Molecular resolution of a complex of potyviruses infecting solanaceous crops at the centre of origin in Peru

C. Spetz1, A. M. Taboada2, S. Darwich1, J. Ramsell1, L. F. Salazar2 and J. P. T. Valkonen1,3

1 Department of Plant Biology and Forest Genetics, Genetics Centre, SLU, PO Box 7080, SE-750 07 Uppsala, Sweden
2 International Potato Center (CIP), Apartado 1558, Lima, Peru
3 Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland

Correspondence
Jari Valkonen (at SLU)
jari.valkonen{at}vbiol.slu.se


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Peru is a centre of origin and domestication of the potato, pepper and tomato (family Solanaceae). Many potyviruses (genus Potyvirus) that infect these crops were described 20–30 years ago. However, definitive classification of these viruses as distinct species remains unresolved for several reasons, including their close serological relationships, similar symptomatology in test plants and lack of genomic sequence data. Using samples collected from Peru, we have determined the complete genomic sequence of two strains of Peru tomato virus (PTV) as well as near-complete sequences for two additional PTV strains. We also obtained partial sequences of four strains of Potato virus V (PVV). Comparisons with genomic sequences of Wild potato mosaic virus (WPMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV), Potato virus A (PVA) and other potyviruses established that all these viruses constitute different taxa (species). Phylogenetic comparisons indicated that PTV, PVV and WPMV are the most closely related species which, together with PepMoV, PVY, Pepper yellow mosaic virus and Pepper severe mosaic virus, constitute a group that is distinguishable from other potyviruses. Therefore, the members of this group may share a common ancestor. PVA does not belong to this group. PVV and PTV were also closely related serologically. However, PTV did not cross-protect against PVV and WPMV in tobacco plants or complement systemic infection of PVV and WPMV in pepper plants. Two biologically and phylogenetically distinguishable strain groups were identified within PTV and PVV. In future studies, the sequence data and virus-specific primers and probes for PTV, PVV and WPMV described in this study will enable accurate indexing of plants with respect to either single or mixed infection with these viruses.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Potyvirus (family Potyviridae) contains ~200 virus species that account for almost 25 % of known plant viruses (Shukla et al., 1994). Despite their prevalence, potyvirus classification is far from complete. Conventional virus classification has relied on particle morphology, serology, cross-protection and biological properties (Shukla et al., 1994). However, these criteria are sometimes insufficient to discriminate strains (defined by having ‘distinct but stable and heritable biological, serological and/or molecular traits’; Fauquet et al., 2003) from species (‘a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche’; Murphy et al., 1995) amongst potyviruses. Intraspecific variability may be large among these viruses and different strains of the same species may have different host ranges that overlap with those of other species. Furthermore, many species are serologically related. Similar difficulties in classification are encountered with other virus genera, such as Begomovirus (family Geminiviridae) (Fauquet et al., 2003). Therefore, genomic sequence data have become indispensable for demarcating virus strains and species (Van Regenmortel et al., 2000; Fauquet et al., 2003).

The potyvirus genome consists of a single-stranded messenger-polarity RNA of ~9700 bases. Sequences within the 3'-proximal portion of the genome are commonly used for species demarcation (Shukla et al., 1994; Berger et al., 1997), including the nucleotide or amino acid sequences of the coat protein (CP) coding region and the nucleotide sequence of the 3'-nontranslated region (3'-NTR). However, intraspecific variability may be larger within the 5'-proximal part of the genome, particularly in the P1 protein coding region and therefore these sequences are also useful for strain identification (Tordo et al., 1995; Aleman-Verdaguer et al., 1997; Kekarainen et al., 1999; Oruetxebarria & Valkonen, 2001; Onshima et al., 2002).

Many potyviruses cause economically significant yield losses in potato (Solanum tuberosum L.), pepper (Capsicum spp.) and tomato (Lycopersicon esculentum Mill.) crops throughout the world (Kyle, 1993; Jeffries, 1998; Stevenson et al., 2001). The coastal regions of Peru and the Andean regions of Peru and Bolivia constitute the centre of origin or the site of domestication for many cultivated potato, tomato and pepper species (Hawkes, 1990; Hancock, 1993; Smartt & Simmonds, 1995; Ochoa, 1999). Many, if not most, of the potyviruses that currently infect these crops elsewhere in the world may have originated in Peru, having been spread via contaminated germplasm (Salazar, 1971; Jones, 1981, 1987; Fribourg & Nakashima, 1984; Kyle, 1993; Jeffries, 1998; Stevenson et al., 2001). However, molecular data on the native Peruvian potyviruses are scarce and their molecular genetic diversity is unknown. Some virus diversity may have resulted from selection of viral progenies that were better adapted to hosts that have undergone genetic changes during domestication (García-Arenal et al., 2001). However, Peru is unique for the great abundance of wild relatives growing in close proximity to potato, tomato and pepper crops (Hawkes, 1990; Hancock, 1993; Smartt & Simmonds, 1995; Ochoa, 1999), which is expected to enhance the diversification of virus species and strains. Consequently, the exchange of viruses between solanaceous crops and their wild relatives (Jones, 1981) may contribute to strain differentiation and the evolution of new viral species. Thus, the study of potyviruses sampled from solanaceous crops in Peru is of particular interest.

