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Generic RT-PCR tests for detection and identification of tospoviruses
Accepted Manuscript Title: Generic RT-PCR tests for detection and identification of tospoviruses Author: A. Hassani-Mehraban M. Westenberg J.T.J. Verhoeven B.T.L.H. van de Vossenberg R. Kormelink J.W. Roenhorst PII: DOI: Reference:
S0166-0934(15)30104-X http://dx.doi.org/doi:10.1016/j.jviromet.2016.03.015 VIRMET 12986
To appear in:
Journal of Virological Methods
Received date: Accepted date:
19-11-2015 28-3-2016
Please cite this article as: Hassani-Mehraban, A., Westenberg, M., Verhoeven, J.T.J., van de Vossenberg, B.T.L.H., Kormelink, R., Roenhorst, J.W., Generic RT-PCR tests for detection and identification of tospoviruses.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2016.03.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Generic RT-PCR tests for detection and identification of tospoviruses
A. Hassani-Mehraban1, M. Westenberg1, J.T.J. Verhoeven1 ,B.T.L.H. van de Vossenberg1, R. Kormelink2 & J.W. Roenhorst1* 1
National Plant Protection Organization, National Reference Centre, Geertjesweg 15, P.O.
Box 9102, 6700 HC Wageningen, the Netherlands; 2Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands
*Corresponding e-mail address: [email protected] Running head: Generic detection of tospoviruses
Highlights New RT-PCR tests have been designed for generic detection and identification of tospoviruses This is the first test design potentially allowing detection and identification of all known and unknown tospovirus species, since primer design is based on conserved regions within different (sub) clades Tests have been successfully applied on diagnostic samples, which allowed identification of seven different species and a non-described tospovirus from alstroemeria plants Tests provide a relevant tool for plant quarantine and diagnostic laboratories
Abstract A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5’ upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all
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species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a nondescribed tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.
Keywords: Bunyaviridae, plant virus; diagnostics; clade-specific primers; nucleocapsid gene
Introduction Tospoviruses are causing serious losses in many economically important crops worldwide, (Prins and Goldbach 1998; Pappu et al., 2009). To date 29 species have been distinguished within the genus Tospovirus (family Bunyaviridae), including 11 assigned (Plyusnin et al., 2012; http://ictvonline.org/virusTaxonomy.asp) and 18 tentative species (Chu et al., 2001b; Meng et al., 2013; De Oliviera et al., 2012; Dong et al., 2013; Plyusnin et al., 2012; Shimomoto et al., 2014; Torres et al., 2012; Yin et al., 2014; Zhou et al., 2011). These species differ in molecular and biological properties, as well as geographical distribution. Tospoviruses consist of enveloped particles, enclosing three (negative or ambisense) ssRNA segments, denoted large (L), medium (M) and small (S), complexed with the nucleocapsid (N) protein (De Haan et al., 1990). Within the genus Tospovirus, species are identified on the criterion that the sequence of the N protein shows less that 90% identity with other species (de Ávila et al., 1993; Plyusnin et al., 2012). In addition, plant host range, vector specificity and serological relationships of the N protein are taken in consideration. At present the majority of
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species can be assigned to the American, Asian and Eurasian clades, remaining a small group of distinct and floating species (Hassani Mehraban et al., 2005; Pappu et al., 2009). To control tospovirus diseases, species identification is important (EFSA, 2012). In many crops, it is not possible to identify the virus just on the basis of symptomatology, as symptoms caused by different species might look similar. Therefore, the availability of reliable and sensitive diagnostic tests is essential to detect and identify the viral etiology of the disease. Mechanical inoculation of test plants is suitable for detection, but does not allow identification at the species level. Serological assays using poly- and monoclonal antisera, mainly against the N protein, are widely used, although a proper identification is often hampered by cross reactions (Supplemental data [S1]). In addition, over the last decades an increasing number of molecular tests has been developed, either targeting individual species or aiming a broader detection. Especially for generic tests, however, data on the scope of detection are lacking and none of them seems to cover all known species (Table 4). Therefore, this study aimed to develop a molecular test for generic detection and identification of all tospoviruses. Based on the alignment of N gene nucleotide sequences from 28 different species, clade-specific primers were designed. In the final test eleven primers were used, which showed to successfully detect different tospoviruses in diagnostic samples and allow their classification and further speciation into any of the American, Asian and Eurasian clades.
Materials and methods Virus isolates and propagation Twenty tospovirus isolates used in this study are listed in Table 1. Virus isolates were propagated by mechanical inoculation on Nicotiana benthamiana plants with inocula prepared 3
from infected leaf tissue ground in PBS buffer (pH 7.0), containing 0.1% Na2SO3. Inoculated plants were grown under greenhouse conditions at 18-25ºC and 14 h illumination) and monitored for symptom expression. Leaves of plants showing systemic symptoms (after 1-2 weeks) were harvested and stored at -80°C until use for RNA extraction. Samples of (freeze-) dried leaf material of CCSV, MeSMV, PCFV and PolRSV were used for RNA extraction directly.
Tospovirus phylogeny Amino acid sequences of the N protein from 28 tospovirus species were obtained from GenBank (see Table 2 for Acc. numbers) and aligned using ClustalW (Larkin et al., 2007) in Geneious R7 (Biomatters Ltd, Auckland, New Zealand) using Bunyamwera virus (Acc. No. NP_047213) as outgroup. The alignment was used to construct a neighbour-joining tree with bootstrap analysis (N=1000) in Mega 5.05 (Tamura et al., 2011).
Sequence alignment and primer design Nucleotide sequences of the N gene and its 5’ untranslated region sequence (UTR) from 28 tospo virus species were obtained from GenBank (see Table 2 for accession numbers) and aligned using ClustalW (Larkin et al., 2007) in Geneious R7 (Biomatters Ltd, Auckland, New Zealand). Note that TNSV was not used for primer design because the sequence was not available at the time the alignment was made. Conserved and (sub) clade-specific N gene sequences were selected from the ClustalW alignment and used for design of (sub) cladespecific primer pairs (Table 2). Primers complementary to the very first eight conserved terminal nucleotides of the N UTR sequence were extended with nine nucleotides (GGGGGATCC) to increase annealing specificity during subsequent PCR amplification. Primers were tested in silico for self-annealing and primer-dimer formation by using 4
OligoCalc software (Kibbe, 2007). The specificity was tested within the Geneious R7 program by allowing a maximum of four mismatches and by perfoming BLAST searches in GenBank (Altschul et al., 1990). To verify the identity of virus isolates, species-specific primers amplifying the entire N gene were designed, based on available sequences in GenBank (Table 2). These primers were designed along with start and stop codons, except for the forward primer of PCFV which was selected from a sequence more upstream of the start codon to obtain a higher GC content.
RNA extraction Leaf material (ca. 0.1-0.5 g) was homogenized in 3.5 ml of GH+ grinding buffer (6 M guanidine hydrochloride, 0.2 M sodium acetate pH 5.2, 25mM EDTA and 2.5% PVP-10) according to a protocol modified from Menzel et al. (2002). Homogenization was performed in extraction bags using a HOMEX 6 homogenizer (Bioreba AG Reinach, Switzerland). Next, plant sap (1 ml) was lysed by incubation in a thermomixer for 10 min at 65°C and 850 rpm and subsequently centrifuged for 2 min at 16.000g. A volume of 500 ul from the supernatant was transferred to a QIAshredder spin column for total RNA purification by the RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands) and following the manufactures protocol from there. RNA was eluted in a final volume of 40 μl elution buffer and stored at -80˚C. Healthy leaf material and extraction buffer have been used as negative isolation controls (NIC).
