Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses
© Zhang et al. 2016
Received: 17 August 2015
Accepted: 21 December 2015
Published: 14 January 2016
Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide.
RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates.
Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5′ regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees.
All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative-sense RNA viruses.
Tomato spotted wilt disease caused by Tomato spotted wilt virus (TSWV) was first described in 1919 in Australia (Brittlebank, 1919)  and has gradually spread to Europe, Africa, North America, and South America [2–11]. Recently, TSWV was detected in middle-eastern and far-eastern countries in Asia including Iran, Japan, South Korea and China [12–17]. TSWV is mainly transmitted in a persistent manner by several species of thrips, especially the western flower thrip Frankliniella occidentalis, which may contribute to the worldwide spread of TSWV as well as to tomato spotted wilt disease [14, 18, 19]. TSWV infectes many important economic plants including tomato and potato, and causes serious damage including ringspot, black streak and tip dieback [20–22].
To date, whole-genome sequences have been determined for 19 TSWV isolates including 14 from South Korea, two from China (YN and CG-1), 1 from Brazil (BR-01) and 2 from Italy (p202/3WT and p105) (Additional file 1: Table S1) [16, 17, 24]. There are currently 26 full-length L, 59 full-length M, and 63 full-length S sequences deposited in the database (Additional file 1: Table S1). Previous studies suggested that both reassortment and recombination have contributed to the molecular diversity and evolution of TSWV based on partial sequences or regional whole-genome isolates [17, 27, 28]. However, only a few recombination events associated with TSWV have been described .
To identify the origin and evolution of TSWV in China, whole-genome sequences of three new TSWV isolates from tobacco, red pepper and green pepper in China were determined in this study. Molecular diversity and phylogenetic analysis revealed that all five Chinese TSWV isolates can be divided into two groups. Reassortment and huge numbers of recombination events were found based on phylogenetic and recombination analysis among all TSWV isolates worldwide.
Sequencing of three new full-length TSWV isolates and examination of molecular diversity of TSWV in China
Three new TSWV isolates (YNta, YNrp and YNgp) originating in three different infected plants (tobacco, red pepper and green pepper) were obtained in Yunnan province (China) in 2013. The genome information of these isolates (YNta, YNrp and YNgp) is listed in Additional file 1: Table S1, which also contains information on previously reported TSWV isolates. RNA L of YNta, YNrp and YNgp were identical lengths (8913 nts), while the genomic sizes of RNAs M and S varied slightly due to size variation in the intergenic region (IGR) (Additional file 1: Table S1).
When two other previously reported Chinese TSWV isolates (YN and CG-1) were included in the analysis, new characteristic of TSWV were observed. In addition to size variations in IGR of the RNA M and S, there were size variations in open reading frames (ORFs) and 5′ and 3′ untranslated regions (UTRs). For RNA L, the ORF in isolate CL4 (YN) is 8637 nt, compared with 8640 nt for other Chinese isolates. The 5′UTR and 3′UTR of isolate CL5 (CG-1) are 34 nt and 243 nt, respectively, compared with 33 nt and 240 nt in other Chinese isolates. These size variations contributed to the size differences among different isolates (CL4, 8910 nt; CL5, 8917 nt; CL1, CL2, CL3, 8913 nt). For RNA S, the 5′UTR of CS5 is 86 nt compared with 88 nt for other Chinese isolates. In addition, the size of the IGR in CS5 (503 nt) is shorter than the IGR in other Chinese isolates (548 to 551 nt) (Additional file 1: Table S1). Based solely on size similarities, TSWV isolates YNta, YNrp and YNgp may be more related than YN and CG-1.
