Characterization of the first complete genome sequence of an Impatiens necrotic spot orthotospovirus isolate from the United States and worldwide phylogenetic analyses of INSV isolates
Zhao et al. BMC Res Notes
Characterization of the first complete genome sequence of an Impatiens necrotic spot orthotospovirus isolate from the United States and worldwide phylogenetic analyses of INSV isolates
Kaixi Zhao 0
Paolo Margaria 0 1
Cristina Rosa 0
0 Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University , University Park, PA 16802 , USA
1 Plant Virus Department, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH , 38124 Brunswick , Germany
Objective: Impatiens necrotic spot orthotospovirus (INSV) can impact economically important ornamental plants and vegetables worldwide. Characterization studies on INSV are limited. For most INSV isolates, there are no complete genome sequences available. This lack of genomic information has a negative impact on the understanding of the INSV genetic diversity and evolution. Here we report the first complete nucleotide sequence of a US INSV isolate. Results: INSV-UP01 was isolated from an impatiens in Pennsylvania, US. RT-PCR was used to clone its full-length genome and Vector NTI to assemble overlapping sequences. Phylogenetic trees were constructed by using MEGA7 software to show the phylogenetic relationships with other available INSV sequences worldwide. This US isolate has genome and biological features classical of INSV species and clusters in the Western Hemisphere clade, but its origin appears to be recent. Furthermore, INSV-UP01 might have been involved in a recombination event with an Italian isolate belonging to the Asian clade. Our analyses support that INSV isolates infect a broad plant-host range they group by geographic origin and not by host, and are subjected to frequent recombination events. These results justify the need to generate and analyze complete genome sequences of orthotospoviruses in general and INSV in particular.
Impatiens necrotic spot orthotospovirus; Orthotospovirus; Complete genome sequence; Phylogenetic analysis; Recombination
Orthotospoviruses cause high economic losses
]. Most of the information about
orthotospoviruses was acquired by studying the type species Tomato
spotted wilt orthotospovirus (TSWV) [
] and their
molecular features are shared among members of the
genus. Orthotospoviruses are transmitted by thrips
(order: Thysanoptera, family: Thripidae) in a circulative
and propagative manner [
]. Four species belonging
to the genus Frankliniella are INSV vectors .
Orthotospoviruses are classified based on
nucleocapsid (N) amino-acid (aa) sequence identity and serological
cross reactivity, plant host range and thrips
transmission specificity [
], and are considered as distinct
species when their nucleocapsid aa identity is less than 90%
]. INSV was first designated as TSWV-I strain [
]. INSV glycoproteins are serologically related to
TSWV, while the N proteins are serologically unrelated
INSV’s host range includes about 300 plant species
]. Even though INSV can considerably affect
vegetables, its economic importance for vegetables was less
than for ornamental plants [
], but in the last few
]. INSV importance is increasing in
vegetables in Europe and North America.
Characterization studies on INSV are quite limited.
Until now, only four complete INSV genomes have
been sequenced. The type isolate (NL-07) was reported
by van Poelwijk et al. in 1997 and consists of an L and
S segments from the Netherlands [
], and an M
segment from the US  (M74904.1; NC_003625.1,
NC_003616.1, NC_003624.1). The M segment from the
US was included in the type isolate because it differed by
only 4 nucleotides from the M segment of NL-07, whose
700 (out of 5000) nucleotides were sequenced at that
time, even though the similarity between the
remaining 4300 nucleotides was unknown. Among the three
remaining INSV full genome sequences, one is from
Italy (DQ425094.1, DQ425095.1, DQ425096.1) and two
from China (GQ336989.1, GQ336990.1, GQ336991.1;
GU112503.1, GU112504.1, GU112505.1). Isolate
GU112505.1 from China contains a non-functional RdRp
due to mutation and is missing a portion of the S
segment, lowering the number of complete INSV sequences
de facto to two. Availability of genomic sequences from
different geographic origin is pivotal to understand INSV
genetic diversity and evolution, especially considering
that orthotospoviruses have a tripartite genome and can
reassort. Furthermore, while for other orthotospoviruses
like TSWV, the aa sequence of N is sufficiently diverse to
confer phylogenetic character, the INSV-N is highly
conserved and it is not phylogenetically informative [
Genetic analysis can be used to characterize the structure
of a virus population in relation to a location or host, and
to probe the origin in a population and gene flow across
time and space. Thus, we suggest that it is important to
fully sequence a larger number of INSV genomes, and
information gained by doing so will generate
understanding of the etiology and aid management of the disease.