RT-PCR assays cDNA synthesis and PCR were performed using the OneStep RT-PCR Kit (Qiagen, Venlo, the Netherlands) in a total volume of 25 μl, containing 1 μl RNA template, 5 μl OneStep RTPCR Buffer, 1 μl OneStep RT-PCR Enzyme Mix, 1 μl dNTP Mix (10 mM) and 1 μl of each primer (10 μM). The RT-step was performed at 50°C for 30 min followed by a PCR 5
consisting of one cycle at 95°C for 15 min, followed by 35 cycles at 94°C for 1 min, 50-58°C for 30 sec and 72°C for 1 min, and a final extension cycle at 72°C for 5 min. The annealing temperatures (Ta) used for the different tospovirus clades are shown in table 2. To verify the quality of RNA extraction, plant NADH dehydrogenase subunit 5 (nad5) mRNA was RTPCR amplified using nad-5F (5’-GATGCTTCTTGGGGCTTCTTGTT-3’) and nad5-R (5’CTCCAGTCACCAACATTGGCATAA-3’) primers (Menzel et al., 2002). Five μl of each PCR reaction was mixed with 1 μl 6x Bromophenol Blue Loading solution (Promega, Leiden, the Netherlands) and products resolved on a 1.5% RESult LE General Purpose Agarose gel (BIOzymTC, Landgraaf, the Netherlands) with SYBR-safe staining (Life Technologies, Bleiswijk, the Netherlands). Amplicons were visualized under a GeneGenious gel imaging system (Syngene, Cambridge, United Kingdom) and their sizes estimated using a 1 kb Plus DNA ladder (Promega). Molecular grade water was used as negative amplification control (NAC) in each run to monitor possible contamination. The RT-PCR on BeNMV has been performed at the Department of Cell Biology, University of Brasília, by using the SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Life Technologies, São Paulo, Brasil). This reaction was performed in a total volume of 25 μl, containing 1 μl RNA template, 12,5 μl Reaction Mix, 1 μl SuperScript® III RT/ Platinum® Taq Mix, and 1 μl of each primer (10 μM). The RT-step was performed at 50°C for 30 min followed by a PCR consisting of one cycle at 94°C for 2 min, 35 cycles of 94°C for 1 min, 58°C for 30 sec, 68°C for 1 min, and a final extension cycle at 68°C for 5 min.
Sequence analysis PCR products were purified using QIAquick PCR Purification Kit (Qiagen) prior to cycle sequencing with both amplification primers in individual runs. Cycle-sequence reactions were 6
based on the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies) and performed according to the manufacturer’s instructions. Cycle-sequence products were purified using the DyeEx 2.0 Spin Kit (Qiagen) and sequenced on a 3500 Genetic Analyzer (Life Technologies). Electropherograms were assembled and edited using Geneious R7. Amplification primers were trimmed from the consensus sequence and, when needed, further trimming was performed to obtain high quality (PHRED > 30) consensus sequence data. The assembled sequences were verified by comparing with available sequences in the GenBank using BLASTn and BLASTp (Altschul et al., 1990).
Results Tospovirus phylogeny Phylogenetic analysis with the amino acid sequences of the N protein of 29 tospovirus species revealed that tospoviruses can be divided in five distinct groups with LNRV as an independent species (Figure 1). The American clade could be divided in two subgroups, clades AM C1 and AM C2. Species originating from Asia fell into two subgroups, clades AS 1 and AS 2. It was noted that the N protein sequence of species belonging to the AS 1 clade are more similar to those of the EuroAsian (EA) clade then to those of the AS 2 clade. The division in (sub) clades was used for the design of clade specific primers.
Primer design and in silico verification of species identification A multiple nucleotide sequence alignment of the N gene and its 5’ UTR from 28 tospovirus species revealed no highly conserved regions shared by all tospoviruses, with the exception of similarities within the very first and conserved eight terminal nucleotides from the RNA segment. Therefore, this sequence was selected for design of a forward primer and extended 7
with a few nucleotides for the introduction of clade-specificity, based on the strategy as described by Hassani-Mehraban et al. (2005). According to this concept, four primers were designed and designated AM1-F, AM2-F, AS-EA-F and LNRV-F, specific to members of the American clade 1, American clade 2, Asian and Eurasian clades (and their subgroups,), and LNRV, respectively (Table 2, Column Generic primers). In addition to these termini-primers clade-specific reverse primers were designed in such a way that the obtained amplicon length after RT-PCR on a virus from each (sub) clade was distinct in size to allow for immediate classification. To ensure that the partial N gene and its 5’ UTR sequence between the terminal–end primer binding site and the internal reverse primer site provided enough resolution for species identification, BLASTn analysis was performed with these sequences from all 28 species used in the alignment. The results revealed that in all cases the intraspecies variation was less than the interspecies variation and were supported by tree views in which all sequences fell within their species-specific clade (data not shown).
RT-PCR and sequence analysis Using the primer sets as described above and designed for RT-PCR amplification of (partial) N gene specific for the various (sub) clades, tests were performed on samples collected from infected plants challenged with isolates from 20 different tospoviruses (Table 1). After some optimization, the results from repeated tests provided consistent results as shown in Figure 2. RT-PCR reactions with clade-specific primer sets were initially performed for each subset of tospoviruses at a Ta of 50˚C, but this only resulted in the expected (size-) specific amplicons for the American clade 1 (760 bp; Figure 2A 1st panel (top)), and Asian clade 2 primer sets (520 bp; Figure 2A, 3rd panel), and non-specific PCR products for the others (data not shown). After the application of gradient PCRs (52-58oC) the optimum Ta turned out to be 8
52˚C for members of the Asian clade 1 (370 bp; Figure 2A, 2nd panel) and Eurasian clade (790 bp; Figure 2A, 2nd panel), and 58oC for those of the American clade 2 (670 bp; Figure 2B, right panel) and LNRV (430 nt; Figure 2A, 4th panel). Attempts to combine all cladespecific RT-PCRs in one experiment failed and were only successful for the combination Asian clade 1 and Eurasian clade (Figure 2A, 2nd panel). Furthermore, the results of the final five separate RT-PCRs show that for all tospoviruses in the same clade the similar, size-expected amplicon was generated, while no amplicon was detected in the negative isolation controls performed with extraction buffer (NIC) and healthy leaf material of N.bentahmiana (Figure 2A). In the case of IYSV two amplicons of the expected size were generated with both the Asian clade 1 and Eurasian clade primers (Figure 2A, 2nd panel IYSV), likely due to the fact that Asian clade 1 reverse primer was able to anneal to the RNA of this IYSV isolate. Nevertheless, the virus could be identified as IYSV by sequencing both amplicons together with the Eurasian clade primers. BeNMV and a selection of species from the other clades (GRSV, IYSV, LNRV, WSMoV; Figure 2B) were tested in a separate test. The results show that the American clade 2 primers were able to detect BeNMV (Figure 2B, right panel), while no amplicons were produced with extraction buffer (Figure 2B, left panel NIC) or RNA from healthy leaf material (Figure 2B, left panel N. benthamiana, right panel S. lycopersicum ) or on the tested species from the other clades (Figure 2B, left panel). In all experiments, the RNA quality of the samples was verified by production of the expected amplicon for nad5 as an internal control (Figure 2A). Subsequent sequencing and analysis of all amplicons by BLAST searches in NCBI GenBank confirmed their viral identity and indicated that amplicon-sequences obtained from diagnostic test samples this way, would immediately allow a provisional identification of the isolate.
Testing of diagnostic samples 9
To verify the validity of the designed primer sets for use in tospovirus diagnostics and classification a set of twenty five samples collected from different crops and origins suspected of tospovirus infections were tested using the generic RT-PCRs. In twenty samples the presence of a tospovirus was detected (Table 3). The majority of the samples originated from Asia and appeared infected with one of the Asian clade members. The others came from various regions and were infected by members of the American clade 1. From the remaining samples, two turned out to be infected with a cucumovirus, one with a potyvirus and two with an unknown virus (data not shown). Unfortunately, for non-infected pepper samples the Asian clade 1/Eurasian clade primer combination gave a plant specific amplicon around the expected 800 bp fragment for the Eurasian clade tospoviruses, as confirmed by sequence analyses. This indicates that sequencing of the obtained amplicons is required to confirm the genuine presence and identification of a tospovirus in field samples. In further support for the usefulness of the designed primers sets, the generic tospovirus tests were successfully applied on a yet non-described tospovirus isolate from alstroemeria plants, provisionally named Alstroemeria yellow spot virus (Hassani-Mehraban et al., manuscript in preparation). As expected from its S RNA nucleotide sequence (unpublished data) an amplicon was produced with the Eurasian primer set (Figure 2A) and the identity confirmed by sequence analysis.
