Arch Virol DOI 10.1007/s00705-014-2256-3

ANNOTATED SEQUENCE RECORD

The first complete genome sequences of two distinct European tomato spotted wilt virus isolates P. Margaria • L. Miozzi • M. Ciuffo H. R. Pappu • M. Turina

•

Received: 4 June 2014 / Accepted: 8 October 2014 Ó Springer-Verlag Wien 2014

Abstract Tomato spotted wilt virus (TSWV) represents a major constraint to the production of important vegetable and ornamental crops in several countries around the world, including those in Europe. In spite of their economic importance, European TSWV isolates have only been partially characterized, and a complete genome sequence has not been determined yet. In this study, we completed the whole genome sequence of two distinct TSWV isolates from Italy, p105 and p202/3WT. The sequences of the L and M segments of p105 and of the L segment of p202/ 3WT were determined using a combined approach of RTPCR and small RNA (sRNAs) contig assembly. Phylogenetic analysis based on RNA-dependent RNA polymerase and GN/GC protein sequences grouped the two isolates in two different clades, showing that different evolutive lineages are present among Italian TSWV isolates. Analysis of possible recombination/reassortment events among our isolates and other available full-length genome TSWV sequences showed a likely reassortment event involving the L segment.

Tospoviruses are among the most serious threats to vegetable and ornamental crops around the world [1]. They Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2256-3) contains supplementary material, which is available to authorized users. P. Margaria  L. Miozzi  M. Ciuffo  M. Turina (&) Istituto per la Protezione Sostenibile delle Piante, Sez. di Torino, CNR, Strada delle Cacce 73, 10135 Turin, Italy e-mail: [email protected] H. R. Pappu Department of Plant Pathology, Washington State University, PO Box 646430, Pullman, WA 99164, USA

belong to the genus Tospovirus, the only genus of plantinfecting viruses in the family Bunyaviridae [2]. In Europe, tomato spotted wilt virus (TSWV), the type member of the genus, was first reported in England in 1929 [3] and thereafter became increasingly important in several vegetables and ornamentals. There was a dramatic increase in TSWV incidence beginning in the late 1980s following the introduction of the thrips vector Frankliniella occidentalis [4]. Today, TSWV is by far the economically most important virus among the tospoviruses in the Mediterranean region, especially in tomato and pepper, historically two staple vegetable crops in this area [4]. A serious threat related to TSWV is the emergence of resistance-breaking strains that are able to overcome the Tsw and Sw5 resistance genes in pepper and tomato, respectively [5–9]. This has serious implications for disease management, since growing virus-resistant pepper and tomato cultivars is an economically feasible control method for this virus; nevertheless, an integrated management approach is available for most tospovirus-caused diseases [10–12]. The population structure of TSWV worldwide and locally has been studied previously, showing distinct phylogenetic lineages [4, 13–17]. Tospovirus particles are quasi-spherical and enveloped by a lipid membrane derived from the host, into which two virus-encoded glycoproteins (GN and GC) are embedded. The genome consists of three single-stranded RNAs, designated small, medium and large (S, M, and L) [18]. The S and M segments have an ambisense coding strategy: the S segment encodes the non-structural protein (NSs) in the viral sense and the nucleocapsid (N) protein in the viralcomplementary sense; the M segment encodes the nonstructural movement protein (NSm) in the viral sense and the glycoprotein precursor in the viral-complementary sense. The L segment has negative polarity, with a single