Potato virus Y (PVY) is a widely studied type member of the genus Potyvirus (De Bokx & Huttinga, 1981), which infects potato, pepper and tomato plants in Peru and elsewhere (Salazar, 1971; Glais et al., 2002). PVY comprises many strains (De Bokx & Huttinga, 1981; Glais et al., 2002) and those infecting potatoes can be classified into at least four strain groups based on their ability to trigger a hypersensitive resistance response in standard potato cultivars (Jones, 1990; Valkonen et al., 1996). These PVY strain groups are largely consistent with the phylogenetic groups established on the basis of CP-encoding and 3'-NTR sequences (Van der Vlugt et al., 1993; Boonham et al., 1999), but there is no experimental data to indicate that either CP or 3'-NTR sequences are directly responsible for the symptomatic phenotypes on which the strain group concept is based. Molecular studies on isolates of PVYC indicate that this strain group (or pathotype) consists of two genetic groups (Blanco-Urgoiti et al., 1998), emphasizing that molecular studies increase the resolution of intraspecific variability. Indeed, molecular characterization is an efficient approach by which PVY isolates from different crops may be compared, since many PVY isolates from pepper and tomato do not infect potato cultivars systemically (Gébré-Sélassié et al., 1985).

Many potyviruses of solanaceous crops are to some extent serologically related to PVY and were initially described as deviant PVY strains but later reclassified as different species. For example, Pepper mottle virus (PepMoV) (Purcifull et al., 1975), Pepper yellow mosaic virus (PepYMV) (Inoue-Nagata et al., 2002) and Potato virus V (PVV) (De Bokx et al., 1975; Fribourg & Nakashima, 1984; Jones & Fribourg, 1986) were originally described as PVY. Sequence comparison was the only method by which these viruses could be correctly classified as unique species (Robaglia et al., 1989; Vance et al., 1992; Oruetxebarria et al., 2000; Inoue-Nagata et al., 2002). However, sequence data are not available for all potyviruses that infect solanaceous crops. Since some of the non-sequenced viruses were the first to be described, redundancies in classification could arise in instances where non-sequenced potyviruses appear to be conspecific with viruses that were later described under another name. Thus, the fact that the taxonomic status of non-sequenced viruses remains confused and/or disputed complicates the classification of potyviruses of known sequence. For example, it was proposed (Alvarez & Fernandez-Northcote, 1996; Stevenson et al., 2001) that PVV, a virus characterized from potato in Peru in the 1980s (Fribourg & Nakashima, 1984), may be a strain of Peru tomato virus (PTV) that was described in the 1970s in tomato and pepper crops grown in the desert valleys of coastal Peru (Raymer et al., 1972; Fribourg, 1979; Fernandez-Northcote & Fulton, 1980). Furthermore, Wild potato mosaic virus (WPMV) was isolated in 1974 from wild potatoes (Solanum chancayense Ochoa) growing in the lomas vegetation of the Peruvian coastal desert at Lachay (Jones & Fribourg, 1979). As with PVV, serological relatedness suggests that WPMV might be a strain of PTV (Adam et al., 1995). The recently determined genomic sequences of PVV (Oruetxebarria et al., 2000) and WPMV (Spetz & Valkonen, 2003) indicate that they are different taxa, but since no sequence data are available for PTV, the aforementioned questions remain open. Similarly, it is not known whether Pepper severe mosaic virus (PepSMV) or PepYMV are conspecific with PTV. The serological relationships between PTV, PVV, WPMV, PVY and PepMoV have been reported in many studies (Fribourg, 1979; Jones & Fribourg, 1979; Fernandez-Northcote & Fulton, 1980; Fribourg & Nakashima, 1984; Adam et al., 1995), further emphasizing the need to resolve the taxonomic positions of the viruses in this ‘potyvirus complex’ infecting solanaceous species. PTV has not been isolated outside Peru, whereas PVV occurs in a few potato cultivars in Western Europe (Jones, 1987; Oruetxebarria & Valkonen, 2001) and shows only limited sequence variability (Oruetxebarria et al., 2000; Oruetxebarria & Valkonen, 2001). Therefore, we undertook a molecular characterization of potyviruses infecting potato and pepper crops in the coastal areas of Peru, with an emphasis on PTV and PVV, with the aim of clarifying these taxonomic questions.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus isolates, test plants and inoculation.
Eighteen virus isolates were obtained from potato and pepper crops grown in the coastal areas of Peru. Viruses were transmitted by mechanical inoculation of infective sap to Nicotiana occidentalis, Nicotiana benthamiana, tobacco (Nicotiana tabacum cv. White Burley) and/or Datura metel in an aphid-proof screenhouse at the International Potato Center (CIP), Lima. Five virus isolates were selected for further analysis due to their differential reaction with PVV antisera in ELISA. Isolates PA10, PA11 and PA13 obtained from potato plants in 1999 reacted readily with PVV polyclonal antibodies (pAbs) obtained from Bioreba (Carroboro, NC), whereas isolates PPK11 and PPK13 obtained from pepper in 1999 reacted weakly with the PVV pAb obtained from Agdia (Elkhart, IN). These isolates were inoculated into tobacco (cv. Samsoun nn) at the Genetics Centre, SLU, Sweden. An isolate of PVV (SE1) was detected by ELISA in a landrace potato accession (Tärendö Oja) at the Nordic Genebank, Alnarp, Sweden, and included in the study as a new European isolate of PVV. The previously described isolate M4 of PTV (Fribourg, 1979; obtained from ATCC), Dv42 of PVV (Fribourg & Nakashima, 1984; Oruetxebarria et al., 2000) and WPMV (Jones & Fribourg, 1979; Spetz & Valkonen, 2003) were included for comparison. PTV-M4 and WPMV have been maintained in Nicotiana spp. since their isolation from pepper and the wild potato (S. chancayense), respectively.