Discussion A RT-PCR approach for generic detection and provisional identification of tospoviruses has been developed, based on five RT-PCR’s targeting tospovirus species from different clades and a floating species within the genus. By using conserved regions from viruses within each (sub) clade, primers have been designed that potentially detect all (known) species from that 10
clade. This was nicely demonstrated by the production and nucleotide sequence confirmation of expected amplicons from twenty available isolates of species from the American clades 1 and 2, Asian clades 1 and 2, the Eurasian clade and the floating species LNRV. The usefulness of the PCR tests to even detect new and unknown tospovirus species was strengthened by the positive results obtained with a yet non-described putative new tospovirus species from Alstroemeria that was not included in the analyses for primer design. Application of the tests on 25 diagnostic samples from mainly peppers and tomatoes suspected from a tospovirus infection, already gave promising results. In 20 out of 25 samples a tospovirus was detected and seven different species within the American clade 1 and Asian clade 1 could be identified. In the five remaining samples, for which the test results were negative, other virus species were found. Unfortunately, the application of RT-PCR tests on samples collected from pepper revealed a non-specific reaction with the Eurasion primers, which could only be prevented when the virus was meachanically transfered to another host like N. benthamiana. This observation stresses the importance to verify the identity of the amplicon by sequence analysis and avoid false interpretations. Although the N gene sequence obtained from amplicons will not only verify the presence but additionally allows the classification of the tospovirus involved, some caution has to be considered in light of the possible occurrence of recombination and reassortment between tospoviruses (Tentchev et al., 2011). The added value of the newly developed generic tests in comparison with those already used, is illustrated by indicating for each test the species that can be detected and identified within the different (sub) clades (Table 4). Test results for the remaining tospovirus species are not reported (or not known), except for PCFV which is not detected by the test described by Chen et al. (2012). From this overview it is clear that the clade-specific primer sets described in this paper provide the only tests detecting species in all clades. Moreover, the fact that their design 11
is based on conserved regions within each (sub) clade, substantially increases the probability of detection of other known and unknown species. Furthermore, by targeting the N gene, sequencing of the amplicons allows a provisional identification of the species, which is an advantage over tests that target the G1/G2, NSm or L genes. In conclusion, these results demonstrate that the newly-developed generic RT-PCR tests allow detection and provisional identification of known and unknown tospoviruses.. In addition, the species-specific primers enable confirmation of the identity by sequence analysis of the Ngene. The fact that the primer sets are based on conserved regions within the different (sub) clades, confirms their potential ability of generic testing on tospoviruses. This makes the tests a relevant tool for plant quarantine and diagnostic laboratories.
Acknowledgements We thank J. Saaijer (Laboratory of Virology, Wageningen University and Research Centre, the Netherlands), L. Hüner (National Plant Protection Organization, the Netherlands) and M. Martins Severo de Almeida (Department of Cell Biology, University of Brasília, Brazil) for technical assistance, and R. Oliveira Resende (Department of Cell Biology, University of Brasília, Brasília, Brazil), T.C. Chen (Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan), Y. Shimomoto (Kochi Agricultural Research Center, Kochi, Japan) and M. Turina (Instituto di Virologia Vegetale del CNR, Torino, Italy) for providing virus isolates.
References
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Figure legends
Figure 1. Neighbour-joining tree with bootstrap (N=1000) analysis of the nucleocapsid protein sequence of 29 tospovirus species and Bunyamwera virus (GenBank Acc. No. NP_047213) as outgroup. The scale bar measures evolutionary distance in substitutions per amino acid. The accession numbers corresponding to each isolate are given in Table 2.
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Figure 2. Agarose gel electrophoresis of amplicons obtained from 20 different tospovirus isolates using the five optimized tospovirus RT-PCR tests: A. Amplicons produced by the primers (from top to bottom) on viral RNA from members belonging to the American clade 1 (AM-C1), combination of Asian clade 1 and Eurasian clade and (AS-C1 and EA-C), Asian clade 2 (AS-C2) members and LNRV. Primers specific for the plant NADH dehydrogenase subunit 5 (nad5) mRNA was used as internal control to verify the quality of RNA; B. Amplicons produced on tospoviruses by American clade 2 primers (AM-C2). NAC: Negative amplification control (molecular grade water). NIC: Negative isolation control (extraction buffer), and healthy leaf material of N. benthamiana and S. lycopersicum. Species indicated by acronyms as in Figure 1; AYSV: Alstroemeria yellow spot virus, (Hassani-Mehraban et al., 2015, manuscript in preparation).
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Table 1. Tospovirus isolates used in this study Species1
Code
Origin
ANSV
Colombia
WUR2
AYSV3
ALS-2000
WUR
BeNMV
Brazil
University of Brasília, Brazil
CaCV
Thailand-
WUR
CCSV
Taiwan
National Chung Hsing University, Taiwan
CSNV
Brazil
WUR
GBNV
India
WUR
GRSV
SA-05
WUR
INSV
NL-07
WUR
IYSV
IYSV-NL
WUR
LNRV
Japan
Kochi Agricultural Research Center, Japan
MeSMV
Mexico
Istituto di Virologia Vegetale del CNR, Italy
MYSV
Thailand-isolate physalis severe mottle virus (PSMV)
WUR
PCFV
Taiwan
National Chung Hsing University, Taiwan
PolRSV
Plg3
Istituto di Virologia Vegetale del CNR, Italy
TCSV
BR-03
WUR
TNRV
Thailand
WUR
TSWV
BR-01
WUR
TYRV
Iran
WUR
WSMoV
Thailand
WUR
1
Species indicated by acronyms as in Figure 1; 2Wageningen University and Research Centre; 3Alstroemeria yellow spot virus, Hassani-
Mehraban et al., 2015 (manuscript in preparation).
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Table 2. Generic and specific primers designed and used in this study
Clade
American 1
American 2
Asian 1
Species1
Eurasian
LNRV
1
Generic Primers (5’‐3’)
Position in S RNA2
Ta (˚C)
Amplicon size (bp) using generic primers
ANSV
F: ATGTCTAAGGCTAAGTTGAC; R: TTAAGCAACACCTGAAATTTTGG
GQ478668
1‐14 / 732‐753
50
762
CSNV
F: ATGTCTAAAGTTAAGCTTAC; R: TTAAACAAGATCTTTAGGAATAAG
AF067068
1‐14 / 732‐753
50
762 761
GRSV
F: ATGTCTAAGGTCAAGCTCAC; R: CCAAGATTGCTGGTGTTGCATG
AF487517
1‐14 / 731‐752
50
INSV
F: ATGAACAAAGCAAAGATTACC; R: TTAAATAGAATCATTTTTCCC
NC_003624
1‐14 / 741‐762
50
762
PNSV
NA3
HE584762
1‐14 / 732‐753
50
762
MeSMV
F: ATGTCTAAGGTCAAGCTCACC; R: TCATGCAACACCAGCAATCTTG
EU275149
1‐14 / 739‐760
50
769
TCSV
F: ATGTCTAAGGTTAAGCTCAC; R: CAAAACTCGCAGAACTTGCTTG
AF413110
1‐14 732‐753
50
762 763
AM1‐F: GGGGGATCCAGAGCAATTGTGTC AM1‐R: CTTTGCTTTTCAGCACAGTGCA4
TSWV
F: ATGGGATCTAATGCACTAAAG; R: TTAGCTCATGAAGCCATGATTTTTGC
NC_002051
1‐14 / 733‐754
50
ZLCV
NA
AF067069
1‐14 / 821‐842
50
851
BeNMV
F: ATGGGATCTAATGCACTAAAG; R: TTAGCTCATGAAGCCATGATTTTTGC
JN587268
1‐11 / 647‐663
58
672
SVNaV
NA
GU722319
1‐11 / 664‐678
58
687
CaCV
F: ATGTCTACCGTCAGGCAACTTAC; R: TCACACTTCAATAGATGTACTAG
NC_008301
1‐13 / 339‐360
52
369
CCSV
F: ATGTCTAACGTCAGAGGTTTAAC; R: TTAAAAAGTTAAATCACCACTC
AY867502
1‐13 / 339‐360
52
369
GBNV
F: ATGTCTAACGTCAAGCAACTC; R: TTACAATTCCAGCGAAGGACC
NC_003619
1‐13 / 339‐360
52
369
NA
KM819706
1‐13/ 339‐360
52
369
MYSV
F: ATGTCTACCGTTGCTAAGCTTAC; R: TTAAACTTCAATGGACTTAGACC
NC_008300
PCSV
NA
TNRV
F: ATGTCTAACGTTAGGAAAACC; R: TTACAGTCCTATACTTCCTCTC
TZSV
NA
WBNV
NA F: ATGTCTAACGTTAAGCAGCTC; R: TTACACTTCCAAAGAAGTGCTG
MVBaV
WSMoV Asian 2
N gene primers (5’‐3’)
Accession no.