Nucleotide and amino acid sequence identity for TSWV isolates in China
Molecular diversity and phylogenetic relationships among TSWV isolates worldwide
Molecular diversity of RNA L, M and S for TSWV isolates worldwide
In the phylogenetic tree of RNA M, 62 isolates were divided into three clades (Fig. 2b). The first clade included two sub-clades, one of which contained 56 isolates from South Korea, China, Japan, Italy, Spain, and the USA; the second sub-clade included two isolates from South Korea and the USA. Both the second and third clades contained isolates from South Korea and Spain (Fig. 2b). M RNAs of the five Chinese isolates were more related than their RNA L since they all belonged to the first sub-clade of the first clade. CM1, CM2 and CM3, which also form a branch, have the closest relationship with Chinese isolate CM4 and four isolates from the USA (UM7, UM8, UM9 and UM10). CM5 is most closely related to one isolate from South Korea (KM20) and two isolates from the USA (UM2 and UM3) (Fig. 2b). Therefore, Chinese isolate RNA M were most closely related to some TSWV isolates from USA.
In the phylogenetic tree of RNA S, 66 isolates were divided into two clades (Fig. 2c). The first clade included four sub-clades, the first one of which included 60 isolates from South Korea, China, Japan, Italy, Bulgaria, Brazil and USA; the second, third and fourth sub-clade contained one isolate from Germany (GS1), Brazil (BS1) and Italy (IS16) respectively. The second clade contained two sub-clades, one of which contained a single Chinese isolate (CS5), and the second contained one isolate from USA (US16) and one isolate from Bulgaria (BuS1) (Fig. 2c). The origin of RNA S of the 5 Chinese isolates differed from that of RNA L and M, with CS1, CS3 and CS4 comprising a branch with Chinese isolate CS6 and one isolate from South Korea (KS4). CS2 was most related to two isolates from South Korea (KS1 and KS6). CS5 formed a separate sub-clade in the second group (Fig. 2c). Therefore, with the exception of CS5, RNA S of Chinese isolates were most closely related to some TSWV isolates from South Korea.
Based on these phylogenetic trees, L, M and S of individual isolates could have different origins, implying the occurrence of frequent reassortment during the evolution of TSWV isolates. Among the Chinese isolates, RNA L (CL5) and S (CS5) of CG-1 were located in separate clades from the other four isolates, which correlated with the identity analysis of nucleotide sequence (Table 1). The three new isolates sequenced in this study (YNta, YNrp and YNgp) were more closely related to each other since their RNAs were contained in single branches in the phylogenetic trees of RNA L, M and S (Fig. 2a, b and c). These three new isolates also have a close relationship with isolate YN since they belonged to the same branch in phylogenetic trees of RNA M and S (Fig. 2b and c).
Recombination analysis of TSWV isolates worldwide
For 29 full-length RNA L fragments of TSWV, 88 recombination events were detected in 27 RNA L fragments (Additional file 2: Table S2). Only 2 TSWV RNA L (CL5 from China and BL2 from Brazil) did not have detectable recombination events. Recombination events in RNA L were located throughout the genome, although most were located in the 5′ half of the RdRp ORF (Fig. 3; Additional file 2: Table S2). Some short recombination events located in the 3′ terminal region are associated with recombination events in the 5′ half of the RdRp ORF, such as those located at 8722–4138 in CL3 (event 9), 8128–1379 in KL19 (event 15), 8746–2865 in JL1 (event 67), 8726–4162 in UL3 (event 77) etc. (Fig. 3 and Additional file 2: Table S2). In addition, separate recombination events that were located in the 3′ half of the RdRp ORF were detected in some RNA L from Chinese, South Korean and Italian isolates, such as recombination events located at 4781–6936 in CL4 (event 16), 5155–5629 in KL12, KL17 and IL1 (event 44, 50 and 85), 5154–5634 in KL1, KL4, KL6, KL7, KL8, KL10, KL16 and KL19 (event 19, 26, 29, 32, 35, 38, 41, 47 and 59), 5154–7248, 6308–6716 and 6910–7248 in KL19 (event 60–62) (Fig. 3 and Additional file 2: Table S2). Interestingly, recombination events having same breakpoints with same minor and major parents were detected in different RNA L, e.g., recombination events located at positions 5155–5629 (events 44/50/85) and ~8912-367 (events 42/49/86) found in KL12, KL17 and IL1 (Fig. 3 and Additional file 2: Table S2). Similar cases included events 69/73 and 70/72 in UL1 and UL2; events 68/71/80;events 1/4/7/17/24/27/30/33/36/39/45; events 2/5/18/25/28/31/34/37/40/46/54/84; events 20/22/51/79; events 64/75; events 43/48; events 19/26/32/35/38/41/47/59; events 6/9 and events 21/23/52 (detailed information are listed in Additional file 2: Table S2).