INSV isolate UP01 was found in a commercial
greenhouse in Pennsylvania in July 2014, in an impatiens
showing ringspots symptom, acquired complying with Penn
State institutional guidelines. The plant was initially
tested for TSWV, INSV, Tobacco mosaic virus (TMV),
and Cucumber mosaic virus (CMV) by ImmunoStrip®
assays (Agdia, Elkhart, IN, USA), and found to be infected
only with INSV. Following four passages by mechanical
inoculation from single lesions on Nicotiana
benthamiana, the virus species was confirmed by ELISA assay
(Agdia, Elkhart, IN, USA). Mechanical inoculations were
used to assess the partial INSV isolate host range.
Inoculated plants were maintained in growth chambers at
25 °C with 16 h photoperiod for symptom development.
All inoculated plants were tested by ELISA for INSV.
This isolate was transmitted from Emilia sonchifolia
to E. sonchifolia by Frankliniella occidentalis (Western
flower thrips, WFT) to verify its vector transmissibility.
Thrips transmission experiments ([
modifications) were conducted with symptomatic leaves from
infected E. sonchifolia as virus source. First-instar larvae
(12 h old) of WFT were given a 24 h acquisition access
period and then reared on virus-free green bean pods
until adulthood. These adult thrips were given a 48-h
inoculation access period to 2 weeks old E.
sonchifolia seedlings (20 thrips per plant). This experiment was
repeated twice. Inoculated plants were maintained in a
growth chamber (25 °C, 16 h photoperiod) for symptom
development and then were tested by ELISA.
Transient agroinfiltration was used to test the
functionality of the INSV NSs protein as silencing
suppressor according to previous protocols [
full-length UP01 NSs was cloned into pBin61 vector and
transiently expressed through agroinfiltration together
with pBin-GFP in 16C N. benthamiana. Vector only
(pBin61) and pBin61-p19, both together with
pBinGFP, were used as negative control and positive control,
respectively. GFP expression of agroinfiltrated plants was
checked with UV light 3 days post-agroinfiltration.
Total RNA was extracted from systemically infected
N. benthamiana leaves using the Spectrum™ Plant-Total
RNA Kit (Sigma-Aldrich, St. Louis, MO, USA), following
the manufacturer’s directions. Reverse transcription was
performed using Superscript IV reverse transcriptase
(Invitrogen, Grand Island, NY, USA), random
primers and 500–1000 ng of RNA as template. Overlapping
amplicons were obtained by PCRs with gene-specific
primers designed on conserved regions of available INSV
isolates (Additional file 1) and the Q5 High Fidelity PCR
Kit (NEB, Ipswich, MA, USA), followed by 5 min
adenylation at 72 °C using GoTaq DNA Polymerase
(Promega, Madison, WI, USA). PCR products were cloned
into pGEM-T Easy vector (Promega, Madison, WI,
USA) and sequenced at the PSU Genomic Core
Facility by Sanger sequencing. Overlapping sequences were
assembled using Vector NTI software (Invitrogen, Grand
Island, NY, USA).
Phylogenetic trees were constructed by
neighborjoining method [
] using MEGA7 software [
1000 bootstrap replicates. Percentages of pairwise
identity among the aligned nucleotide and protein sequences
were calculated using MatGAT v.2.03 [
reassortment and recombination events were predicted by
Recombination Detection Program (RDP4 v.4.80) [
using several algorithms on the MUSCLE alignment file
of concatenated full-length genome sequences, created
Symptoms, hosts and vector
INSV-UP01 produced typical INSV symptoms of
chlorotic blotches and mottling on local leaves of N.
benthamiana at 4–5 days post-inoculation and curling of newly
emerged leaves; and chlorotic spots, mosaic and mottling
on the systemic leaves of E. sonchifolia. On both hosts
the isolate produced occasional ringspots on the
systemic leaves. The isolate was successfully transmitted by
F. occidentalis. N. benthamiana, N. tabacum, impatiens,
pepper, Datura stramonium and E. sonchifolia, could be
infected with UP01, but not Arabidopsis thaliana.
The three genomic INSV RNA segments were 8776, 4975
and 3010 nt in length, respectively (Additional files 2, 3,
4, NCBI accession numbers MH171172–MH171174).
The L segment was predicted to contain an ORF of 8598
nt in position 8747-150 and to encode a putative RdRp
protein of 2865 aa, in the negative sense. The M segment
encoded the putative NSm protein in the viral sense in
position 86-997 and the putative Gn/Gc protein
precursor in the complementary sense in position 4805-1473,
separated by an intergenic region of 475 nt. The M
segment had 85 and 170 nt in its 5′ and 3′ UTR,
respectively. The S segment encoded a putative N protein in
position 2861-2073, and a putative NSs protein in
position 80-1429, with an intergenic AU-rich region of 643
nt. Multifunctional properties of the NSs protein have
been shown for orthotospoviruses [
]. Since the NSs of
TSWV has been demonstrated to function as silencing
], we performed in planta transient
Agrobacterium tumefaciens silencing suppression assays
 to test this activity for UP01 and demonstrated that
UP01 NSs is a strong silencing suppressor (Additional
Several amino acid substitutions distributed along the
whole RdRp protein sequence were observed between
UP01 and other INSV isolates (Additional file 6).