Initial propagation of the new Peruvian virus isolates at SLU, Sweden, was carried out in a growth chamber in the laboratory (Weiss Umweltstechnik, Reiskirchen, Germany; photoperiod 18 h, light intensity 250 µE s-1 m-2, 17–19 °C). Two leaves from each 5-week-old tobacco plant were mechanically inoculated. Carborundum (silicon carbide, <400 mesh; Sigma-Aldrich) was used as an abrasive. At 16 days post-inoculation (p.i.), systemically infected, symptomatic leaves were collected and stored at -70 °C until use.

Potato clone A6 (Solanum demissumxS. tuberosum cv. Aquila) and potato cvs Pentland Dell, Pentland Ivory and King Edward were propagated as pathogen-free clones in tissue culture and transferred to soil for experiments. Pepper (Capsicum annuum cv. RNaky) and tobacco (cv. Samsoun nn) plants were grown from seeds. Experiments were performed in Fi-totron 600H growth chambers (Fisons Environmental Equipment, Loughborough, UK; photoperiod 16 h, light intensity 230 µE s-1 m-2, 17–19 °C, relative humidity 75 %). Inoculum was prepared by grinding 1 g frozen virus-infected leaves in 5 ml distilled water using a pestle and mortar. Leaves of 5-week-old plants were mechanically inoculated using Carborundum as an abrasive. After 14–16 days p.i., inoculated lower leaves and non-inoculated upper leaves of potato and pepper plants as well as the non-inoculated upper leaves of tobacco plants were sampled and tested by ELISA or RNA dot-blot hybridization. The plants were monitored for symptoms as above up to 21 days p.i. A total of six to eight plants of each species were inoculated with each virus in at least two experiments.

Cross-protection and complementation assays.
For testing cross-protection, groups of four tobacco plants (5 weeks old) were mechanically inoculated with PPK13 as above. At 21 days p.i., systemically infected leaves were challenge-inoculated with PVV-Dv42 or WPMV. Healthy tobacco plants (controls) were also inoculated with PVV-Dv42 and WPMV. At 29 days post-challenge inoculation, the uppermost fully expanded, non-inoculated leaves were collected and tested for PPK13, WPMV and PVV by RT-PCR (see below). Complementation assays of viruses in pepper plants were carried out similarly to the cross-protection tests. Both types of experiments were carried out twice.

Virus detection.
PVV, PVY, PVA and Potato virus X (PVX) were detected by double antibody sandwich ELISA (DAS-ELISA) as described (Oruetxebarria et al., 2000) using the following antibodies: monoclonal antibody (mAb) 53/8 to PVV (Adgen, Ayr, Scotland); pAb to PVV (Bioreba), PVY and PVX (Boehringer GmbH and Adgen); and a mixture of two mAbs to PVA (mAb 58/0 and mAb 58/6 from Adgen; Rajamäki et al., 1998). Leaf samples were weighed and ground in ELISA sample buffer (3 ml per g of leaves). Known amounts of purified virions (~200 ng) of PVV-Dv42, PVA-B11 and PVYO-UK (Table 1) were included for comparison. Absorbance was recorded at 405 nm with a Benchmark microtitre plate reader (Bio-Rad) at the time-point when 200 ng of purified virions generated an A405 value of 2·50.


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Table 1. Primers used for amplification of viral sequences

 
PPK11, PPK13 and WPMV were detected by RNA dot-blot hybridization. Total RNA was extracted from leaves using the Trizol reagent (Gibco BRL Life Technologies). RNA concentrations were measured at 260 nm using a GeneQuant II spectrophotometer (Amersham Pharmacia Biotech). RNA quality was checked by agarose gel electrophoresis. Similar amounts of RNA (2 µg) were dotted on to membranes (Hybond-C nitrocellulose; Amersham Pharmacia Biotech) and hybridized with RNA probes specific for WPMV, PPK13 and PPK11. The RNA probes were generated by amplifying the CP and 3'-NTR coding regions of PPK11, PPK13 and WPMV by PCR using the primer pairs PPK11-FHIND/PPK11-R1APA, PPK13-3HIND/PPK13-R4APA and WPMV-1HIND/WPMV-R1APA, respectively (Table 1). The amplified fragments were ligated into TA cloning vector pCRII (Invitrogen) previously digested with HindIII and ApaI. After linearization with HindIII, the viral sequences were transcribed and labelled using the DIG RNA labelling system (Boehringer). Dot-blot hybridization was carried out as recommended by the manufacturer (Boehringer).

