA) and transported to the
University of KwaZulu-Natal (South Africa) for pathogen
characterization.
Serological detection
Eight leaf samples were tested for IYSV in duplicate wells using a
commercial kit supplied by Loewe® Biochemica GmbH (Sauerlach,
Germany) following the manufacturer’s instructions. Briefly,
microtiter plates were coated with IYSV-specific antibodies. About
0.5 g of garlic leaf tissue showing disease symptoms were excised
and ground in liquid nitrogen in a pestle and mortar. Macerated
tissues were mixed with Conjugate Buffer at 1:20 dilution, and 0.2
ml mixture added to each microtiter well before overnight
incubation. After four washes, an enzyme-labelled antibody-APconjugate was applied to the plate wells. In the final step, 0.2 ml of
the Substrate Buffer Solution containing the dissolved substrate
was applied to the microtiter plate. After 2 h of incubation, the
reaction was visually assessed for yellow color development which
is characteristic of a positive test.
RNA extraction and RT-PCR
Total RNA was extracted from the garlic leaves using the Quick
RNA MiniPrep Kit (Zymo Research, USA) according to
manufacturer’s instructions. RT-PCR was performed using the
RevertAid RT Reverse Transcription Kit (Thermo Fischer Scientific,
USA) targeting the N gene. The resultant cDNA was used for PCR
amplification using the KAPA2G Fast Hot Start ReadyMix Kit (KAPA
Biosystems, USA) and N gene-specific primers (IYSV_2F: 5'GGCGGTCCTCTCATCTTACTG-3'
and
IYSV-NCP2_R:
5'GAAGTTCCAGGAGTGCATTTAGTC-3') (Lee et al., 2011) under
the following cycling conditions: initial denaturation at 95°C for 2
min; 35 cycles of 94°C for 15 s (denaturation step), 57°C for 15 s
(annealing step), and 72°C for 15 s (extension step), followed by a
5-min incubation period at 72°C. PCR products were analyzed by
*Corresponding author. E-mail: ckaravina@gmail.com. Tel: +263-772335845.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
930
Afr. J. Biotechnol.
1.5% agarose gel electrophoresis. The amplicons were excised,
purified using a ZymocleanTM Gel DNA Recovery Kit (Zymo
Research, USA) and directly sequenced in both directions at
Inqaba Biotechnical Industries (Pty) Ltd (Pretoria, South Africa).
Sequences generated were blasted into MEGA 6.06 program.
positive samples turned yellow within 2 h of incubation
after the final wash step. The 236-bp bands were
visualized from samples that were IYSV-positive after
electrophoresis on 1.5% agarose gel stained with SYBR
Safe Gel stain (Life Technologies, USA).
Sample preparation, sequencing and data analysis for NGS
A sample that was IYSV-positive by both DAS-ELISA and RT-PCR
was randomly selected and RNA extracted for NGS at the
Agricultural Research Council’s Biotechnology Platform (ARC-BTP)
(Pretoria, South Africa). RNA quality and quantity were assessed
using a nanodrop 2000c spectrophotometer (Thermo Fisher
Scientific, USA). Total RNA samples were pre-treated with RiboZero (NEB, UK) prior to library preparation.
NGS was performed on the Illumina HiSeq platform using
paired`-end chemistry 125 × 125 bp reads. Subsequent sample
demultiplexing was done using the CASAVA pipeline software
(Illumina, USA). Read lengths of less than 25 nucleotides were
trimmed and pair-end sequence libraries were generated. FastQC
version 0.11.5 (https://www.bioinformatics.babraham.ac.uk/projects/
fastqc/ ) was used to assess quality of the raw reads generated by
NGS before and after trimming with the Trimmomatic v 0.36 with
the following settings:
(ILLUMINACLIP:
TrueSeq3-PE-2.fa:2:30:9:1:true
LEADING:3
TRAILING:3 SLIDINGWINDOW:4:15:MINLEN:36) (Bolger et al.,
2014).
The pair-end sequences were subsequently used for both de novo
assembly and reference-based mapping. De novo assembly was
performed using the SPAdes Genome Assembler version 3.10.1
with the parameters: careful mode; only assembler; –k 21, 33, 55,
77, 99 (Bankevich et al., 2012), while referenced-based genome
mapping was done using the BBMap Short Read Aligner version
37.28 (Bushnell, 2014) using IYSV genomes as references. All
contigs generated by the de novo assembly method were subjected
to BLAST using ncbi-blast 2.6.0+ (www.ncbi.nih.gov).
IYSV sequence analysis and phylogeny
All contigs that matched IYSV genomes were selected and aligned
using the Clustal W embedded in MEGA 6.06 software (Tamura et
al., 2013) to generate the consensus sequences of the N, NSs and
NSm genes. The open reading frames (ORFs) on the IYSV genes
were
identified
using
ORF
Finder
(https://www.ncbi.nlm.nih.gov/orffinder). Molecular weight (Mw) of
proteins was determined using the online ExPASy bioinformatics
tool (Gasteiger et al., 2005). Phylogenetic trees of the nucleotide
sequences of the three IYSV ORFs were inferred by Maximum
Likelihood method based on the best evolutionary models as
determined by MEGA 6.06. The trees were rooted using the TSWV
sequence as an outgroup. Bootstrap analyses were conducted
using 1000 replicates. Details of isolates used in the phylogenetic
analysis are shown in Table 3. Nucleotide and amino acid
sequence compositions and sequence identities were calculated
using SIAS program (www.imed.ucm.es/Tools/sias.html).