123

P. Margaria et al.

open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp) in the viral-complementary strand. At the moment, the complete genome sequences of 15 TSWV isolates are available: one from Brazil, one from China and 13 from South Korea [14, 19, 20]. The whole or partial sequences of individual viral genomic segments of several European TSWV isolates are available in GenBank, but a full-length genome sequence is still not available. In this work, we report the complete genome sequences of two Italian TSWV isolates, p105 and p202/3WT. Isolate p105 was originally isolated from pepper during field epidemics in Ligury, Northern Italy, in the late 1990s, and isolate p202/3WT was obtained in 1999 from pepper crops in Sicily, Southern Italy. The original isolates, after singlelesion passage on Nicotiana tabacum [6], were stored in liquid nitrogen. We minimized mechanical inoculation passages using the original samples kept in liquid nitrogen, but since the experimental host for maintenance is N. benthamiana, we cannot exclude host adaptation phenomena in the process of isolation. Nevertheless, competence to infect pepper was maintained for both isolates, but neither isolate was able to overcome the Tsw resistance gene in pepper [6, 21]. We previously characterized the S genomic segment of p105 (accession number DQ376178) and the S and M genomic segments of p202/3WT (accession number HQ830187 and HQ830188) [6, 21]. Our partial biological comparison, which included host range analysis and thrips transmission experiments with F. occidentalis, did not reveal any obvious difference in a controlled environment [21]. Two approaches were used to obtain the complete L RNA sequence: RT-PCR amplification of specific regions of the L RNA and assembly of contigs from small-RNA (sRNA) analysis. Specifically, the sequence of the L segment of p105 and p202/3WT was determined by sequencing the RT-PCR products and confirmed by alignment with the contigs obtained by sRNA analysis. Characterization of the M segment of p105 was instead performed first by sRNA analysis to obtain different viral contigs, and the gaps were then covered by sequencing the RT-PCR products. Terminal sequences were obtained by 50 –30 RACE. RNA was extracted from two infected hosts, N. benthamiana and Solanum lycopersicum, using a Directzol RNA extraction kit (Zymoresearch, Irvine, CA, USA). The RNA quality was assessed using a NanoDrop 2000 Spectrophotometer (Thermoscientific, Waltham, MA USA) and by electrophoresis on 1 % agarose TAE gels. A ThermoscriptTM RT-PCR kit (Invitrogen, Grand Island, NY, USA) and the high-fidelity ‘‘Phusion’’ enzyme (Fisher Scientific, Pittsburgh, PA, USA) were used for reverse transcription and PCR, respectively, according to manufacturer’s instructions. PCR fragments were ligated into

123

pCRBlunt plasmid (Invitrogen), cloned in Escherichia coli DH5a competent cells according to standard protocols, and sequenced. Overlapping clones were assembled to obtain the complete sequence using Vector NTI software (Invitrogen, Grand Island, NY, USA). For sRNA profile analysis, 20 lg of total RNA from each host was used and separately processed (Beijing Genomics Institute, Guangzhou, China) in order to obtain a consensus sequence for each host. Viral sRNA libraries were used to assemble the viral genomes using the Inchworm assembler belonging to the Trinity package [22]. Phylogenetic trees were constructed by the neighbor-joining method [23] using MEGA4 software [24], allowing 1000 bootstrap replicates. Evolutionary distances were computed using the p-distance method [25] and were expressed in units of the number of amino acid differences per site. Percentages of pairwise identities among the aligned RdRp and GN/GC protein sequences were calculated using MatGAT v.2.03 software [26]. The L RNA of p105 (GenBank accession number KJ575620) and p202/3WT (GenBank accession number KJ575619) shared the same length and ORF organization. Both L segments were 8914 nt in length and contained a single ORF in the antiviral sense 8640 nt in length (position 8881-242), encoding a 2879-aa protein with a predicted molecular mass of 331.6 kDa. The 50 and 30 termini contained two untranslated regions (UTR) of 241 and 33 nt, respectively, with conserved terminal ends, a typical feature of tospoviruses [18]. Alignment of the two RdRp amino acid sequences showed several aa substitutions distributed along the whole protein sequence (Supplementary Fig. 1). The predicted amino acid sequences of the RdRps derived from the sequences of p105 and p202/3WT were 97.9 % identical (Supplementary Table 2). Phylogenetic analysis of the available RdRp sequences showed that the two Italian TSWV isolates grouped in two different clades: p105 grouped with isolates K2, K3 and K18 from South Korea; p202/3WT grouped instead in a separate clade, which consisted of two branches: a subclade with isolates from Korea and the isolate from Hawaii, and a different subclade with the isolates from Brazil and China (Fig. 1a). To sequence the M RNA of p105 (deposited in GenBank with accession number KJ575621), we first obtained five fragments of 1006, 195, 391, 669 and 2068 nt by sRNA analysis, corresponding to about 90 % of the expected total size. Completion of the gaps by RT-PCR allowed the assembly of a genomic segment of 4766 nt in length, with the expected ambisense arrangement. The viral-sense ORF was 909 nt in length (position 101–1009), encoding the 302-aa NSm protein, with a predicted molecular mass of 33.8 kDa. The complementary-sense ORF was 3408 nt in length (4682–1275), encoding the 1135-aa glycoprotein precursor protein, with a predicted molecular mass of 127.5 kDa. The 50 and 30 termini contained two UTRs, of

European tomato spotted wilt virus isolates

(a)

CA-5-USA

60

(b)