AM2‐F: GGGGGATCCAGAGCAATCGG AM2‐R1: GCAACTCTACCAGCTTG AM2‐R2: GCAACTTTAGCAGCTTG
1‐13 / 342‐363
52
372
1‐13 / 351‐372
52
381
FJ946835
1‐13 / 350‐371
52
380
EF552433
1‐13 / 339‐360
52
369
GU584184
1‐13 / 339‐360
52
369
KF383956
AS‐EA‐F: GGGGGATCCAGAGCAATCGAGG AS1‐R: GCTTCAGTCCTCTTAAATGTCC
NC_003843
1‐13 / 337‐358
52
367
PCFV
F: CCCATGTCTAAAACCAAAGTCAAG; R: CCCGTGAATAACATCGATATATAG
AF080526
1‐13 / 493‐512
50
521
GYSV
NA
AF013994
1‐13 / 493‐512
50
521
HCRV
NA
KC290943
1‐13 / 765‐784
52
793
IYSV
F: ATGTCTACCGTTAGGGTGAAAC; R: TTAATTATATCTATCTTTCTTG
AF001387
1‐13 / 764‐783
52
792
F: ATGTCTACCACAGGGTTGAG; R: CAAAATCCTTGATTCTTTTTGGAGGC
EF445397
1‐13 / 766‐785
52
794
TYRV
F: ATGGCTACCGCACGAGTGAGC; R: TTAAAATGCATCATTCTTTTTGG
AY686718
1‐13 / 765‐784
52
793
LNRV
F: ATGTCTACCGTCAGGCAACTTAC; R: TCACACTTCAATAGATGTACTAG
AB852525
27‐46 / 437‐458
58
432
PolRSV
AS‐EA‐F: GGGGGATCCAGAGCAATCGAGG AS2‐R: CTTTGAAGATGACCTCATCT
AS‐EA‐F: GGGGGATCCAGAGCAATCGAGG EA‐R: TTGTTCAATGAAGCAGCACC LNRV‐F: CCAGTAAAAGACGAATCCC LNRV‐R: GATTCAAATCGCCCAGCAGTCC
Species indicated by acronyms as in Figure 1; 2Position of the generic forward/reverse primer in the sense RNA; 3NA: Not applicable, since these species were not used in this study or in case of BeNMV the specimen was not available for verification by sequencing of the entire N gene; 4Hassani-Mehraban, A., Botermans, M.,
Verhoeven, J.Th.J., Meekes, E., Saaijer,J., Peters, D., Goldbach, R., Kormelink, R. 2010. A distinct tospovirus causing necrotic streak on Alstoemeria sp. in Colombia. Arch. Virol. 155, 423-428.
20
Table 3. Tospovirus species identified in diagnostic samples Species1
Crop species
Origin (number of samples)
CaCV
Capsicum annuum
China (1), Vietnam (1)
CaCV
Solanum lycopersicum
Vietnam (2)
GBNV
Solanum lycopersicum
India (3)
GRSV
Capsicum annuum
Brazil (1)
INSV
Leontopodium sp.
Switzerland (1)
TCSV
Capsicum annuum
Brazil (1)
TCSV
Capsicum frutescens
Dominican Republic (1)
TNRV
Capsicum annuum
Thailand (6), Vietnam (1)
TSWV
Solanum lycopersicum
Croatia (1)
TSWV
Capsicum annuum
South Africa (1), Turkey (1)
1
Species indicated by acronyms as in Figure 1
21
Table 4. Molecular tests for generic detection (and identification) of tospoviruses1 Reference
Method
Target(gene)
Type of primers
AM-C1
Mumford et al.,
RT-PCR
S RNA (N)
Universal
GRSV, INSV,
1996 Dewey et al.,
AS-C1
AS-C2
WSMoV
PCFV
EA
LNRV
TCSV, TSWV RT-PCR + RFLP
S RNA (N)
Universal
1996 Chu et al.,2001a
AM-C2
GRSV, TCSV, TSWV
RT-PCR
L RNA (L)
Degenerate
GRSV, INSV, TSWV
Eiras et al., 2001
RT-PCR
S RNA (N);
Universal primers
CSNV, GRSV,
M RNA (G1/G2);
3 primers sets
INSV, TCSV,
M-RNA (NSm)
targeting the
TSWV, ZLCV2,3
IYSV2
indicated genes Eiras et al., 2001
Dot blot
L RNA (L)
Specific probes
hybridization
CSNV, GRSV,
IYSV
INSV, TCSV, TSWV, ZLCV
Okuda & Hanada,
RT-PCR + RFLP
S RNA (N)
RT-PCR
S RNA (N)
Degenerate
INSV, TSWV
MYSV, WSMoV
IYSV
Universal
INSV, TSWV
MYSV, WSMoV
IYSV
CSNV, INSV,
CaCV
IYSV
2001 Uga & Tsuda, 2004
degenerate combined with species specific
Kuwabara et al.,
RT-PCR
S RNA (N)
2010 Chen et al., 2012
TSWV, ZLCV RT-PCR
M RNA (NSm);
Degenerate
L RNA (L)
GRSV, INSV,
CaCV, CCSV,
TCSV, TSWV
GBNV, MYSV,
PCFV (negative)
IYSV, TYRV
PCFV
IYSV, PolRSV,
WBNV, WSMoV Charoenvilaisiri
RT-PCR (+
et al., 2014
ELISA)
This paper
RT-PCR
S RNA (N)
Universal
CaCV, MYSV,
S RNA (N)
Universal clade
ANSV, AYSV,
specific
CSNV, GRSV,
GBNV, MYSV,
INSV, MeSMV,
TNRV, WSMoV
TNRV, WSMoV BeNMV
CaCV, CCSV,
LNRV
TYRV
TCSV, TSWV 1
Species indicated by acronyms as in Figure 1. Acronyms represent positive result with particular test(s), unless indicated differently. Test results for other tospovirus species are not reported or unknown;
2
Amplicons only obtained when RT-PCR was performed with purified RNA; 3For total RNA amplicons only obtained with primer set directed to G1/G2 gene.
22
S0166-0934(15)30104-X http://dx.doi.org/doi:10.1016/j.jviromet.2016.03.015 VIRMET 12986
To appear in:
Journal of Virological Methods
Received date: Accepted date:
19-11-2015 28-3-2016
Please cite this article as: Hassani-Mehraban, A., Westenberg, M., Verhoeven, J.T.J., van de Vossenberg, B.T.L.H., Kormelink, R., Roenhorst, J.W., Generic RT-PCR tests for detection and identification of tospoviruses.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2016.03.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Generic RT-PCR tests for detection and identification of tospoviruses
A. Hassani-Mehraban1, M. Westenberg1, J.T.J. Verhoeven1 ,B.T.L.H. van de Vossenberg1, R. Kormelink2 & J.W. Roenhorst1* 1
National Plant Protection Organization, National Reference Centre, Geertjesweg 15, P.O.