For 62 full-length TSWV RNA M fragments worldwide, 143 recombination events were totally detected from 56 RNA M fragments (Additional file 3: Table S3). Only in 6 RNA Ms (CM3, KM3 and KM18, JM1, UM1, SM11), no recombination event was detected. Preeminently within TSWV RNA M, recombination events are located at 5′ one-third of genome with only one exception, which occurred at position of 7–2626 in SM13 (event 99) (Fig. 4; Fig. 4d). In addition, there are three recombination events (including event 21, 33 and 99) bridging two ORF-coding regions (Fig. 4b and d). Similar to the recombination events of RNA L, many short recombination events of RNA M located at 3′ terminal region are simultaneously connected with those at 5′ one-third region of genome, such as recombination at 4743–1110 and 4750–1125 in CM5 (event 8–9), 4742–1212 for UM3 (event 45), 4770–1115 for UM12 (event 66) and so on (Fig. 4; Additional file 3: Table S3). However, different from the recombination events of RNA L, no separate recombination event was detected at the 3′ half of genomic RNA M. Recombination events with same breakpoints were also detected in different RNA M as the case of RNA L, for example, recombination located at position of 37–809 (events 133/138), 123–874 (events 134/139) and 974–1251(events 135/141) in IM1 and IM2 (Fig. 4 and Additional file 3: Table S3). Similar cases included events 82/122, 83/123 and 85/124 in SM4 and SM20; events 107/111, 108/112 and 110/114 in SM16 and SM17; events 5/53; events 51/56/60; events 52/54/57/58; events 55/70; events 1/4; events 101/109; events 42/45/47/142; events 137/143; events 105/119; events 6/59 (detailed information are listed in Additional file 3: Table S3).
For 66 full-length TSWV RNA S fragments, 174 recombination events were detected in 61 RNA S (Additional file 4: Table S4). Only 5 TSWV RNA S (US16, BS2 and BS3, IS1, BuS1) did not have detectable recombination events. Similar to the recombination events of RNA M, recombination events in TSWV RNA S were located within the 5′ 2000 nt of the RNA with one exception: position 999–2833 in US8 (event 70) (Fig. 5; Fig. 5c; Additional file 4: Table S5). Also, many short recombination events located in 3′ terminal regions were similarly associated with these 5′ region recombination events, such as 2899–1772 in CS3 (event 5), 2908–1772 in CS6 (event 10), 2883–1760 in KS5 (event 21), 2813–1616 in KS16 (event 35), 2943–1652 in BuS3 (event 96), etc. (Fig. 5; Additional file 4: Table S4). Furthermore, as found in RNAs L and M, recombination events with identical breakpoints were detected in different RNA S, e.g., recombination events located at position 78–1622, 1234–1761 and 2770–1784 in SS3 and SS4 (Fig. 5 and Additional file 4: Table S4). Similar cases included events 169/172, 167/171 and 166/170 in SS3 and SS4; events 109/115/121/126/154 and 104/110/117/123/149 in IS4, IS5, IS6, IS7 and IS14; events 58/63; events 80/87; events 62/66/68; events 107/113; events 112/119; events 33/37; events 161/106/111/118/124/132/141/150; events 108/120; events 116/122/155; events 84/91/93 (detailed information are listed in Additional file 4: Table S4).