76

CA-6-USA

61

CA-4-USA CA-3-USA

27

63

K5-South Korea

73

K3-South Korea

48

A-USA

K6-South Korea

95 71

TSWV-YN-China YN-China

K7-South Korea 73

GRAU-Spain

K4-South Korea

100 100

K4-South Korea

92

100

Ab1NL2-Spain

92

K17-South Korea

100

K5-South Korea

81

K12-South Korea

K1-South Korea

62

Hawaii-Hawaii Hawaii-USA

99

p105-Italy 100

K2-South Korea

100 99

K6-South Korea

NC-6-USA

K18-South Korea

100

GA-1L-Spain

100

p202/3WT-Italy 99

K2-South Korea

99

K16-South Korea

K10-South Korea 100

K18-South Korea

6946 70

K1-South Korea

100

CA-7-USA p105-Italy

K8-South Korea

38

64

99

NC-8-USA

Br01-Brazil

K3-South Korea

D-191-Australia

Br01-Brazil TSWV-YN-China YN-China

100 78

K17-South Korea 97

0.005

K12-South Korea

49

Oller1IL3-Spain D-The Netherlands

72 64

Pujol1TL3-Spain p202/3WT-Italy

0.005

Fig. 1 Consensus phylogenetic tree obtained from alignments of deduced amino acid sequences of the RNA-dependent RNA polymerase proteins (a) and GN/GC glycoproteins (b) of different TSWV isolates using the neighbor-joining method. Bootstrap values on the branches were derived from a 1,000-bootstrap replicate analysis. The

evolutionary distances were computed using the p-distance method and are expressed in units of the number of amino acid differences per site. Accession numbers of the sequences used are given in Supplementary Table 1

72 and 52 nt, respectively; the intergenic region (IGR) consisted of 442 nt, with a high A-U content predicted to form a hairpin structure. Phylogenetic analysis of GN/GC protein sequences available in the databases grouped the two isolates in two different clades: p105 clustered with the TSWV isolates from the USA, including isolate TSWV-A, and the Korean isolates K2, K3, K18 (as for the L protein), whereas p202/3WT belonged to a separate clade with isolates TSWV-D, Br01, an Australian, two Korean and two Spanish isolates (Fig. 1b). The same alignment was used to determine the amino acid sequence identities of GN/GC precursor proteins among all of the represented isolates (Supplementary Table 3): the GN/GC sequences of the two sequenced Italian isolates were 95.9 % identical, whereas sequence identities inside the same clade were over 99 %. These results further support that p105 and p202/3WT belong to two phylogenetically distinct clades. The occurrence of two distinct TSWV subgroups in Italy was reported previously, wherein a set of TSWV isolates from the Apulia region (southern Italy) clustered with

A-like or D-like TSWV reference isolates based on the NSm protein sequence, providing strong evidence for the presence of two evolutive lineages [13]. The presence of two distinct isolates in the same area increases the potential for further variability through recombination and reassortment. For this reason, given the availability of the full genome sequence of two Italian isolates, possible recombination and reassortment events involving known TSWV isolates were investigated using Recombination Detection Program 4.16 (RDP4) [27], using as input file the Clustal W alignment of the concatenation of L, M and S genomic segments as described previously [14]. The analysis was done using several recombination detection algorithms (GENECONV, Bootscan, Chimaera, MaxChi, SiScan, 3Seq and RDP) with default settings, so as to obtain a consensus results. A putative recombination event was assumed to have occurred only when it was consistently identified by at least three of the above-mentioned seven algorithms, at a probability value threshold of 0.05. A

123

– 6.27E-20 5.53E-45 5.53E-45 5.53E-45 5.53E-45 5.53E-45 5.53E-45 2.40E-40 3Seq

5.53E-45

1.97E-08

9.63E-07 3.21 E-08 7.17E-18 2.90E-07

6.00E-14 2.53E-22

3.83E-26 3.14E-42 3.83E-26 3.14E-42

2.53E-22 2.53E-22

3.83E-26 3.14E-42 3.83E-26 3.14E-42

2.53E-22 2.53E-22

3.83E-26 3.14E-42 3.83E-26 3.14E-42

2.53E-22 2.53E-22 1.83E-22

2.66E-24 4.50E-39

MaxChi

Chimaera Siscan

3.83E-26 3.14E-42

–

– 9.48E-07 2.71 E-28 2.71 E-28 2.71 E-28 2.71 E-28 2.71E-28 2.71 E-28 2.59E-28 Bootscan