UP01′s RdRp shared 97.6 and 97.2% nt identity, and
98.8 and 98.4% aa identity with NL-07 (X93216.1) and
DQ425094.1, respectively (Additional file 7), and showed
motifs conserved in the RdRp of this genus: motif A
(DXXKW), motif B (QGXXXXXSS), motif C (SSD),
motif E (EXXS) [
], motif F2 (KXQRTK) and motif
F3 (DREIY) [
]. Motif F1 (TDF), [
] absent in all
sequenced INSVs, was also not present in UP01. UP01
NSm predicted protein sequence had the ‘D-motif ’ ,
which is a conserved region in the majority of viral
movement proteins belonging to the ‘30K superfamily’ and ‘the
P/D-L-X motif ’ [
], ‘DSRK motif ’ and ‘HH motif ’, which
play essential roles in the subcellular distribution and
tubule formation of TSWV NSm protein [
A recombination event in the L segment is predicted among INSV isolates
Analysis of putative reassortment/recombination events
using INSV concatenated full-length genome sequences
predicted the occurrence of a recombination event
involving isolates UP01, NL-07 and the Italian isolate
(Additional file 8). The event involved the L segment and
was predicted by different algorithms with significance
level set at P ≤ 0.05.
UP01 is consistently placed into the same Western
Hemisphere clade with other US isolates and NL-07, and is
more distantly related to isolates in the Asian clade,
where the Italian isolate also belongs (Figs. 1, 2, 3). As
indicated by Elliott et al. [
] and Nekoduka et al. [
our result confirms that INSV isolates do not group
phylogenetically based on host species (Figs. 1, 2; Additional
UP01 RdRp ORF is overall more related to NL-07
than to other isolates (Additional file 8) but it shares
different degrees of similarities with all isolates based
on the region of the RdRp examined, suggesting a
possible recombination event for this segment involving the
region of 2850-8690. The resolution of the RdRp
phylogeny is penalized by having only 5 sequences available.
Phylogenetic analyses of the M segment and its two
ORFs (Gn/Gc and NSm) (Fig. 1a–c) and IGR (Fig. 1d)
show again that INSV isolates are divided into Western
Hemisphere and Asian clades, with UP01 in the
Western Hemisphere clade, and the isolates from Italy and
Asia in the Asian clade. In the Asian clade are
unexpectedly grouped also one A. thaliana (NSm JX138532.1,
Gn/Gc JX138530.1) (Fig. 1a, b) and one lettuce isolates
(KF745141.1) (Fig. 1b) from the US, suggesting that these
isolates might be of European/Asian origin and have
been introduced recently in the US. While this segment
is better represented, still not many sequences are
available to resolve some of the phylogenetic relationships for
members of the two clades.
Phylogenetic analyses using N protein nucleotide
sequences (Fig. 2a) indicate that UP01 grouped in the
Western Hemisphere clade. This clade contains isolates
from the US and the Netherlands, but also one isolate
from Japan (AB894565.1), again indicating that INSV was
probably introduced into different regions via import of
infected plant material. UP01N protein shares very high
aa identity with other INSV isolates (Additional file 10).
The division into Western Hemisphere and Asian clade
is also congruent when looking at the phylogenetic
analyses of the S segment (Fig. 2c), where UP01 belongs to the
Western Hemisphere clade and is distantly related to the
Chinese isolate (GU112504.1). But while for the M
segment UP01 is closely related to the reference sequence
(M74904.1, NC_003616.1), with whom it shares a more
recent origin (bootstrap value > 90%), and it is less related
with USA WA Basil isolate (KX790322.1) (bootstrap
value > 90%) (Fig. 1c), the phylogenetic study of the S
segment (Fig. 2c) revealed that UP01 is more related to
the USA WA basil isolate than to the reference sequence.
This observation, for the first time, questions combining
in a reference genome sequences that superficially seem
to belong to the same isolate, but that could belong to
distinct clades, when analyzed using a larger number of
sequences. An alternative explanation to our result could
indicate a reassortment event between isolates from in
different geographic regions that led to the emergence
of the reference genome. Interestingly, the USA CA
lettuce isolate SV-L1 (NSs KF745142.1 and N KF745140.1,
respectively) that was isolated from an INSV outbreak in
Coastal California clustered with other US isolates when
its NSs (Fig. 2b) and N ORFs (Fig. 2a) were analyzed,
but its NSm (KF745141.1) sequence grouped with the
Asian clade with high bootstrap support (Fig. 1b),
indicating a possible reassortment or recombination event.