Box 9102, 6700 HC Wageningen, the Netherlands; 2Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands
*Corresponding e-mail address: [email protected] Running head: Generic detection of tospoviruses
Highlights New RT-PCR tests have been designed for generic detection and identification of tospoviruses This is the first test design potentially allowing detection and identification of all known and unknown tospovirus species, since primer design is based on conserved regions within different (sub) clades Tests have been successfully applied on diagnostic samples, which allowed identification of seven different species and a non-described tospovirus from alstroemeria plants Tests provide a relevant tool for plant quarantine and diagnostic laboratories
Abstract A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5’ upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all
1
species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a nondescribed tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.
Keywords: Bunyaviridae, plant virus; diagnostics; clade-specific primers; nucleocapsid gene
Introduction Tospoviruses are causing serious losses in many economically important crops worldwide, (Prins and Goldbach 1998; Pappu et al., 2009). To date 29 species have been distinguished within the genus Tospovirus (family Bunyaviridae), including 11 assigned (Plyusnin et al., 2012; http://ictvonline.org/virusTaxonomy.asp) and 18 tentative species (Chu et al., 2001b; Meng et al., 2013; De Oliviera et al., 2012; Dong et al., 2013; Plyusnin et al., 2012; Shimomoto et al., 2014; Torres et al., 2012; Yin et al., 2014; Zhou et al., 2011). These species differ in molecular and biological properties, as well as geographical distribution. Tospoviruses consist of enveloped particles, enclosing three (negative or ambisense) ssRNA segments, denoted large (L), medium (M) and small (S), complexed with the nucleocapsid (N) protein (De Haan et al., 1990). Within the genus Tospovirus, species are identified on the criterion that the sequence of the N protein shows less that 90% identity with other species (de Ávila et al., 1993; Plyusnin et al., 2012). In addition, plant host range, vector specificity and serological relationships of the N protein are taken in consideration. At present the majority of
2
species can be assigned to the American, Asian and Eurasian clades, remaining a small group of distinct and floating species (Hassani Mehraban et al., 2005; Pappu et al., 2009). To control tospovirus diseases, species identification is important (EFSA, 2012). In many crops, it is not possible to identify the virus just on the basis of symptomatology, as symptoms caused by different species might look similar. Therefore, the availability of reliable and sensitive diagnostic tests is essential to detect and identify the viral etiology of the disease. Mechanical inoculation of test plants is suitable for detection, but does not allow identification at the species level. Serological assays using poly- and monoclonal antisera, mainly against the N protein, are widely used, although a proper identification is often hampered by cross reactions (Supplemental data [S1]). In addition, over the last decades an increasing number of molecular tests has been developed, either targeting individual species or aiming a broader detection. Especially for generic tests, however, data on the scope of detection are lacking and none of them seems to cover all known species (Table 4). Therefore, this study aimed to develop a molecular test for generic detection and identification of all tospoviruses. Based on the alignment of N gene nucleotide sequences from 28 different species, clade-specific primers were designed. In the final test eleven primers were used, which showed to successfully detect different tospoviruses in diagnostic samples and allow their classification and further speciation into any of the American, Asian and Eurasian clades.
Materials and methods Virus isolates and propagation Twenty tospovirus isolates used in this study are listed in Table 1. Virus isolates were propagated by mechanical inoculation on Nicotiana benthamiana plants with inocula prepared 3
from infected leaf tissue ground in PBS buffer (pH 7.0), containing 0.1% Na2SO3. Inoculated plants were grown under greenhouse conditions at 18-25ºC and 14 h illumination) and monitored for symptom expression. Leaves of plants showing systemic symptoms (after 1-2 weeks) were harvested and stored at -80°C until use for RNA extraction. Samples of (freeze-) dried leaf material of CCSV, MeSMV, PCFV and PolRSV were used for RNA extraction directly.
Tospovirus phylogeny Amino acid sequences of the N protein from 28 tospovirus species were obtained from GenBank (see Table 2 for Acc. numbers) and aligned using ClustalW (Larkin et al., 2007) in Geneious R7 (Biomatters Ltd, Auckland, New Zealand) using Bunyamwera virus (Acc. No. NP_047213) as outgroup. The alignment was used to construct a neighbour-joining tree with bootstrap analysis (N=1000) in Mega 5.05 (Tamura et al., 2011).
Sequence alignment and primer design Nucleotide sequences of the N gene and its 5’ untranslated region sequence (UTR) from 28 tospo virus species were obtained from GenBank (see Table 2 for accession numbers) and aligned using ClustalW (Larkin et al., 2007) in Geneious R7 (Biomatters Ltd, Auckland, New Zealand). Note that TNSV was not used for primer design because the sequence was not available at the time the alignment was made. Conserved and (sub) clade-specific N gene sequences were selected from the ClustalW alignment and used for design of (sub) cladespecific primer pairs (Table 2). Primers complementary to the very first eight conserved terminal nucleotides of the N UTR sequence were extended with nine nucleotides (GGGGGATCC) to increase annealing specificity during subsequent PCR amplification. Primers were tested in silico for self-annealing and primer-dimer formation by using 4
OligoCalc software (Kibbe, 2007). The specificity was tested within the Geneious R7 program by allowing a maximum of four mismatches and by perfoming BLAST searches in GenBank (Altschul et al., 1990). To verify the identity of virus isolates, species-specific primers amplifying the entire N gene were designed, based on available sequences in GenBank (Table 2). These primers were designed along with start and stop codons, except for the forward primer of PCFV which was selected from a sequence more upstream of the start codon to obtain a higher GC content.
RNA extraction Leaf material (ca. 0.1-0.5 g) was homogenized in 3.5 ml of GH+ grinding buffer (6 M guanidine hydrochloride, 0.2 M sodium acetate pH 5.2, 25mM EDTA and 2.5% PVP-10) according to a protocol modified from Menzel et al. (2002). Homogenization was performed in extraction bags using a HOMEX 6 homogenizer (Bioreba AG Reinach, Switzerland). Next, plant sap (1 ml) was lysed by incubation in a thermomixer for 10 min at 65°C and 850 rpm and subsequently centrifuged for 2 min at 16.000g. A volume of 500 ul from the supernatant was transferred to a QIAshredder spin column for total RNA purification by the RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands) and following the manufactures protocol from there. RNA was eluted in a final volume of 40 μl elution buffer and stored at -80˚C. Healthy leaf material and extraction buffer have been used as negative isolation controls (NIC).
RT-PCR assays cDNA synthesis and PCR were performed using the OneStep RT-PCR Kit (Qiagen, Venlo, the Netherlands) in a total volume of 25 μl, containing 1 μl RNA template, 5 μl OneStep RTPCR Buffer, 1 μl OneStep RT-PCR Enzyme Mix, 1 μl dNTP Mix (10 mM) and 1 μl of each primer (10 μM). The RT-step was performed at 50°C for 30 min followed by a PCR 5
consisting of one cycle at 95°C for 15 min, followed by 35 cycles at 94°C for 1 min, 50-58°C for 30 sec and 72°C for 1 min, and a final extension cycle at 72°C for 5 min. The annealing temperatures (Ta) used for the different tospovirus clades are shown in table 2. To verify the quality of RNA extraction, plant NADH dehydrogenase subunit 5 (nad5) mRNA was RTPCR amplified using nad-5F (5’-GATGCTTCTTGGGGCTTCTTGTT-3’) and nad5-R (5’CTCCAGTCACCAACATTGGCATAA-3’) primers (Menzel et al., 2002). Five μl of each PCR reaction was mixed with 1 μl 6x Bromophenol Blue Loading solution (Promega, Leiden, the Netherlands) and products resolved on a 1.5% RESult LE General Purpose Agarose gel (BIOzymTC, Landgraaf, the Netherlands) with SYBR-safe staining (Life Technologies, Bleiswijk, the Netherlands). Amplicons were visualized under a GeneGenious gel imaging system (Syngene, Cambridge, United Kingdom) and their sizes estimated using a 1 kb Plus DNA ladder (Promega). Molecular grade water was used as negative amplification control (NAC) in each run to monitor possible contamination. The RT-PCR on BeNMV has been performed at the Department of Cell Biology, University of Brasília, by using the SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Life Technologies, São Paulo, Brasil). This reaction was performed in a total volume of 25 μl, containing 1 μl RNA template, 12,5 μl Reaction Mix, 1 μl SuperScript® III RT/ Platinum® Taq Mix, and 1 μl of each primer (10 μM). The RT-step was performed at 50°C for 30 min followed by a PCR consisting of one cycle at 94°C for 2 min, 35 cycles of 94°C for 1 min, 58°C for 30 sec, 68°C for 1 min, and a final extension cycle at 68°C for 5 min.