For Western blot analysis, systemically infected upper leaves of tobacco plants inoculated with PVV-Dv42, PPK13 and WPMV were ground in extraction buffer (0·25 M Tris/HCl pH 7·0) at 1 g ml-1. The extract was diluted 1 : 1 with sample buffer (0·25 M Tris/HCl pH 6·8, 40 % glycerol, 15 % {beta}-mercaptoethanol, 2·6 mM SDS, 0·7 mM urea, 0·03 % bromophenol blue). Samples were boiled for 5 min and separated by 10 % SDS-PAGE. Proteins in the gel were then transferred to a Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech). Similar amounts of protein in samples were verified by staining the membrane with Ponceau S solution (Sigma Diagnostics). The membrane was probed with the PVV mAb 53/8 diluted in a buffer (1x TBS containing 2·5 % non-fat milk) followed by a secondary antibody (anti-mouse sheep pAb conjugated with horseradish peroxidase; Amersham Pharmacia Biotech) diluted in the same buffer. Bands were detected by chemiluminescence using the ECL method (Amersham Pharmacia Biotech).

Cloning and sequencing.
Virions were purified from infected tobacco leaves as described by Oruetxebarria et al. (2000). Purified virions were solubilized in a denaturing buffer (10 mM Tris/HCl, 1 mM EDTA) containing 1 % SDS (v/v), heated to 55 °C for 30 min and extracted with 1 vol. Tris/HCl (pH 7·5)-equilibrated phenol/chloroform/isoamyl alcohol (25 : 24 : 1). Extracted RNA was further purified with the RNeasy total RNA kit (Qiagen) according to the manufacturer's instructions. cDNA synthesis was performed with the Superscript II reverse transcriptase (Gibco BRL Life Technologies) primed with a poly(dT)18 oligonucleotide according to the manufacturer's instructions.

With PTV, the strategy described for the cloning and sequencing of the WPMV genome (Spetz & Valkonen, 2003) was used. This strategy is based on three degenerate primers (YCD, VAT and RKL; Table 1) designed on the basis of conserved sequence motifs in potyviruses (Spetz & Valkonen, 2003). First, the 3'-proximal region (NIb–3'NTR) of the virus genome was amplified and sequenced. The remainder of the viral genome sequence was obtained by the combined use of the degenerate primers and virus-specific primers designed on the basis of the sequence of the preceding clone. The order and orientation of cloning is depicted in Fig. 1. At least two clones from independent PCR reactions were sequenced in both directions for each of the genomic regions analysed in all viruses, unless specified otherwise.



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Fig. 1. Schematic presentation of the Peru tomato virus (PTV) genome structure and the strategy used for amplifying and sequencing the genomic fragments. Cloning was carried out in the 3'->5'orientation. Solid lines depict amplified fragments of PTV isolate PPK13; dashed lines represent amplified fragments of PTV isolate PPK11; dotted lines represent the regions amplified from PTV isolates V2 and M4. The primers indicated for each amplified fragment are described in Table 1. The amino acids that demarcate the putative cleavage sites for the viral-encoded proteinases P1, HCpro and NIapro of isolates PPK13 and PPK11 are shown below the polyprotein. The size (kDa) of each putative mature protein processed from the polyprotein is indicated within each box. P1, the first protein; HCpro, helper component proteinase; P3, the third protein; 6K1 and 6K2, 6 kDa proteins; CI, cylindrical inclusion protein; NIa, nuclear inclusion protein a; VPg, viral genome-linked protein; NIapro, the main viral proteinase; NIb, RNA-dependent RNA polymerase; CP, coat protein; 5'-NTR and 3'-NTR, nontranslated regions.

 
The 5'RACE (rapid amplification of cDNA ends) system (Life Technologies) was used for the amplification of the 5' end of the genome of the PPK13 isolate. cDNA synthesis was carried out using 2 µg total RNA primed with a PTV-specific reverse primer (PPK13-GSP1; Table 1), after which PCR amplifications were carried out with a nested PTV-specific primer (PPK13-GSP2; Table 1) and the 5'RACE-abridged anchor primer (5'AAP). Four clones from two independent PCR reactions were sequenced to determine the 5' end. All PCR reactions were carried out using the Expand high fidelity PCR system (Roche). PCR fragments were cloned into the TA vector pCRII and sequenced using a primer-walking strategy. Sequencing reactions were performed using the DYEnamic ET Terminator Cycle sequencing kit (Amersham Pharmacia Biotech) and analysed on an automated ABI PRISM XL377A DNA sequencer (Perkin-Elmer).

Amplification and sequencing of the complete genome of PPK11 were accomplished as described for PPK13. The 5'NTR-CI region was cloned using the forward primer T7-A4 (Table 1), designed on the basis of the first 28 nucleotides of the 5'-NTR of PPK13, and the reverse primer PPK11-CISR (Table 1). Cloning of the 5'-NTR, P1, P3, 6K1, CI, CP and 3'-NTR regions of the PTV isolates M4 and V2 was achieved using primers designed on the basis of the PPK13 sequence (Table 1).

Sequences of the PVV isolates PA10, PA11, PA13 and SE1 were amplified from total RNA extracts of infected tobacco or potato (SE1) leaves by RT-PCR with primer pairs ASC-T7/PVV-35 (5'-NTR+P1 region) and PVV-37/DT18 (CP+3'-NTR region) (Table 1) as described by Oruetxebarria et al. (2001). PCR fragments were cloned and sequenced as described above for PTV.

