Background
The viruses of
Tospovirus, the only plant-infecting genus in the family
Bunyaviridae, cause severe damage to several agricultural crops worldwide [
1,
2]. Tospoviruses have enveloped quasi-spherical particles that are 80–120 nm in diameter, and a tripartite-segmented single-stranded RNA (ssRNA) genome [
3]. The large (L) RNA is in negative sense and contains one single open reading frame (ORF) encoding an RNA-dependent RNA polymerase for replication and transcription [
4,
5]. Both middle (M) and small (S) RNAs are ambisense, each consisting of two bi-directional ORFs flanked by an AU-rich intergenic region. The M RNA encodes a movement protein (NSm) from the viral sense and the envelope glycoproteins Gn and Gc from the viral complementary sense [
6,
7]. The S RNA encodes a suppressor of plant gene silencing (NSs) from the viral sense and an RNA-encapsidating nucleocapsid protein (NP) from the viral-complementary sense [
8‐
10].
According to the International Committee on Taxonomy of Viruses, the criteria for demarcating a tospovirus species include thrips-vector specificity, specific host range, serology of NP, and lower than 90 % amino acid identity of the NP [
3]. Tospoviruses can be serologically grouped with the aid of antisera against the NPs. The original demarcation consisted of four serogroups (I to IV) with six known tospoviruses [
11]. However, a type member-based serological classification system was recommended as increasing characterized tospoviruses [
12]. Currently, a more comprehensive serological grouping has been established through experimental evidence. Most of the known tospoviruses are now classified into four serogroups using
Groundnut yellow spot virus (GYSV),
Iris yellow spot virus (IYSV),
Tomato spotted wilt virus (TSWV) and
Watermelon silver mottle virus (WSMoV) as type members [
13‐
15]. The serological grouping of tospoviruses matches well with their phylogenetic clustering, in which tospoviruses sharing more than 51.8 % similarity at the NP amino acid sequence level are serologically related [
13,
16]. Because of the high degree of sequence identity within the same serogroup, distinguishing and diagnosing tospoviruses rely on monoclonal antibodies (MAbs) with a higher specificity to a particular species. However, tospoviruses, such as Capsicum chlorosis virus (CaCV),
Groundnut bud necrosis virus (GBNV),
Watermelon bud necrosis virus (WBNV) and WSMoV, sharing 80 % or higher NP amino acid sequence similarity are still difficult to distinguish even when MAbs are used [
17]. Therefore, when generating MAbs, it is critical to validate the serological assays to prevent false diagnosis.
Tospoviruses are causing significant losses in yield and quality of several economic crops in China [
18,
19]. Two new tospoviruses Tomato necrotic spot associated virus (TNSaV) and Tomato zonate spot virus (TZSV) infecting tomato were first discovered in Guizhou and Yunnan provinces, respectively [
19,
20]. The serological relationship between TNSaV and TZSV was demonstrated by the cross reaction with the antiserum against the TZSV NP [
19]. TZSV currently becomes the important threat infecting tomato, tobacco and ornamentals in southwestern China, and
Frankliniella occidentalis (Pergande) is its main transmissible vector [
18,
20‐
22]. Calla lily chlorotic spot virus (CCSV), first collected from calla lily in Taiwan, is occurring in Yunnan Province that infects tobacco and spider lily [
23,
24]. The transmissible vector of CCSV and TNSaV in China remains unknown.
Symptomatology is insufficient for identification of virus species due to the fact that similar symptoms on the same crop may be caused by different tospoviruses. Indeed, both TNSaV and TZSV induce yellow and necrotic ringspots on tomato fruits [
19,
20] and all of CCSV, TNSaV and TZSV cause chlorotic and necrotic spots on tobacco leaves [
19,
21,
24]. The NPs of CCSV, TNSaV and TZSV share high degrees of amino acid identity (80.9–85.8 %) with each other [
19,
20,
23], and their serological relationship was recently demonstrated through the serological assays using the MAbs against the NP of CCSV (MAb-CCSV-NP) [
25] and the NSs protein of WSMoV (MAb-WNSs) [
26]. Although the virus-specific primers for reverse transcription-polymerase chain reaction (RT-PCR) can be used to identify tospovirus species when antibodies are unavailable or indistinguishable, the need of professional skill and equipment and the cost of manpower and time limit the application of RT-PCR for a large amount of samples in epidemiological investigation. Enzyme-linked immunosorbent assay (ELISA) is an efficient serological method for field survey of viruses, and the titer and specificity of antibodies are very important for successful assays. CCSV, TNSaV and TZSV induce similar symptoms on their common natural hosts in southwestern China [
19,
24], the production of virus-specific antibodies for identification of these tospoviruses is essential to improve field surveys.