Sequence analysis PCR products were purified using QIAquick PCR Purification Kit (Qiagen) prior to cycle sequencing with both amplification primers in individual runs. Cycle-sequence reactions were 6
based on the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies) and performed according to the manufacturer’s instructions. Cycle-sequence products were purified using the DyeEx 2.0 Spin Kit (Qiagen) and sequenced on a 3500 Genetic Analyzer (Life Technologies). Electropherograms were assembled and edited using Geneious R7. Amplification primers were trimmed from the consensus sequence and, when needed, further trimming was performed to obtain high quality (PHRED > 30) consensus sequence data. The assembled sequences were verified by comparing with available sequences in the GenBank using BLASTn and BLASTp (Altschul et al., 1990).
Results Tospovirus phylogeny Phylogenetic analysis with the amino acid sequences of the N protein of 29 tospovirus species revealed that tospoviruses can be divided in five distinct groups with LNRV as an independent species (Figure 1). The American clade could be divided in two subgroups, clades AM C1 and AM C2. Species originating from Asia fell into two subgroups, clades AS 1 and AS 2. It was noted that the N protein sequence of species belonging to the AS 1 clade are more similar to those of the EuroAsian (EA) clade then to those of the AS 2 clade. The division in (sub) clades was used for the design of clade specific primers.
Primer design and in silico verification of species identification A multiple nucleotide sequence alignment of the N gene and its 5’ UTR from 28 tospovirus species revealed no highly conserved regions shared by all tospoviruses, with the exception of similarities within the very first and conserved eight terminal nucleotides from the RNA segment. Therefore, this sequence was selected for design of a forward primer and extended 7
with a few nucleotides for the introduction of clade-specificity, based on the strategy as described by Hassani-Mehraban et al. (2005). According to this concept, four primers were designed and designated AM1-F, AM2-F, AS-EA-F and LNRV-F, specific to members of the American clade 1, American clade 2, Asian and Eurasian clades (and their subgroups,), and LNRV, respectively (Table 2, Column Generic primers). In addition to these termini-primers clade-specific reverse primers were designed in such a way that the obtained amplicon length after RT-PCR on a virus from each (sub) clade was distinct in size to allow for immediate classification. To ensure that the partial N gene and its 5’ UTR sequence between the terminal–end primer binding site and the internal reverse primer site provided enough resolution for species identification, BLASTn analysis was performed with these sequences from all 28 species used in the alignment. The results revealed that in all cases the intraspecies variation was less than the interspecies variation and were supported by tree views in which all sequences fell within their species-specific clade (data not shown).
RT-PCR and sequence analysis Using the primer sets as described above and designed for RT-PCR amplification of (partial) N gene specific for the various (sub) clades, tests were performed on samples collected from infected plants challenged with isolates from 20 different tospoviruses (Table 1). After some optimization, the results from repeated tests provided consistent results as shown in Figure 2. RT-PCR reactions with clade-specific primer sets were initially performed for each subset of tospoviruses at a Ta of 50˚C, but this only resulted in the expected (size-) specific amplicons for the American clade 1 (760 bp; Figure 2A 1st panel (top)), and Asian clade 2 primer sets (520 bp; Figure 2A, 3rd panel), and non-specific PCR products for the others (data not shown). After the application of gradient PCRs (52-58oC) the optimum Ta turned out to be 8
52˚C for members of the Asian clade 1 (370 bp; Figure 2A, 2nd panel) and Eurasian clade (790 bp; Figure 2A, 2nd panel), and 58oC for those of the American clade 2 (670 bp; Figure 2B, right panel) and LNRV (430 nt; Figure 2A, 4th panel). Attempts to combine all cladespecific RT-PCRs in one experiment failed and were only successful for the combination Asian clade 1 and Eurasian clade (Figure 2A, 2nd panel). Furthermore, the results of the final five separate RT-PCRs show that for all tospoviruses in the same clade the similar, size-expected amplicon was generated, while no amplicon was detected in the negative isolation controls performed with extraction buffer (NIC) and healthy leaf material of N.bentahmiana (Figure 2A). In the case of IYSV two amplicons of the expected size were generated with both the Asian clade 1 and Eurasian clade primers (Figure 2A, 2nd panel IYSV), likely due to the fact that Asian clade 1 reverse primer was able to anneal to the RNA of this IYSV isolate. Nevertheless, the virus could be identified as IYSV by sequencing both amplicons together with the Eurasian clade primers. BeNMV and a selection of species from the other clades (GRSV, IYSV, LNRV, WSMoV; Figure 2B) were tested in a separate test. The results show that the American clade 2 primers were able to detect BeNMV (Figure 2B, right panel), while no amplicons were produced with extraction buffer (Figure 2B, left panel NIC) or RNA from healthy leaf material (Figure 2B, left panel N. benthamiana, right panel S. lycopersicum ) or on the tested species from the other clades (Figure 2B, left panel). In all experiments, the RNA quality of the samples was verified by production of the expected amplicon for nad5 as an internal control (Figure 2A). Subsequent sequencing and analysis of all amplicons by BLAST searches in NCBI GenBank confirmed their viral identity and indicated that amplicon-sequences obtained from diagnostic test samples this way, would immediately allow a provisional identification of the isolate.
Testing of diagnostic samples 9
To verify the validity of the designed primer sets for use in tospovirus diagnostics and classification a set of twenty five samples collected from different crops and origins suspected of tospovirus infections were tested using the generic RT-PCRs. In twenty samples the presence of a tospovirus was detected (Table 3). The majority of the samples originated from Asia and appeared infected with one of the Asian clade members. The others came from various regions and were infected by members of the American clade 1. From the remaining samples, two turned out to be infected with a cucumovirus, one with a potyvirus and two with an unknown virus (data not shown). Unfortunately, for non-infected pepper samples the Asian clade 1/Eurasian clade primer combination gave a plant specific amplicon around the expected 800 bp fragment for the Eurasian clade tospoviruses, as confirmed by sequence analyses. This indicates that sequencing of the obtained amplicons is required to confirm the genuine presence and identification of a tospovirus in field samples. In further support for the usefulness of the designed primers sets, the generic tospovirus tests were successfully applied on a yet non-described tospovirus isolate from alstroemeria plants, provisionally named Alstroemeria yellow spot virus (Hassani-Mehraban et al., manuscript in preparation). As expected from its S RNA nucleotide sequence (unpublished data) an amplicon was produced with the Eurasian primer set (Figure 2A) and the identity confirmed by sequence analysis.