Sequence analyses.
Identities of viral nucleotide and amino acid sequences characterized in this study or obtained from databases (Table 2) were calculated as described by Aleman et al. (1996). Multiple alignments of sequences were made using the DNASTAR computer software package. Phylogenetic relationships were analysed with PHYLIP (Felsenstein, 1993). Protein distance matrices were calculated with PROTDIST using the dayhoff PAM matrix amino acid replacement model (Dayhoff et al., 1978). Phylogenetic trees were constructed using NEIGHBOR (Saitou & Nei, 1987) with 100 bootstrapped data sets to test statistical significance. A consensus tree was obtained using CONSENSE. Clustal X (Thompson et al., 1997) was used as a second method to evaluate the confidence of the phylogenetic trees. The trees were constructed using a neighbour-joining majority-rule consensus with 1000 bootstrapped replicates. Trees were visualized using TREEVIEW.


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Table 2. Virus isolates and database accession numbers

 
RNA secondary structures for the 3'-NTR of PTV, PVV, WPMV, PepMoV and PVY were predicted using the mFOLD program version 3.1 (Mathews et al., 1999) with parameters of 37 °C and ionic concentration corresponding to 1 M NaCl. The four most stable structures for each virus were analysed.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Serological relationships, host responses and virus–virus interactions
Isolates PA10, PA11 and the reference strain PVV-Dv42 (Oruetxebarria et al., 2000) were readily detected by ELISA using antibodies to PVV (Table 3). In contrast, the PVV antibodies reacted only weakly, albeit reproducibly, with isolates PPK11, PPK13 and the reference strain PTV-M4. The WPMV reference strain was not detected. Western blot analyses using the mAb 58/3 to PVV CP were consistent with the results of ELISA, indicating strong detection of PVV CP, relatively weaker but specific detection of PPK13 CP and no detection of WPMV. Antibodies to PVY, PVA and PVX did not react with any of the above viruses in ELISA (Table 3).


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Table 3. Serological and biological properties of virus isolates obtained from pepper and potato in Peru

 
PA10, PA11 and PVV-Dv42 produced similar mild mosaic and vein-clearing symptoms in tobacco plants (Table 3). However, PA10 and PA11 differed from PVV-Dv42 in that they caused vein necrosis on the lower side of systemically infected leaves in potato clone A6. PA10 and PA11 also differed from PVV-Dv42 based on infection of potato cv. Pentland Ivory. All three isolates infected the inoculated leaves of P. Ivory, but only PVV-Dv42 caused localized necrotic lesions (Fig. 2D). Neither of the isolates infected P. Ivory plants systemically and no infection or symptoms were detected in the inoculated or non-inoculated upper (new growth) leaves of potato cvs P. Dell and King Edward or in pepper cv. RNaky (Table 3). PA13 and SE1 were readily detected with the antibodies to PVV, but testing of these isolates on indicator plants was not continued due to mixed infections with other viruses (PA13 with PVX and SE1 with PVY). Thus, PA13 and SE1 were only included in the sequence analyses.



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Fig. 2. Detection of Peru tomato virus (PTV) isolates PPK13 (A) and PPK11 (B) and Wild potato mosaic virus (WPMV) (C) in inoculated leaves (IL) and upper (new growth) uninoculated leaves (UL) by RNA dot-blot hybridization using virus-specific RNA probes complementary to the viral CP coding region. Lane 1, potato clone A6; lane 2, potato cv. Pentland Dell; lane 3, potato cv. King Edward; lane 4, pepper cv. RNaky; lane 5, tobacco cv. Samsoun nn. The weak signals observed in the inoculated potato (lanes 1–3, all viruses) and pepper leaves (lower panel, WPMV) are indicative of limited local infection and/or residual amounts of viral inoculum. (D) Induction of localized necrotic lesions by the European Potato virus V (PVV) isolate Dv42 (a) and symptomless infection with the Peruvian PVV isolate PA10 (b) in inoculated leaves of potato cv. Pentland Ivory, which carries the gene Nvtbr for hypersensitive resistance to PVV. PVV infection in both leaves was confirmed by ELISA.

 
PPK11 and PPK13 and the reference strain PTV-M4 caused systemic infection of pepper plants and could therefore be readily distinguished from other viruses used in this study (Table 3; Fig. 2). Severe mosaic symptoms were observed and PPK13 also induced epinasty in pepper leaves (Table 3). In tobacco plants, the mosaic symptoms induced by PPK11 were mild, whereas PPK13 and PTV-M4 caused severe mosaic and ringspot symptoms. None of the isolates infected potato plants systemically, but RNA dot-blot hybridization revealed minute amounts of viral RNA in inoculated leaves (Fig. 2). While the possibility that the dot-blot detected residual amounts of inoculum cannot be excluded, these data may suggest limited local infections (Table 3).

Cross-protection by PPK13 against either PVV-Dv42 or WPMV was tested in tobacco plants (susceptible to all three viruses). Leaves systemically infected with the protector virus (PPK13) were subsequently challenged via inoculation with WPMV or PVV-Dv42. At 29 days after the challenge inoculations, the symptoms in the plants co-infected with PPK13 and either PVV-Dv42 or WPMV were no different from the plants infected with PPK13 alone. Additionally, upper (new growth) leaves that had not been inoculated were tested by RT-PCR using virus-specific primers (Table 1) and the results indicated that PPK13 did not provide cross-protection against PVV-Dv42 or WPMV (data not shown).