RESULTS
Serology and RT-PCR analyses
Of the eight samples that were tested, six were positive
for IYSV by both DAS-ELISA and RT-PCR. All IYSV-
RNA quality assessment and NGS data analysis
The concentration of the RNA sample that was sent for
NGS was 194.38 ng/µL, with an absorbance A260/A280
ratio of 1.95. The size of the NGS data generated was
3.3 GB and consisted of 6 921 806 raw reads. The
average reads length after trimming was 119 bp (Table
1). A total of 107 102 contigs were generated by de novo
analysis. Of these, 18 matched to the L (2), M (8) and S
(8) RNA segments of the IYSV genome. From the
reference-based mapping, a total of 671 reads were
mapped to the IYSV genome (Table 1). Although the
results obtained using both de novo assembly and
reference-based mapping methods were consistent, full
IYSV genome segments of the garlic Zimbabwe (garlicZim) isolate were not recovered. However, three genes
(N and NSs found on the S RNA segment and the NSm
present on the M RNA segment) were found to be
complete after visual inspection and analysis on the ORF
Finder. Consequently, these genes were considered for
phylogenetic analyses. The nucleotide sequences of
these genes were deposited in GenBank under the
accession numbers shown in Table 2.
Sequence characteristics and phylogenetic analyses of
the N, NSs and NSm genes were from the IYSV garlicZim isolate
The N gene of the IYSV garlic-Zim isolate was 822 nt
long and coded for a protein with a molecular weight of
30.46 kDa. The NSs and NSm proteins had molecular
weights of 50.11 and 34.73 kDa, respectively (Table 2).
The N gene of the garlic-Zim isolate had a sequence
identity of 93.06% to the onion-infecting Serbian isolate
(KT272878) at the nucleotide level, while at the protein
level; it was most identical (95.25%) to the onion-infecting
Sri Lankan isolate (GU901211). It shared the lowest
nucleotide and protein sequence identity with the Iranian
isolate (HQ148173). The NSs gene sequence of the
garlic-Zim isolate was 91.66% identical to The
Netherlands isolate (AF001387) at the nucleotide level.
As for the NSm gene, it was most identical to the USA
isolate (FJ361359) at both nucleotide and amino acid
levels (Table 3).
Phylogenetic analysis of the N genes produced two
distinct clusters (A and B; Figure 1). The N gene of the
IYSV garlic-Zim isolate was in cluster A along with
homologous sequences of isolates from Australia, Brazil,
Egypt, India, Israel and Sri Lanka. Cluster B composed of
N sequences of isolates from Iran, Japan, Serbia, The
Netherlands and The UK (Figure 1).
The NSs gene of the garlic-Zim isolate clustered with
the homologous sequences of isolates from India and
Karavina et al.
931
Table 1. Characteristics of the NGS data generated from total RNA infected by IYSV.
Characteristics
Number of raw reads
Average read length
Number of reads after trimming
Average reads length after trimming and adapter removal
Number of contigs generated
Length of contigs
Contigs matching IYSV
Number of reads mapped to L RNA
Number of reads mapped to M RNA
Number of reads mapped to S RNA
Statistics
6 921 806
125 bp
5 758 205
119 bp
107 102
100-31303 bp
18 (S: 2, M: 8, L: 8)
336
133
202
Table 2. Sequence characteristics of the N, NSs and NSm genes of the garlic-Zim isolate.
Segment
S
M
ORF
polarity
(-)
(+)
Accession
number
MF359019
MF359021
Protein
coded
N
NSs
(+)
MF359020
NSm
ORF length
(nt)
822
1332
Number of amino
acids
273
443
Protein weight
(kDa)
30.46
50.11
983
311
34.73
Table 3. Percentage nucleotide (nt) and protein (aa) sequence identities between the IYSV N, NSs and NSm genes and other IYSV
isolates.
Accession No.
Isolate
AF001387
AF067070
AF271219
KT272878
JQ973066
AB180919
AM900393
AY345226
EU310295
EU477515
EU586203
GU901211
HQ148173
KF171105
KT225547
KJ868797
FJ361359
AF213677
AF214014
KM035409
163-14
Washington
SgOniD1
NSW-1
IYSV-On-Vir
New Zealand
605-SRB
5
IYSV-Egyptian
DOGR
DOGR
N
Nt
41.36
92.33
91.11
93.06
92.94
42.09
41.22
89.9
91.97
92.7
41.96
92.57
41.05
92.21
85.76
-
NSs
Aa
17.14
93.79
92.7
93.23
94.52
16.08
17.62
92.7
93.79
93.23
16.18
95.25
15.82
94.52
86.49
-
The Netherlands (Figure 2A). As for the NSm gene, it also
clustered with homologous sequences of IYSV isolates
nt
91.66
91.51
-
NSm
aa
82.02
83.59
-
nt
97.11
95.19
89.63
87.07
aa
99.67
95.83
94.55
89.42
from Brazil (AF213677) and The USA (FJ361359) (Figure
2B).