Unfortunately, the Gn/Gc sequences for these isolates are
not available to support these hypotheses.
The phylogenetic analysis of the N protein (Fig. 2a)
is the one for which more sequences are available, and
highlights how having a large number of sequences can
resolve better the INSV phylogeny and can be
epidemiologically informative. In fact, in the case of the INSV
sequences reported in a recent outbreak in lettuce in
Costal California [
], phylogeny shows that all lettuce
strains responsible for the outbreak were identical or
highly related, but they differed from isolates found in the
surrounding weeds and crops.
Result of the analysis of putative
reassortment/recombination suggests that a recombination event involving
UP01 might have happened. As mentioned above,
phylogenetic analysis also supports the predicted
recombination event (Fig. 3) and further confirms the occurrence of
Fig. 3 Neighbor-joining phylogenetic tree derived from the alignment of breakpoint for recombination different INSV isolates. a Sequences on L
segment from 1 to 2849 nt and b sequences on L segment from 2850 to 8690 nt. Bootstrap values were derived from 1000 bootstrap replicates.
Scale bar represents number of substitution per site. Accession numbers and plant host species of the sequences are shown in the figure
genetic exchanges in the evolution of orthotospoviruses.
Reassortment is also biologically important because it
could result in new resistant-breaking strains [
45, 53, 54
or emergence of new viruses [
Additional complete genome sequences from the INSV
outbreak in Coastal California would be needed to
confirm reassortment and recombination events between
Additional file 1. List of primers. The table contains the list of primers
used for RT-PCR and Sanger sequencing of isolate UP01, and includes the
segment name, sequence from 5′ to 3′, and name for each of the primers
Additional file 2. Sequence of INSV UP01 L segment in fasta format.
Additional file 3. Sequence of INSV UP01 M segment in fasta format.
Additional file 4. Sequence of INSV UP01 S segment in fasta format.
Additional file 5. Bioassays for silencing suppression activity of UP01
NSs allelic variant. Full-length UP01 NSs was cloned into pBin61 vector
and transiently expressed through agroinfiltration together with
pBinGFP in 16C transgenic Nicotiana benthamiana. Vector only (pBin61)
andpBin61-p19 were used as negative control and positive control,
respectively. Pictures were taken by using a hand-held UV light 3 days
Additional file 6. Alignment of INSV RdRp amino acid sequences. Figure
shows the amino acid substitutions distributed along the 2221–2760
amino acids of the Query: UP01 and Sbjct: NC_003625.1 RdRp protein
Additional file 7. (a) pairwise comparison of RdRp predicted amino acid
sequences and (b) pairwise comparison of RdRp nucleotide sequences.
The table contains the RdRp nt pairwise comparison among INSV isolates
listed in the first column. Values in blue cells indicate percentage identity.
INSV isolates are identified by their accession numbers in NCBI.
Additional file 8. Putative recombination event among full-length INSV
isolates of different geographic origin, predicted by the RDP software.
Analysis of putative reassortment/recombination events using INSV
concatenated full-length genome sequences predicted a possible
recombination event involving isolates UP01, NL-07 (from the Netherlands) and
the Italian isolate. The event involved the L segment and was predicted by
different algorithms, with significance level set at P ≤ 0.05.
Additional file 9. Neighbor-joining phylogenetic tree derived from the
alignment of INSV RdRp coding sequence (cds) of different INSV isolates.
Bootstrap values were derived from 1000 bootstrap replicates. Accession
numbers and plant host species of the sequences are shown in the figure.
Additional file 10. Pairwise comparison of N protein predicted amino
acid sequences. Table contains the N protein amino acid pairwise
comparison among INSV isolates listed in the first column. Values in blue cells
indicate percentage identity. Values in green cells indicate percentage
similarity. INSV isolates are identified by their accession numbers in NCBI.
INSV: Impatiens necrotic spot orthotospovirus; TSWV: Tomato spotted wilt
orthotospovirus; aa: amino-acid; RDP: Recombination Detection Program; UTR
: untranslated region; RdRp: RNA-dependent RNA polymerase; IGR: intergenic
region; ORF: open reading frame.
KZ contributed to the design of the study, cloning and sequencing of the new
INSV isolate, data analysis and preparing the manuscript. PM and CR
contributed to design of the study, data analysis and preparing the manuscript. All
authors read and approval the final manuscript.
We sincerely thank to Dr. Gary W. Moorman (Pennsylvania State University) for
providing us the infected plant tissue.
The authors declare that they have no competing interests.
Availability of data and materials
Sequence data will be available from the additional files and other data will be
available from the corresponding author upon request.
Consent for publication
Ethics approval and consent to participate
C. Rosa’s salary is in part supported by USDA HATCH Accession Number:
1009992. Project Number: PEN04604.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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