Because pepper cv. RNaky was resistant to PVV-Dv42 and WPMV and susceptible to PPK13, we tested whether co-infection with PPK13 could assist PVV-Dv42 or WPMV to overcome the resistance. Leaves systemically infected with PPK13 were inoculated with PVV-Dv42 or WPMV and the new upper, non-inoculated leaves were tested as above by RT-PCR at 29 days after the challenge inoculation. No systemic infection by PVV-Dv42 or WPMV was observed in the presence of PPK13, indicating no complementation or synergy between the viruses.

Genomic sequences of PTV
The complete genomic sequences were determined from viral RNAs that had been extracted from purified virions and cloned by RT-PCR. The genomes of PPK13 (EMBL database accession no. AJ437280) and PPK11 (AJ516010) were 9881 and 9892 nucleotides, respectively, excluding the poly(A) tail. Computer-assisted analysis revealed an optimal translation initiation region with a purine (A) at position -3 and a guanine at position +4 (AXXAUGG) (Fütterer & Hohn, 1996) and a single ORF was predicted to encode a polyprotein of 3061 amino acids in both viruses (Fig. 1). The putative polyprotein cleavage sites specific for the three viral proteinases P1, HCpro and NIa-Pro (Riechmann et al., 1992) were detected based on comparison with other potyviral polyproteins (Fig. 1).

To verify that PPK11 and PPK13 constituted de facto PTV isolates, the 5'-NTR–CI and NIb–3'-NTR portions of the genome (~75 %) of the PTV-M4 reference strain were determined and used for comparison. Furthermore, sequence analysis revealed that the plants infected with PPK11 also contained another virus isolate designated V2 that showed significant similarity to PPK13 (see below). The same regions as for M4 were cloned and sequenced from V2. Comparison of the sequences among PPK11, PPK13, M4 and V2 revealed high levels of identity within the CP, P3 and 6K1 regions and the portion of the CI region that was determined in M4 and V2. The nucleotide and amino acid sequence identities for any of these regions were >81·7 and >87·4 %, respectively (for CP, see Table 4). In contrast, the sequences of the 5'-NTR (59·2–94·8 %), P1 (61·1–98·8 %) and 3'-NTR (75·3–95·1 %) showed more variation (Table 4) but were within the range of intraspecific variability reported for isolates of other potyviruses such as Yam mosaic virus (Aleman-Verdaguer et al., 1997), PVA and PVY (Kekarainen et al., 1999). The polyprotein sequence identity of PPK11 and PPK13 was 88·6 %, which is comparable to the polyprotein identities among different strains of other potyviruses for which database sequences are available (e.g. Pea seed-borne mosaic virus, 85·5 %; Zucchini yellow mosaic virus, 87·5 %; PVY, 88·2 %; Sugarcane mosaic virus, 89·3 %; PVA, 91·6 %). These data indicated that PPK11, PPK13 and V2 are PTV.


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Table 4. Percentage sequence identity between Potato virus V (PVV), Peru tomato virus (PTV) and Wild potato mosaic virus (WPMV) isolates

 
Sequence analysis at the whole genome level showed that PPK11 was the most unique of all the PTV isolates (Table 4). One of the specific and distinct features of PPK11 was the 5'-NTR (223 nucleotides), which was 10 nucleotides longer than that of PPK13, V2 and M4. The P1 protein of PPK11 contained a stretch of amino acids (residues 20–40) that deviated substantially from the consensus sequence of other PTV isolates due to many nucleotide substitutions (data not shown). The polyprotein cleavage site between CI and 6K2 differed between PPK11 and PPK13 (Fig. 1). The 3'-NTR of PPK11 (474 nucleotides) had one additional nucleotide compared with the other three PTV isolates (data not shown).

Comparison of PPK11 and PPK13 polyprotein amino acid sequences with those of other viruses revealed the typical motifs conserved in potyvirus proteins (motifs were identical to those found in WPMV; see Spetz & Valkonen, 2003). The highest overall amino acid identity was found with WPMV (74·8 and 75·6 %, respectively) and PVV (73·6 and 74·4 %) and a lower identity with PepMoV (PPK11: 58·8 and 59·3 %; PPK13: 59·3 and 59·9 %) and PVY (PPK11: 53·4–53·8 %; PPK13: 53·7–54·3 %). These data support the classification of PTV as an independent virus species. However, the amino acid sequence identity of several mature homologous proteins among PTV, PVV and WPMV was much higher (Table 5).


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Table 5. Percentage sequence identity of Peru tomato virus (PPK13 and PPK11) compared with Wild potato mosaic virus (WPMV) and isolate Dv42 of Potato virus V (PVV)

 
The 3'-NTR region of the PTV strains PPK13 (473 nucleotides) and PPK11 (474 nucleotides) as well as PVV (446 nucleotides; Oruetxebarria & Valkonen, 2001) and WPMV (472 nucleotides; Spetz & Valkonen, 2003) are unusually long compared with other potyviruses. Secondary structure predictions of the 3'-NTR revealed a conserved structure formed by four major stem–loops in both PTV isolates (free energy value -143·5 kcal mol-1) and WPMV (-128·4 kcal mol-1) (data not shown). These secondary structures were similar to those predicted in the 3'-NTR of PVV (Oruexebarria & Valkonen, 2001) but were not found in PepMoV and PVY. They may be involved in the initiation of negative-strand synthesis of the viral RNA (Teycheney et al., 2000) as well as genome amplification (Haldeman-Cahill et al., 1998).