932
Afr. J. Biotechnol.
JQ973066_USA
EU477515_New_Zealand
KT272878_Serbia
KF171105_Pakistan
AF067070_Brazil
MF359019_garlic-Zim
A
KT225547.1_Egypt
GU901211_Sri_Lanka
AF271219_Israel
AY345226_Australia
EU310295_India
AB180919_Japan
EU586203_Serbia
HQ148173_Iran
B
AF001387_Netherlands
AM900393_UK
DQ915947_TSWV
Figure 1. Phylogenetic analysis of the N gene of the garlic-Zim isolate by the Maximum Likelihood method based on the
Tamura 3-parameter model.
AF001387_IYSV_Netherlands
FJ361359_IYSV_USA
KJ868797_IYSV_India
AF213677_IYSV_Brazil
MF359021_garlic-Zim
MF359020_garlic-Zim
AY686718_TYRV
AF214014_IYSV_Netherlands
KC290943_HCRSV
JX833564_SLNSV
KM035409_IYSV_India
JN560177_TYRV
KX468765_PoRSV
JX833565_SLNSV
KM589495_CaCV
A
KX468766_PoRSV
B
Figure 2. Molecular phylogenetic analyses of the NSs (A) and NSm (B) genes by Maximum Likelihood method based on the
Tamura 3-parameter model.
Karavina et al.
DISCUSSION
The genomic organization of the garlic-Zim isolate is
typical of tospoviruses in being tripartite (Pappu et al.,
2009). Though no full genomic segments were
recovered, NGS enabled the simultaneous recovery of
the two full genes on the S RNA segment and one gene
on the M segment. This is not possible with Sanger
sequencing. This study lays the foundation for future
studies on the full genome of IYSV in Zimbabwe.
Despite the global importance of IYSV, only a few full
genome and gene sequences have been characterized
(Gawande et al., 2015). This greatly compromises
studies to understand pathogen evolution and
management. The current study is first in Africa to
characterize more than one full gene sequence of the
same IYSV isolate. The only other full IYSV gene
sequences from Africa deposited in public databases are
from Egypt (Accessions KT225547.1 and KC161369.1).
Phylogenetic analysis of the N gene showed no specific
clustering patterns based on geographical locations. This
could suggest the possibilities of long-distance migration,
recombination and reassortment events in IYSV. Such
events are highly prevalent in tospoviruses (Margaria et
al., 2015; Zhang et al., 2016). Being a pathogen of some
internationally-traded ornamental plants (Bag et al., 2015;
CABI, 2018), it is possible that IYSV and its vectors have
been unintentionally distributed worldwide in live plant
shipments. Also, the smuggling of live host plants across
borders could have contributed to pathogen’s worldwide
distribution. The S segment is known to be substantially
more prone to recombination than the M and L segments
(Gawande et al., 2015). For the occurrence of either
recombination or reassortment to be confirmed in the
Gar-Zim isolate, full genomic segments must be
recovered and analyzed.
In addition to DAS-ELSIA and serology, NGS was
employed in further confirming the occurrence of IYSV in
Zimbabwe. Knowledge of IYSV presence is crucial in
epidemiological studies towards developing effective
disease control strategies. When compared to serology
and RT-PCR, NGS is a more rapid procedure and it also
produces nucleic acid sequences for substantial parts of
the viral genome. Another major advantage of NGS over
DAS-ELISA and RT-PCR is that the latter approaches
require reagents designed exclusively to detect their viral
target and any variation in the virus genome may cause
the assay to fail. NGS is non-targeted and requires no
prior knowledge of the target. Therefore, it can detect
existing strains, new variants and even new strains
(Adams et al., 2009).
To maximize the chances of detecting IYSV in the
sequenced data, two different methods (de novo
assembly and reference-based mapping) were employed.
De novo assembly recreates the original genome
sequence through overlapping reads while referencebased mapping requires a previously assembled genome
933
to be used as a reference. A major advantage of de novo
assembly over reference-based mapping is that it gives
the virome of the host(s) studied. It also detects other
viruses not targeted by the study (Martin and Wang,
2011). In this study, Garlic common latent virus, Garlic
virus B, Garlic virus C and Shallot virus X were detected
(data not shown). There were no significant differences in
the IYSV genes that were recovered by both de novo and
reference-based mapping.
The fact that viruses other than IYSV were detected in
the sample that was sent for NGS shows that mixed and
multiple infections are common in nature. This implies
that symptom expression cannot be conclusively relied
upon for disease diagnosis.
Conclusion
The characterized IYSV genes of the garlic-Zim isolate
are the foundation for future full genome studies of this
important pathogen. Knowledge of the full genome is
critical in understanding the evolutionary patterns of
IYSV. Pathogen genomic information is also important in
developing IYSV disease management strategies.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
ACKNOWLEDGEMENTS
The authors would like to thank The Kellogg Foundation
Southern African Scholarship Program for financial
support.
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