Molecular diversity among 5 TSWV Chinese isolates
International trade of seeds and vegetables, and the presence or importation of vector thrips have allowed for the rapid spread of TSWV and associated diseases worldwide [20, 21]. During this period of expansion, TSWV has been continuing to evolve, possibly depending on host and environmental factors. For the current study, three new isolates of TSWV were cloned from different hosts (tobacco, red pepper and green pepper) grown in Chinese fields and then compared with two previously cloned TSWV isolates from tomato and lettuce in China . Among these five TSWV isolates in China, only slight variations were found in the sizes of L, M and S RNA (Additional file 1: Table S1). When 29 RNA L, 62 RNA M and 66 RNA S were compared (Additional file 1: Table S1), the range of variation was considerable but mainly in untranslated regions (Table 2). For the 5 ORFs encoded by the TSWV (−) strand or (+) strand, only RdRp and NSs vary in size (15 and 204 nt, respectively) due to mutations that introduce nonsense codons (Table 2). Currently, it is not known if these size changes affect infectivity of TSWV, or if the clones represent viable viral RNAs (there is no reverse genetic system available for TSWV). CG-1 (CL5, CM5 and CS5 RNAs), isolated from lettuce in China may be considered a new TSWV species based on molecular diversity (Table 1), and phylogenetic analyses (Fig. 2). In addition, the RdRp amino acid sequence of TSWV-YN from tomato in China differs from other 4 Chinese isolates (Table 1). In contrast, YNta, YNrp and YNgp are relative stable and are found together in one branch of the RNA L and M phylogenetic trees.
The phylogenetic trees revealed that the three RNAs of the 5 Chinese TSWV isolates do not group together in the same clades, suggesting that these viruses have undergone reassortment during their evolution, as has been reported to occur for TSWV [17, 28]. RNA L of CL1, CL2, CL3 and CL4 are related to the RNA L of isolates from South Korea, while CL5 is a new species with an unclear origin (Fig. 2a). For RNA M, the origin of CM1, CM2, CM3, and CM4 is RNA M from the USA, while the origin of CM5 is RNA M from either the USA or South Korea (Fig. 2b). For RNA S, the origin of CS1, CS2, CS3 and CS4 is RNA S from South Korea, while the origin of CS5 is RNA S from either the USA or Bulgaria (Fig. 2c).
High levels of recombination events have occurred among TSWV isolates worldwide
Besides reassortment of fragments L, M and S in different TSWV isolates, many recombination events were detectable in nearly all RNA L, M and S examined, differing from a previous report using fewer isolates that suggested only a low level of recombination events have occurred . There are currently two major hypotheses concerning the contribution of recombination to the evolution of modern RNA viruses. One suggests that recombination is a major evolutionary mechanism for single-stranded RNA viruses and a minor mechanism for the evolution of multipartite RNA viruses [29, 30], which is supported by reports on Soybean mosaic virus and CMV [31–33]. The second hypothesis is that both recombination and reassortment are important evolutionary mechanisms for multipartite RNA viruses, which is supported by studies of Brome mosaic virus . Our study provides additional support for the second hypothesis, although previous studies have shown that homologous recombination seems to be very rare or even absent in most negative-sense RNA viruses [35, 36].
For all three RNAs of TSWV isolates, recombination events were mainly detected in the 5′ half of the RNA. The reason for this preference is not clear. One possibility is that RdRp firstly bound RNA template, synthesized complementary strands and then had chance to facilitate recombination among different complementary strands. For bi-cistronic RNA M and S, the vast majority of recombination spanned only the ORF located at 5′, suggesting that recombination preferred to occur at ORF-coding region instead of untranslated regions and different ORFs are undergoing separate evolution. In addition, recombination events involving short fragments in the 3′ terminal region were connected with 5′ region recombination events in all three RNAs, which may cause by closed structure of TSWV RNAs resulting from the long base-pairing between 5′ and 3′ terminal sequences (5′-GAGCAAUUGUGUCA------UGACACAAUUGCUCU-3′) .