Intraspecific genetic variability of PVV
Isolate SE1 from this study was the first PVV isolate from Sweden to be characterized. Similar to other new PVV isolates in this study, the 5'-NTR–P1 and NIb–3'-NTR portions of the SE1 genome were cloned and sequenced. SE1 showed high sequence identity with PVV-Dv42 (Table 4) and other previously characterized PVV isolates from Europe (Oruetxebarria et al., 2000), thus further emphasizing the low variability of PVV in Europe. In contrast, the three PVV isolates from Peru possessed higher sequence variability. Isolates PA10 and PA11 differed more significantly from SE1 and PVV-Dv42 than did isolate PA13. The largest differences in nucleotide sequence identity in comparisons of PA10 and PA11 with SE1 and PVV-Dv42 were found within the P1 protein-encoded region (84·3–85·6 %) and the 5'-NTR (81·9–82·4 %) (Table 4). The respective nucleotide sequence identities of PA13 as compared with SE1 and PVV-Dv42 were much higher (~94 % for the P1 region and ~89 % for the 5'-NTR) but not as high as the respective identities between SE1 and PVV-Dv42 (98 % and 95 %, respectively).

Phylogenetic relationships
Phylogenetic relationships among PTV, WPMV, PVV and other potyviruses that infect solanaceous plants were analysed using the amino acid sequences of P1 (Fig. 3A) and CP (Fig. 3B). Isolates that in previous studies were considered to be the same species (e.g. PVY, PepMoV or PVV) (Van der Vlugt et al., 1993; Oruetxebarria et al., 2000) were likewise placed in highly supported, virus-specific groups in our analysis, regardless of the genomic region used. Thus, the phylogenetic analysis provided clear virus species demarcation consistent with the aforementioned pairwise sequence comparisons and putative identification of isolates as either PTV or PVV.



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Fig. 3. Phylogenetic relationships of potyviruses that infect solanaceous species, determined on the basis of the amino acid sequence of either P1 (A), CP (B) or the entire polyprotein (C). Bootstrap values exceeding 90 are shown. See Table 2 for sequence accession numbers. Plum pox virus (PPV) and Pea seed-borne mosaic virus (PSbMV) are not commonly found in the species of Capsicum and Solanum in the field but may infect other species of Solanaceae in the wild and thus were included as outgroups.

 
The PTV and PVV isolates described in this study belong to more than one strain based on the molecular data and phylogenetic analysis. PPK11 differed from the other three PTV isolates based on CP sequence analysis (Fig. 3B), but an additional strain group was identified using P1 sequences due to the separation of M4 from the other isolates (Fig. 3A). On the basis of CP sequences, PVV isolates PA10 and PA11 were placed into a group different from the European isolates and PA13 (Fig. 3B). However, PA13 was distinguished from all other PVV isolates based on P1 sequence data (Fig. 3B). Thus, sequence analysis of P1 proved more valuable than that of CP with respect to achieving a higher resolution of intraspecific variability.

PepSMV (Rabinowicz et al., 1993) and PepYMV (Inoue-Nagata et al., 2002) are two potyviruses isolated from pepper plants in Argentina and Brazil, respectively. Their CP sequences were included in our analysis but neither virus was grouped with PTV, PepMoV or any other virus tested (Fig. 3B). However, they belonged to the highly supported main cluster comprised of PVV, PTV, WPMV, PepMoV and PVY. This group was clearly separated from other potyviruses that infect solanaceous hosts (Fig. 3B), also supported by the analysis of the entire polyprotein sequence (Fig. 3C).


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The complete genomic sequence of PTV determined in this study resolves the taxonomic relationships among the closely related potyviruses known to infect potato, tomato and pepper in Peru since the 1970s. After PVY (Salazar, 1971), PTV was the next potyvirus to be described (Raymer et al., 1972; Fribourg, 1979; Fernandez-Northcote & Fulton, 1980; Fribourg & Fernandez-Northcote, 1982), but until now no sequence data were available on PTV. This lack of sequence data prevented the final assignment of the taxonomic positions of several closely related potyviruses that were described after PTV, including PepMoV (Purcifull et al., 1975; Vance et al., 1992), PepSMV (Feldman & Gracia, 1977; Rabinowicz et al., 1993), WPMV (Jones & Fribourg, 1979; Spetz & Valkonen, 2003), PVV (Fribourg & Nakashima, 1984; Oruetxebarria et al., 2000) and PepYMV (Inoue-Nagata et al., 2002). Our data show that PTV is related to, but not conspecific with, other described potyviruses (genus Potyvirus). Consequently, the taxonomic status of the aforementioned potyviruses of known sequence is now validated. With respect to virus species demarcation, our data emphasize that true relatedness can be established more accurately by comparing entire polyprotein sequences as opposed to segments therein (e.g. only CP sequences; Berger et al., 1997). This is illustrated by the fact that apparent relatedness among polyproteins in our study differed significantly from that among individual proteins or non-coding regions.