Discussion A RT-PCR approach for generic detection and provisional identification of tospoviruses has been developed, based on five RT-PCR’s targeting tospovirus species from different clades and a floating species within the genus. By using conserved regions from viruses within each (sub) clade, primers have been designed that potentially detect all (known) species from that 10
clade. This was nicely demonstrated by the production and nucleotide sequence confirmation of expected amplicons from twenty available isolates of species from the American clades 1 and 2, Asian clades 1 and 2, the Eurasian clade and the floating species LNRV. The usefulness of the PCR tests to even detect new and unknown tospovirus species was strengthened by the positive results obtained with a yet non-described putative new tospovirus species from Alstroemeria that was not included in the analyses for primer design. Application of the tests on 25 diagnostic samples from mainly peppers and tomatoes suspected from a tospovirus infection, already gave promising results. In 20 out of 25 samples a tospovirus was detected and seven different species within the American clade 1 and Asian clade 1 could be identified. In the five remaining samples, for which the test results were negative, other virus species were found. Unfortunately, the application of RT-PCR tests on samples collected from pepper revealed a non-specific reaction with the Eurasion primers, which could only be prevented when the virus was meachanically transfered to another host like N. benthamiana. This observation stresses the importance to verify the identity of the amplicon by sequence analysis and avoid false interpretations. Although the N gene sequence obtained from amplicons will not only verify the presence but additionally allows the classification of the tospovirus involved, some caution has to be considered in light of the possible occurrence of recombination and reassortment between tospoviruses (Tentchev et al., 2011). The added value of the newly developed generic tests in comparison with those already used, is illustrated by indicating for each test the species that can be detected and identified within the different (sub) clades (Table 4). Test results for the remaining tospovirus species are not reported (or not known), except for PCFV which is not detected by the test described by Chen et al. (2012). From this overview it is clear that the clade-specific primer sets described in this paper provide the only tests detecting species in all clades. Moreover, the fact that their design 11
is based on conserved regions within each (sub) clade, substantially increases the probability of detection of other known and unknown species. Furthermore, by targeting the N gene, sequencing of the amplicons allows a provisional identification of the species, which is an advantage over tests that target the G1/G2, NSm or L genes. In conclusion, these results demonstrate that the newly-developed generic RT-PCR tests allow detection and provisional identification of known and unknown tospoviruses.. In addition, the species-specific primers enable confirmation of the identity by sequence analysis of the Ngene. The fact that the primer sets are based on conserved regions within the different (sub) clades, confirms their potential ability of generic testing on tospoviruses. This makes the tests a relevant tool for plant quarantine and diagnostic laboratories.
Acknowledgements We thank J. Saaijer (Laboratory of Virology, Wageningen University and Research Centre, the Netherlands), L. Hüner (National Plant Protection Organization, the Netherlands) and M. Martins Severo de Almeida (Department of Cell Biology, University of Brasília, Brazil) for technical assistance, and R. Oliveira Resende (Department of Cell Biology, University of Brasília, Brasília, Brazil), T.C. Chen (Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan), Y. Shimomoto (Kochi Agricultural Research Center, Kochi, Japan) and M. Turina (Instituto di Virologia Vegetale del CNR, Torino, Italy) for providing virus isolates.
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12
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Mumford, R.A., Barker, I., Wood, K.R., 1996. An improved method for the detection of Tospoviruses using the polymerase chain reactions. J. Virol. Methods 57, 109-115. Okuda, M., Hanada, K., 2001. RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template. J. Virol. Methods 96, 149–156. Pappu, H.R. Jones, R.A.C., Jain, R.K., 2009. Global status of tospovirus epidemics in diverse cropping systems: Successes gained and challenges that lie ahead. Virus Res. 141, 219-236. Plyusnin, A., Beaty, B.J., Elliott, R.M., Goldbach, R., Kormelink, R., Lundkvist, A., Schmaljohn, C.S., Tesh, R.B., 2012. Bunyaviridae. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J., eds. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego, USA: Elsevier Academic Press, 725–741. Prins, M., Goldbach, R., 1998. The emerging problem of tospovirus infection and nonconventional methods of control. Trends Microbiol. 6, 31-35. Shimomoto, Y., Kobayashi, K., Okuda, M., 2014. Identification and characterization of Lisianthus necrotic ringspot virus, a novel distinct tospovirus species causing necrotic disease of lisianthus (Eustoma grandiflorum). J. Gen. Plant Pathol. 80, 169-175. Tamura, K, Peterson, D., Peterson, N., Stecher, G., Nei, M. Kumar, S., 2011. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evo. 28, 2731-2739. Tentchev, D., Verdin, E., Marchal, C., Jacquet, M., Aguilar, J.M., Moury, B., 2011. Evolution and structure of Tomato spotted wilt virus populations: evidence of extensive reassortment and insights into emergence processes. J. Gen. Virol. 92, 961–973. Torres, R., Larenas, J., Fribourg, C., Romero, J., 2012. Pepper necrotic spot virus, a new tospovirus infecting solanaceous crops in Peru. Arch. Virol. 157, 609-615. 15
Uga, H., Tsuda, S., 2005. A one-step reverse transcription-polymerase chain reaction system for the simultaneous detection and identification of multiple tospovirus infections. Phytopathol. 95, 166-171. Yin, Y., Zheng, K., Dong, J., Fang, Q., Wu, S., Wang, L., Zhang, Z., 2014. Identification of a new tospovirus causing necrotic ringspot on tomato in China. Virol. J. 11, 213. Zhou, J., Kantartzi, S.K., Wen, R.H., Newman, M., Hajimorad, M.R., Rupe J.C., Tzanetakis, I.E., 2011. Molecular characterization of a new tospovirus infecting soybean. Virus Genes 43, 289-295.
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Figure legends
Figure 1. Neighbour-joining tree with bootstrap (N=1000) analysis of the nucleocapsid protein sequence of 29 tospovirus species and Bunyamwera virus (GenBank Acc. No. NP_047213) as outgroup. The scale bar measures evolutionary distance in substitutions per amino acid. The accession numbers corresponding to each isolate are given in Table 2.
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Figure 2. Agarose gel electrophoresis of amplicons obtained from 20 different tospovirus isolates using the five optimized tospovirus RT-PCR tests: A. Amplicons produced by the primers (from top to bottom) on viral RNA from members belonging to the American clade 1 (AM-C1), combination of Asian clade 1 and Eurasian clade and (AS-C1 and EA-C), Asian clade 2 (AS-C2) members and LNRV. Primers specific for the plant NADH dehydrogenase subunit 5 (nad5) mRNA was used as internal control to verify the quality of RNA; B. Amplicons produced on tospoviruses by American clade 2 primers (AM-C2). NAC: Negative amplification control (molecular grade water). NIC: Negative isolation control (extraction buffer), and healthy leaf material of N. benthamiana and S. lycopersicum. Species indicated by acronyms as in Figure 1; AYSV: Alstroemeria yellow spot virus, (Hassani-Mehraban et al., 2015, manuscript in preparation).
18
Table 1. Tospovirus isolates used in this study Species1
Code
Origin
ANSV
Colombia
WUR2
AYSV3
ALS-2000
WUR
BeNMV
Brazil
University of Brasília, Brazil
CaCV
Thailand-
WUR
CCSV
Taiwan
National Chung Hsing University, Taiwan
CSNV
Brazil
WUR
GBNV
India
WUR
GRSV
SA-05
WUR
INSV
NL-07
WUR
IYSV
IYSV-NL
WUR
LNRV
Japan
Kochi Agricultural Research Center, Japan
MeSMV
Mexico
Istituto di Virologia Vegetale del CNR, Italy
MYSV
Thailand-isolate physalis severe mottle virus (PSMV)
WUR
PCFV
Taiwan
National Chung Hsing University, Taiwan
PolRSV
Plg3
Istituto di Virologia Vegetale del CNR, Italy
TCSV
BR-03
WUR
TNRV
Thailand
WUR
TSWV
BR-01
WUR
TYRV
Iran
WUR
WSMoV
Thailand
WUR
1
Species indicated by acronyms as in Figure 1; 2Wageningen University and Research Centre; 3Alstroemeria yellow spot virus, Hassani-
Mehraban et al., 2015 (manuscript in preparation).