The similar recombination events in different RNA L, M and S of TSWV imply a connection between recombination and the phylogenetic tree. Isolates with similar or identical recombination breakpoints are grouped together. For example, for RNA L, three isolates (KL12, KL17, IL1) have recombination events located at positions 5155–5629 and ~8912-367 (Additional file 2: Table S2, Fig. 3), forming a cluster in the phylogenetic tree of RNA L (Fig. 2a). The second characteristic is that isolates in which no recombination was detected are usually clustered in separate groups or branches. For example, CL5 in which no recombination was detected belonged to a separate group from the other 28 isolates (Fig. 2a).
Whole-genome sequences of three new TSWV isolates from different hosts in China were determined. Based on molecular diversity on 29 RNA L, 62 RNA M and 66 RNA S of TSWV, it is suggested that the entire TSWV genome, especially the M and S RNAs, have undergone extreme variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments. In addition, all five Chinese TSWV isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. Significant numbers of recombination events were detected among TSWV isolates worldwide with apparent regional preference and showed inherent connection with phylogenetic trees. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses.
Whole-genome cloning and sequencing of three new TSWV isolates from China
Tobacco, red pepper and green pepper plants with typical symptoms of TSWV infection were collected from Luxi county, Honghe city (Yunnan province) in China in 2013 (specific permission was not required). Three new TSWV isolates (YNta, YNrp and YNgp) were identified following cloning and sequencing. In brief, total RNA was extracted from leaves of putatively infected tobacco, red pepper or green pepper using TRIzol Reagent (TransGen). For cDNA synthesis, total RNA was combined with dNTPs and primers corresponding to the 3′ ends of L, M or S RNAs (Additional file 5: Table S5). After 5 min incubation at 65 °C, 5 units of PrimeScript reverse transcriptase and 5× buffer (Takara) was added and incubation continues at 42 °C for 1.5 h. Following cDNA synthesis, PCR amplification was performed using LA Taq DNA polymerase and pairs of primers corresponding to internal and terminal regions of L, M or S RNAs (Additional file 5: Table S5). Three fragments were amplified for L (positions 1–2722, 2512–5927 and 5906–8913), two fragments were amplified for M (positions 1–3401 and 2215–4772), and full-length cDNA was amplified for S. All PCR products were cloned into pMD18-T (Takara) and sequenced using universal vector primers and specific primers designed for TSWV components (Additional file 5: Table S5). At least three clones for each PCR product were sequenced to avoid experimental errors.
Sequence assembly, construction of phylogenetic trees and recombination analysis
Sequence assembly was accomplished using DNAMAN, which was also used to analyze nucleotide and amino acids sequences among TSWV isolates. After sequence assembly, complete sequences of L, M and S for the three new TSWV isolates were deposited into GenBank with accession numbers from KM657114 to KM657122 (http://www.ncbi.nlm.nih.gov/nucleotide/).
The phylogenetic tree was constructed using the MEGA 5.0 software package  based on methods of neighbor-joining and Kimura 2-parameter. Bootstrap resampling (1000 replicates) was used to ensure reliability of individual nodes in the phylogenetic tree. Values lower than 70 % were hidden. Searches for recombination events employed six methods including RDP, GENECONV, Bootscan, Maxchi, Chimaera and SiScan, implemented in RDP4 , with likely parental isolates and recombination break points determined using default settings. Recombination events were noted if supported by at least three different methods (P-values <1.0 × 10−6). Sequences of additional full-length TSWV isolates were obtained from http://www.ncbi.nlm.nih.gov/nucleotide/ (See Additional file 1: Table S1 for detailed information).
This work was supported by grants from The National Natural Science Foundation of China (31370179), and The Shandong Province Natural Sciences Foundation of China (ZR2013CM015). We are grateful to Prof. Anne E. Simon, University of Maryland (USA) for valuable suggestions and critical modification on the manuscript.
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