In this study, MAbs against the NP of the TZSV isolate 13YV639, which was collected from spider lily (Crinum asiaticum L.) in Yunnan Province, China, were screened. Two MAbs with distinct serological reactivity were obtained. Using the newly generated MAbs and previously reported MAbs, we proposed an efficient serological assay to differentiate CCSV, TNSaV and TZSV in field samples.
Discussion
There is a need to develop tools to identify and diagnose tospoviruses in the field earlier to prevent a disease from becoming an epidemic. In eastern Asian countries, including mainland China, India, Japan, Taiwan and Thailand, tospoviruses cause severe agricultural problems. Most of tospoviruses known to prevail in these countries are clustered in the WSMoV serogroup that can be detected by serological assays using the group-broad antiserum RAs-WSMoV-NP against the WSMoV NP [
13] and MAb-WNSs produced from the NSs protein of WSMoV [
26]. The NP MAbs are useful for differentiation of most tospovirus species in a serogroup [
13,
25]. However, the tospovirus species sharing over 80 % NP amino acid identity are still difficult to distinguish even when MAbs were used [
17], and an additional RT-PCR analysis using species-specific primers is required.
CCSV, TNSaV and TZSV also share higher than 80 % NP amino acid identity with each other [
19,
20]. Their close serological relationship was experimentally demonstrated by reacting with MAb-WNSs and MAb-CCSV-NP in both indirect ELISA and immunoblotting (Additional file
2: Figure S1). Here, we attempted to produce MAbs specific to TZSV for developing a method to specifically diagnose each virus without the need of additional RT-PCR analysis. Eighteen tospovirus species representing the four major serogroups were used to test the serological reaction of the two prepared MAbs. Moreover, the identity of viruses was verified by RT-PCR analyses using the newly designed primer pairs specific to CCSV, TNSaV or TZSV. The reaction of the obtained MAb-TZSV-NP(S15) supports a closer serological relatedness between TZSV and CCSV rather than TNSaV. We noticed a weak reaction with a two-fold mean reading (0.27) higher than those of the negative controls WSMoV-infected (0.13) and healthy (0.11) plants when MAb-TZSV-NP(S15) incubated with TNSaV in indirect ELISA (Fig.
2a), but no signal was found in immunoblotting (Fig.
2b). The loading and transfer of proteins were confirmed by Ponceau S staining, therefore it is unlikely that this resulted from an experimental error, and suggests that the protein region recognized by MAb-TZSV-NP(S15) is conserved in CCSV and TZSV, but not in the NP of TNSaV. According to the results of the epitope assay of MAb-TZSV-NP(S18) and multiple alignments of NPs, the epitope of MAb-TZSV-NP(S15) could be near the V5 and V6 regions of the TZSV NP, in which shares higher homology between CCSV and TZSV than TNSaV (Fig.
5). The antigenic epitope of MAb-TZSV-NP(S15) needs to be further characterized.
In contrast, MAb-TZSV-NP(S18) is TZSV-specific. The deduced antigenic epitope of MAb-TZSV-NP(S18) is aa 78–86 region of TZSV NP, which is unique to TZSV. Most residues of aa 78–86 are conserved in the NPs of the reported TZSV isolates, except the aa-84 residue that is glycine in the TZSV-13YV639 isolate used in this study or serine in the original TZSV-Tomato-YN isolate [
20]. Serological results showed that MAb-TZSV-NP(S18), as well as MAb-TZSV-NP(S15), can be used to react with different TZSV isolates collected from fields in Yunnan. This includes the Tomato-YN isolate (Fig.
7) with the difference in aa-84 residue, and suggests that the MAb-TZSV-NP(S18) is a valuable tool for detecting TZSV in field surveys. We detected higher tospovirus infections when MAb-TZSV-NP(S15) was used (Table
2), which may have resulted from the higher titer of MAb-TZSV-NP(S15). The presence of other tospoviruses serologically related with TZSV, such as CCSV and TNSaV, could be excluded from the collected field samples by RT-PCR.