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Table 2. Generic and specific primers designed and used in this study
Clade
American 1
American 2
Asian 1
Species1
Eurasian
LNRV
1
Generic Primers (5’‐3’)
Position in S RNA2
Ta (˚C)
Amplicon size (bp) using generic primers
ANSV
F: ATGTCTAAGGCTAAGTTGAC; R: TTAAGCAACACCTGAAATTTTGG
GQ478668
1‐14 / 732‐753
50
762
CSNV
F: ATGTCTAAAGTTAAGCTTAC; R: TTAAACAAGATCTTTAGGAATAAG
AF067068
1‐14 / 732‐753
50
762 761
GRSV
F: ATGTCTAAGGTCAAGCTCAC; R: CCAAGATTGCTGGTGTTGCATG
AF487517
1‐14 / 731‐752
50
INSV
F: ATGAACAAAGCAAAGATTACC; R: TTAAATAGAATCATTTTTCCC
NC_003624
1‐14 / 741‐762
50
762
PNSV
NA3
HE584762
1‐14 / 732‐753
50
762
MeSMV
F: ATGTCTAAGGTCAAGCTCACC; R: TCATGCAACACCAGCAATCTTG
EU275149
1‐14 / 739‐760
50
769
TCSV
F: ATGTCTAAGGTTAAGCTCAC; R: CAAAACTCGCAGAACTTGCTTG
AF413110
1‐14 732‐753
50
762 763
AM1‐F: GGGGGATCCAGAGCAATTGTGTC AM1‐R: CTTTGCTTTTCAGCACAGTGCA4
TSWV
F: ATGGGATCTAATGCACTAAAG; R: TTAGCTCATGAAGCCATGATTTTTGC
NC_002051
1‐14 / 733‐754
50
ZLCV
NA
AF067069
1‐14 / 821‐842
50
851
BeNMV
F: ATGGGATCTAATGCACTAAAG; R: TTAGCTCATGAAGCCATGATTTTTGC
JN587268
1‐11 / 647‐663
58
672
SVNaV
NA
GU722319
1‐11 / 664‐678
58
687
CaCV
F: ATGTCTACCGTCAGGCAACTTAC; R: TCACACTTCAATAGATGTACTAG
NC_008301
1‐13 / 339‐360
52
369
CCSV
F: ATGTCTAACGTCAGAGGTTTAAC; R: TTAAAAAGTTAAATCACCACTC
AY867502
1‐13 / 339‐360
52
369
GBNV
F: ATGTCTAACGTCAAGCAACTC; R: TTACAATTCCAGCGAAGGACC
NC_003619
1‐13 / 339‐360
52
369
NA
KM819706
1‐13/ 339‐360
52
369
MYSV
F: ATGTCTACCGTTGCTAAGCTTAC; R: TTAAACTTCAATGGACTTAGACC
NC_008300
PCSV
NA
TNRV
F: ATGTCTAACGTTAGGAAAACC; R: TTACAGTCCTATACTTCCTCTC
TZSV
NA
WBNV
NA F: ATGTCTAACGTTAAGCAGCTC; R: TTACACTTCCAAAGAAGTGCTG
MVBaV
WSMoV Asian 2
N gene primers (5’‐3’)
Accession no.
AM2‐F: GGGGGATCCAGAGCAATCGG AM2‐R1: GCAACTCTACCAGCTTG AM2‐R2: GCAACTTTAGCAGCTTG
1‐13 / 342‐363
52
372
1‐13 / 351‐372
52
381
FJ946835
1‐13 / 350‐371
52
380
EF552433
1‐13 / 339‐360
52
369
GU584184
1‐13 / 339‐360
52
369
KF383956
AS‐EA‐F: GGGGGATCCAGAGCAATCGAGG AS1‐R: GCTTCAGTCCTCTTAAATGTCC
NC_003843
1‐13 / 337‐358
52
367
PCFV
F: CCCATGTCTAAAACCAAAGTCAAG; R: CCCGTGAATAACATCGATATATAG
AF080526
1‐13 / 493‐512
50
521
GYSV
NA
AF013994
1‐13 / 493‐512
50
521
HCRV
NA
KC290943
1‐13 / 765‐784
52
793
IYSV
F: ATGTCTACCGTTAGGGTGAAAC; R: TTAATTATATCTATCTTTCTTG
AF001387
1‐13 / 764‐783
52
792
F: ATGTCTACCACAGGGTTGAG; R: CAAAATCCTTGATTCTTTTTGGAGGC
EF445397
1‐13 / 766‐785
52
794
TYRV
F: ATGGCTACCGCACGAGTGAGC; R: TTAAAATGCATCATTCTTTTTGG
AY686718
1‐13 / 765‐784
52
793
LNRV
F: ATGTCTACCGTCAGGCAACTTAC; R: TCACACTTCAATAGATGTACTAG
AB852525
27‐46 / 437‐458
58
432
PolRSV
AS‐EA‐F: GGGGGATCCAGAGCAATCGAGG AS2‐R: CTTTGAAGATGACCTCATCT
AS‐EA‐F: GGGGGATCCAGAGCAATCGAGG EA‐R: TTGTTCAATGAAGCAGCACC LNRV‐F: CCAGTAAAAGACGAATCCC LNRV‐R: GATTCAAATCGCCCAGCAGTCC
Species indicated by acronyms as in Figure 1; 2Position of the generic forward/reverse primer in the sense RNA; 3NA: Not applicable, since these species were not used in this study or in case of BeNMV the specimen was not available for verification by sequencing of the entire N gene; 4Hassani-Mehraban, A., Botermans, M.,
Verhoeven, J.Th.J., Meekes, E., Saaijer,J., Peters, D., Goldbach, R., Kormelink, R. 2010. A distinct tospovirus causing necrotic streak on Alstoemeria sp. in Colombia. Arch. Virol. 155, 423-428.
20
Table 3. Tospovirus species identified in diagnostic samples Species1
Crop species
Origin (number of samples)
CaCV
Capsicum annuum
China (1), Vietnam (1)
CaCV
Solanum lycopersicum
Vietnam (2)
GBNV
Solanum lycopersicum
India (3)
GRSV
Capsicum annuum
Brazil (1)
INSV
Leontopodium sp.
Switzerland (1)
TCSV
Capsicum annuum
Brazil (1)
TCSV
Capsicum frutescens
Dominican Republic (1)
TNRV
Capsicum annuum
Thailand (6), Vietnam (1)
TSWV
Solanum lycopersicum
Croatia (1)
TSWV
Capsicum annuum
South Africa (1), Turkey (1)
1
Species indicated by acronyms as in Figure 1
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Table 4. Molecular tests for generic detection (and identification) of tospoviruses1 Reference
Method
Target(gene)
Type of primers
AM-C1
Mumford et al.,
RT-PCR
S RNA (N)
Universal
GRSV, INSV,
1996 Dewey et al.,
AS-C1
AS-C2
WSMoV
PCFV
EA
LNRV
TCSV, TSWV RT-PCR + RFLP
S RNA (N)
Universal
1996 Chu et al.,2001a
AM-C2
GRSV, TCSV, TSWV
RT-PCR
L RNA (L)
Degenerate
GRSV, INSV, TSWV
Eiras et al., 2001
RT-PCR
S RNA (N);
Universal primers
CSNV, GRSV,
M RNA (G1/G2);
3 primers sets
INSV, TCSV,
M-RNA (NSm)
targeting the
TSWV, ZLCV2,3
IYSV2
indicated genes Eiras et al., 2001
Dot blot
L RNA (L)
Specific probes
hybridization
CSNV, GRSV,
IYSV
INSV, TCSV, TSWV, ZLCV
Okuda & Hanada,
RT-PCR + RFLP
S RNA (N)
RT-PCR
S RNA (N)
Degenerate
INSV, TSWV
MYSV, WSMoV
IYSV
Universal
INSV, TSWV
MYSV, WSMoV
IYSV
CSNV, INSV,
CaCV
IYSV
2001 Uga & Tsuda, 2004
degenerate combined with species specific
Kuwabara et al.,
RT-PCR
S RNA (N)
2010 Chen et al., 2012
TSWV, ZLCV RT-PCR
M RNA (NSm);
Degenerate
L RNA (L)
GRSV, INSV,
CaCV, CCSV,
TCSV, TSWV
GBNV, MYSV,
PCFV (negative)
IYSV, TYRV
PCFV
IYSV, PolRSV,
WBNV, WSMoV Charoenvilaisiri
RT-PCR (+
et al., 2014
ELISA)
This paper
RT-PCR
S RNA (N)
Universal
CaCV, MYSV,
S RNA (N)
Universal clade
ANSV, AYSV,
specific
CSNV, GRSV,
GBNV, MYSV,
INSV, MeSMV,
TNRV, WSMoV
TNRV, WSMoV BeNMV
CaCV, CCSV,
LNRV
TYRV
TCSV, TSWV 1
Species indicated by acronyms as in Figure 1. Acronyms represent positive result with particular test(s), unless indicated differently. Test results for other tospovirus species are not reported or unknown;
2
Amplicons only obtained when RT-PCR was performed with purified RNA; 3For total RNA amplicons only obtained with primer set directed to G1/G2 gene.
22