2.71 E-28

9.75E-03 1.41E-08

– 3.24E-06

3.28E-28 3.28E-28

3.24E-06 3.24E-06

3.28E-28 3.28E-28

3.24E-06 3.24E-06

3.28E-28 3.28E-28 3.28E-28

2.85E-05

3.24E-06

1.61E-27 RDP

GENECONV

3.24E-06

K18

K12 Br01

K2 K3

p202/3WT p202/3WT

K3 K3

p202/3WT p202/3WT

K3 K3

p202/3WT p202/3WT

Major parent

p202/3WT

K3

p202/3WT

K3

Minor parent

K3

S L L L L L L L L Genome position

L

13719

16799 8886 8908 8908 8908 8908 8908 8908 8956 Ending breakpoint

8908

3 2

102 24

1 1

3074 3074

1 1

3074 24 24

1

123

24 24 Initial breakpoint

1 1 Event

1

China K16 K10 K8 K7 K6 K5 K4 K1 Recombinant

Table 1 Summary of possible recombination events identified by Recombination Detection Program v.4.16 (RDP4), with significance values for each algorithm used

p105

P. Margaria et al.

consensus score above 60 % indicates that the identified sequence is almost certainly a recombinant; a score between 40 and 60 % indicates a fair likelihood that a recombinant was truly identified and could actually be the original parent. As for the convention used by the software, the minor parent is defined as the one contributing the smaller fraction of the recombinant sequence, while the major parent is the one contributing the larger fraction [27]. A previous investigation on possible recombination events was performed on the Korean TSWV isolates. In that study, two recombination events were predicted for the M segment (isolates K12 and K17), and a reassortment event involving the L segment was predicted for three isolates (K2, K3 and K18) [14]. Identical results were obtained when the analysis was repeated using the same isolates, but when the newly sequenced Italian isolates were included along with the Chinese and Brazilian isolates available in the databases, a different reassortment event involving the L segment was predicted by several methods at high stringency for isolates K1, K4, K5, K6, K7, K8, K10, and K16, with K3 as the major parent and p202/3WT as a minor parent (Table 1). Even if there is a likelihood of misidentification of recombinant parents, (as score was between 40 and 60 %), with K3 and p202/3WT possibly being the actual recombinant, an exchange event between these isolates was predicted by seven methods at low P-value (high confidence). Specifically, for isolates K7, K8 and K10, a recombination event was predicted within the L segment, while for isolates K1, K4, K5, K6 and K16 the recombination event involved the whole L segment, which might have originated through reassortment (Table 1). Reassortment of the whole L segment was previously shown to occur both in nature and under controlled environment conditions [5, 14, 21, 28, 29]. The absence of the K2, K3 and K18 isolates in this prediction plot is consistent with the results of the phylogenetic analysis, which grouped the L segment of these isolates with p105, in a different clade from p202/3WT. From our results, it appears that reassortment between Asiatic and the p202/ 3WT isolates could indeed have played a role in the emergence of new isolates, or alternatively, the Korean strains could have contributed to the appearance of the p202/3WT strain. Exchange of full-length genomic segments is known to play an important role in TSVW evolution: analysis of more than 200 isolates evidenced the absence of intrasegment recombination, whereas about 1/5 of the isolates did not belong to the same clade for the three RNA segments and were shown to be reassortants [15]. The same study also suggested genetic exchanges between Asiatic and European TSWV populations, with the former possibly introduced into Europe rather than the reverse [15]. This

European tomato spotted wilt virus isolates

analysis did not include a large portion of the L segment, and therefore the rate of recombination events in the L segment could not be assessed. In our analysis, two more recombination events were predicted: the first involving the L segment between the isolate from China, K2 and Br01, and the second involving the whole S segment between isolates p105, K12 and K18 (Table 1). Our results show that future studies with more whole genomic sequences will be necessary to confirm this hypothesis, given that adding four more isolates to a recently done analysis [14] changed the reassortment events scenario. The availability of the whole genomic sequence of several TSWV isolates collected in different parts of the world would facilitate further studies on the origin and evolution of TSWV, and on the mechanism(s) of genetic exchange in this economically important virus. In this context, this study, in addition to providing the complete genomic sequence of two TSWV Italian isolates, will be useful for providing a better understanding of the possible contribution of European TSWV isolates in the evolution of new isolates in the future.