Some antibodies have higher titer due to the nature of the epitope. When the aa sequence of TZSV-13YV639 NP is used to predict antibody epitope using the B cell epitope prediction tool of IEDB Analysis Resource (
http://tools.immuneepitope.org/bcell), the results suggested that part of the MAb-TZSV-NP(S18) epitope (aa 84–86) conforms with the result of antibody epitope prediction. Several epitopes can be predicted in the aa 88–278 region of TZSV NP with higher scores than that of aa 78–86. The titer difference between MAb-TZSV-NP(S18) and MAb-TZSV-NP(S15) could have resulted from the B cell-targeting property of antigenic epitopes. The epitope of MAb-TZSV-NP(S15) is likely to be in aa 88–278 position of the NP, and the IEDB tool predicts a higher score compared to the epitope of MAb-TZSV-NP(S18).
Both MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18) are successfully used to detect TZSV in natural diseased plant samples. This is the first report to show that approximately 30 % TZSV incidence can be detected in a one-year field survey. Since TZSV was first reported in 2008, several TZSV isolates have been identified in numerous crops in Yunnan Province by RT-PCR [
20‐
22]. Actually, the detection of TZSV in field plant samples was also conducted by ELISA using the antiserum against the TZSV NP, which was also used to react with TNSaV [
19]. Taken together with our previous results and the field survey conducted in 2015, we indicate that TZSV is prevailing in Yunnan Province infecting numerous important economic crops, such as pepper, tobacco and tomato, and the ornamental spider lily.
Although CCSV, TNSaV and TZSV all occur in mainland China, only CCSV has been found in Taiwan [
19,
20,
23,
24]. The quarantine of imported agricultural products for TNSaV and TZSV is important to prevent their invasion in Taiwan. We proposed an efficient serological detection platform for virus inspection that MAb-CCSV-NP [
25] is used to detect all CCSV, TNSaV and TZSV; MAb-TZSV-NP(S15) is used to exclude TNSaV from TZSV and CCSV; and MAb-TZSV-NP(S18) is used to identify TZSV (Additional file
3: Table S2). This assay should provide a method that relies on protein analysis only, and will improve the speed at which tospovirus infections can be detected.
Conclusions
In this study, the close serological relatedness of CCSV, TNSaV and TZSV clustered in the WSMoV serogroup is experimentally demonstrated from the cross reaction with the previous reported MAb-CCSV-NP [
25] and MAb-WNSs [
26]. Two new MAbs against the TZSV NP, MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18), with distinct serological reactivity were obtained. Epitope mapping analyses revealed that the MAb-TZSV-NP(S18) targets a highly conserved region, the residues of aa
78HKIVASGAD
86, at the NPs of known TZSV isolates that is highly specific and suitable for identifying the TZSV species. MAb-TZSV-NP(S15) reacting with both CCSV and TZSV can be used to exclude TNSaV. The TZSV MAbs were applied in field survey in 2015, showing that TZSV is prevailing on economic crops including pepper, tobacco and tomato and the ornamental spider lily in Yunnan Province. All MAb-CCSV-NP, MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18) do not react with other tested tospoviruses ANSV, CaCV, CSNV, GBNV, GCFSV, GRSV, HCRV, INSV, IYSV, MYSV, TCSV, TSWV, TYRV, WBNV and WSMoV. Here we proposed a serological detection platform using these three MAbs to allow researchers and quarantine staff to efficiently diagnose the infections of CCSV, TNSaV and TZSV in China and other countries.
Acknowledgments
The authors thank Dr. Lei Wan (China Medical University Hospital, Taichung, Taiwan) for providing Spll/0-ag/14 myeloma cells and the permission of virus import by the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan. This study was supported by grants provided by the Ministry of Education, Taiwan under the ATU plan; the program (NSC 101-2911-I-005-301) of NCHU-UCD Plant and Food Biotechnology Center, National Chung Hsing University; Provincial Natural Science Foundation of Yunnan Province, China (2012CH007); and Yunnan Provincial Key Laboratory of Agricultural Biotechnology (YBRI201505).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Designed experiments and critically revised the manuscript: TCC. Performed experiments: YHC, JD, WCC, KZ, KW, JHS, YCW. Collected samples: JD, KZ, KW. Analyzed data: YHC, JD, TCC. Drafted manuscript: YHC, JD, SDY, TCC. All authors read and approved the final manuscript.