Phylogenetic analyses of CP, P1 and the entire polyprotein sequences unambiguously indicated that PVV, PTV and WPMV are the most closely related potyvirus species that infect solanaceous hosts. These viruses are grouped together with PepMoV and PVY in the phylogenetically defined ‘PVY group’. Based on the CP sequence data available for PepSMV and PepYMV, these viruses also belong to the phylogenetically defined ‘PVY group’. Viruses of the ‘PVY group’ commonly infect pepper, potato and/or tomato crops in the field, and all of these viruses can infect a number of tobacco cultivars under experimental conditions (see references above). These viruses have apparently evolved through a common genetic lineage and may share a common ancestor. Surprisingly, PVA does not belong to the ‘PVY group’ (Fig. 3) even though it occurs in Peru (Jones, 1981) and is the second most common potyvirus infecting potatoes in the northern hemisphere (Rajamäki et al., 1998).

Our data do not allow us to single out specific hosts that may constitute a driving force for selection and evolution (García-Arenal et al., 2001) of the potyviruses included in this study. Therefore, future detailed studies are required to define the genetic variability within the ‘PVY group’ and PVA, as well as the host response of cultivated forms and wild relatives of potato, tomato and pepper at the centre of origin in Peru and other Andean regions. The sequence data and virus-specific primers and probes described here will help us to address such diversity in future studies. However, as few as four PTV isolates, including the reference strain M4 originally described in Peru (Fribourg, 1979), were sufficient to reveal the wide genetic variability of PTV. Furthermore, comparison of the three isolates of PVV from potatoes in Peru revealed much larger genetic variability than found among the nine PVV isolates described from five European countries (Oruetxebarria et al., 2000; Oruetxebarria & Valkonen, 2001) including isolate SE1 from Sweden (this study).

No biological variability has been previously reported in PVV isolates from Europe. It is therefore significant that the two Peruvian PVV isolates PA10 and PA11 that were tested on potato cultivars belong to a different strain group than the European PVV isolates. The PVV isolates described in Europe induce cell death in potato cultivars such as Pentland Ivory, which carry the gene Nvtbr conferring a hypersensitive resistance response specifically to PVV (Jones & Fribourg, 1986; Jones, 1990; Oruetxebarria et al., 2000). This operational definition characterizes the PVV strain group 1 (PVV-1). Peruvian PVV isolates PA10 and PA11 infected the inoculated leaves of P. Ivory but caused no symptoms. Consequently, these strains belong to PVV strain group 2 (PVV-2). PVV-1 strains do not cause vein necrosis in leaves of the potato clone A6 (Oruetxebarria et al., 2000), in contrast to the original report on PVV from Peru (Fribourg & Nakashima, 1984). In this study, the differential response of potato clone A6 to PVV-1 from Europe and PVV-2 from Peru was demonstrated by direct comparison of the isolates from the two continents. The placement of different PVV isolates into strain groups 1 and 2 is supported by differential responses of potato plants to the viruses as well as phylogenetic analyses. Currently, PVV is uncommon in most potato crops in Europe, due most likely to the fact that Nvtbr and other PVV-specific resistance genes are widespread in currently grown cultivars (Jones, 1987, 1990; Barker, 1997). Introduction of PVV-2 to Europe might pose a risk to potato crops since it is not known whether the cultivars that are PVV-1 resistant would likewise be resistant to PVV-2, as was the case for the three cultivars tested in this study.

None of the PVV isolates, including those described in this study, has been shown to infect pepper plants systemically (Fribourg & Nakashima, 1984; Oruetxebarria et al., 2000). On the other hand, PTV is frequently detected in pepper (this study; Fernandez-Northcote & Fulton, 1980) but does not infect cultivated potatoes systemically (Fribourg, 1979; Fribourg & Fernandez-Northcote, 1982). Only limited PTV infection was apparent in inoculated potato leaves in the present study. WPMV infects neither pepper plants nor cultivated potato systemically, but these data are based on a single virus isolate (Jones & Fribourg, 1979; Spetz & Valkonen, 2003). In light of the original reports on the host range of PVV and PTV and the genomic sequences and serological data from this study, it is apparent that the provisional occurrence of PVV in pepper crops and the purported PTV infections in cultivated potatoes in the field (Alvarez & Fernandez-Northcote, 1996; Stevenson et al., 2001) may be misleading. It is more likely that the reported infections in pepper crops were actually caused by PTV and that the viruses found in potatoes were PVV. Cross-reaction of widely used anti-PVV antibodies with PTV in past studies probably caused confusion with respect to species identity, leading to the misconception that PTV and PVV were conspecific. In future studies, the data and diagnostic tools (Table 1, Fig. 2) described in this study will enable accurate indexing of plants with respect to either single or mixed infection with PTV and PVV.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr R. A. C. Jones for many valuable comments on the manuscript. Financial support from SJFR/Formas, Sweden (grant 22.0/2001-1223) is gratefully acknowledged.


   REFERENCES
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ABSTRACT
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METHODS
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DISCUSSION
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Received 7 March 2003; accepted 7 May 2003.