References 1. Pappu HR, Jones RAC, Jain RK (2009) Global status of tospovirus epidemics in diverse cropping systems: successes achieved and challenges ahead. Virus Res 141:219–236 2. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) (2012) Virus taxonomy: classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego 3. Smith KM (1932) Studies on plant virus diseases. Ann Appl Biol 19:305–330 4. Turina M, Tavella L, Ciuffo M (2012) Tospoviruses in the Mediterranean area. In: Loebenstein G, Lecoq H (eds) Viruses and virus diseases of vegetables in the Mediterranean Basin, Advances in Virus Research. Elsevier Academic Press Inc, San Diego, pp 403–437 5. Hoffmann K, Qiu WP, Moyer JW (2001) Overcoming host- and pathogen-mediated resistance in tomato and tobacco maps to the mRNA of tomato spotted wilt virus. Mol Plant Microbe Interact 14:242–249 6. Margaria P, Ciuffo M, Pacifico D, Turina M (2007) Evidence that the nonstructural protein of tomato spotted wilt virus is the avirulence determinant in the interaction with resistant pepper carrying the Tsw gene. Mol Plant Microbe Interact 20:547–558 7. Aramburu J, Marti M (2003) The occurrence in north-east Spain of a variant of tomato spotted wilt virus (TSWV) that breaks resistance in tomato (Lycopersicon esculentum) containing the Sw-5 gene. Plant Pathol 52:407 8. Ciuffo M, Finetti-Sialer MM, Gallitelli D, Turina M (2005) First report in Italy of a resistance breaking strain of Tomato spotted wilt virus infecting tomato cultivars carrying the Sw5 resistance gene. Plant Pathol 54:564 9. Margaria P, Ciuffo M, Turina M (2004) Resistance breaking strain of tomato spotted wilt virus (Tospovirus; Bunyaviridae) on resistant pepper cultivars in Almeria, Spain. Plant Pathol 53:795

10. Jones RAC (2004) Using epidemiological information to develop effective integrated virus disease management strategies. Virus Res 100:5–30 11. Culbreath AK, Srinivasan R (2011) Epidemiology of spotted wilt disease of peanut caused by tomato spotted wilt virus in the southeastern U.S. Virus Res 159:101–109 12. Momol MT, Olson SM, Funderburk JE, Stavisky J, Marois JJ (2004) Integrated management of tomato spotted wilt on fieldgrown tomatoes. Plant Dis 88:882–890 13. Finetti-Sialer MM, Lanace V, Padula M, Vovlas C, Gallitelli D (2002) Occurrence of two distinct tomato spotted wild virus (TSWV) subgroups in Southern Italy. J Plant Pathol 84:145–152 14. Lian S, Lee J-S, Cho WK, Yu J, Kim M-K et al (2013) Phylogenetic and recombination analysis of tomato spotted wilt virus. PLoS ONE 8(5):e63380 15. Tentchev D, Verdin E, Marchal C, Jacquet M, Aguilar J et al (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 16. Tsompana M, Abad J, Purugganan M, Moyer JW (2005) The molecular population genetics of the tomato spotted wilt virus (TSWV) genome. Mol Ecol 14:53–66 17. Zindovic J, Ciuffo M, Turina M (2014) Molecular characterization of tomato spotted wilt virus in Montenegro. J Plant Pathol 96:201–205 18. Goldbach R, Peters D (1996) Molecular and biological aspects of tospoviruses. In: Elliot RM (ed) The Bunyaviridae. Plenum Press, New York, pp 129–157 19. Hu Z, Feng Z, Zhang Z, Liu Y, Tao X (2011) Complete genome sequence of a tomato spotted wilt virus isolate from China and comparison to other TSWV isolates of different geographic origin. Arch Virol 156:1905–1908 20. Lee J, Cho W, Kim M, Kwak H, Choi H et al (2011) Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates. Arch Virol 156:725–728 21. Margaria P, Bosco L, Vallino M, Ciuffo M, Mautino GC, Tavella L, Turina M (2014) The NSs protein of tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis. J Virol 88:5788–5802 22. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat Biotechnol 29:644–652 23. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 24. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 25. Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New York 26. Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinform 4:29 27. Martin D, Rybicki E (2010) RDP: detection of recombination amongst aligned sequences. Bioinformatics 26:2462–2463 28. Qiu WP, Geske SM, Hickey CM, Moyer JW (1998) Tomato spotted wilt tospovirus genome reassortment and genome segment-specific adaptation. Virology 244:186–194 29. Sin SH, McNulty BC, Kennedy GG, Moyer JW (2005) Viral genetic determinants for thrips transmission of tomato spotted wilt virus. Proc Natl Acad Sci 102:5168–5173

123